(Mycology Series, v. 29) José Ruiz-Herrera - Fungal Cell Wall - Structure, Synthesis, and assembly-CRC Press (2012)

Fungal Cell Wall
Structure, Synthesis, and Assembly

Second Edition
MYCOLOGY SERIES
Editor
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29. Fungal Cell Wall: Structure, Synthesis, and Assembly, Second Edition, José Ruiz-Herrera
Fungal Cell Wall
Structure, Synthesis, and Assembly
Second Edition
José Ruiz-Herrera
Centro de Investigación y de Estudios Avanzados
Irapuato, Mexico
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Dedication
This book, as was the first edition, is dedicated first to my wife, Carmen, with
love, for all the support, help, and understanding that she has given to me during
the last 51 years. I should also include in this dedication all those who have
brought joy to my life, my daughters and sons, Dr. Ethelia Ruiz-Medrano, José
Javier Ruiz-Medrano (in memoriam), Dr. Roberto Ruiz-Medrano, Dr. Carlos
Rubén Ruiz-Medrano, Psychoanalyst Carmiña Ruiz-Medrano, M.Sc., and Dr.
Salvador Francisco Ruiz-Medrano, plus all the members of the G2 generation:
Ivan, Carlos Adrián, Diego, Aurora, Roberto, Gilberto, Mariana Mercedes, and Ethelia
Contents
Preface...............................................................................................................................................xi
Acknowledgments....................................................................................................................... xiii
The author.......................................................................................................................................xv
Chapter 1 Introduction...............................................................................................................1
References.........................................................................................................................................5
Chapter 2 Cell wall composition.............................................................................................. 7

2.1 Introduction............................................................................................................................ 7
2.2 Cell wall composition...........................................................................................................8
2.2.1 Cell wall polysaccharides.......................................................................................8
2.2.2 Cell wall proteins................................................................................................... 11
2.2.3 Lipids from the cell walls..................................................................................... 12
2.2.4 Pigments................................................................................................................. 13
2.2.5 Inorganic salts in the walls.................................................................................. 14
2.3 Differentiation of the cell wall chemical composition................................................... 15
References....................................................................................................................................... 21
Chapter 3 Structure of the fungal cell wall..........................................................................29

3.1 Introduction.......................................................................................................................... 29
3.2 Methods for the study of cell wall structure................................................................... 29
3.3 Organization of the cell wall.............................................................................................. 31
3.4 The cell wall surface............................................................................................................ 32
3.5 Microfibrilar structure of the cell wall............................................................................. 35
3.6 Associations of the components of the cell wall............................................................. 37
3.7 Mechanical characteristics of the fungal cell wall.......................................................... 39
References....................................................................................................................................... 40
Chapter 4 Chitin........................................................................................................................45
4.1 Introduction.......................................................................................................................... 45
4.2 Structure of chitin................................................................................................................ 45
4.3 Chitin distribution in nature............................................................................................. 46
4.4 Chitin biosynthesis.............................................................................................................. 47
4.4.1 General properties of fungal chitin synthases.................................................. 47
4.4.2 CHS gene multiplicity in fungi............................................................................ 49
4.4.3 Classification and possible evolution of fungal chitin synthases................... 51
4.4.4 Structure of chitin synthases............................................................................... 52
vii
viii Contents
4.4.5 Regulation of chitin synthases............................................................................ 53

4.4.6 The mechanism of chitin biosynthesis............................................................... 55
4.5. Inhibitors of chitin synthases............................................................................................ 56
4.6 Cytology of chitin biosynthesis......................................................................................... 57
References....................................................................................................................................... 61
Chapter 5 Chitosan....................................................................................................................69
5.1 General characteristics and distribution of chitosan..................................................... 69
5.2 The functions of chitosan................................................................................................... 69
5.3 Chitosan biosynthesis and genetic control and phylogenetic relationships of
chitin deacetylases............................................................................................................... 70
References....................................................................................................................................... 72
Chapter 6 Fungal glucans........................................................................................................73

6.1 Introduction.......................................................................................................................... 73
6.2 Structure of glucans............................................................................................................ 73
6.3 Role of fungal glucans........................................................................................................ 76
6.4 Synthesis of glucans............................................................................................................77
6.4.1 Introduction............................................................................................................77
6.4.2 Introduction to the synthesis of β-1,3-glucans.................................................. 78
6.4.3 Stimulators and inhibitors of β-1,3-glucan synthases......................................80
6.4.4 Purification of β-1,3-glucan synthases................................................................ 81
6.4.5 Genes encoding β-1,3-glucan synthases............................................................. 82
6.4.6 Structure and phylogenetic characteristics of β-1,3-glucan synthases..........84
6.4.7 Activation of β-1,3-glucan synthases by a small GTPase................................. 85
6.4.8 Cytological aspects of the synthesis of β-1,3-glucans....................................... 86
6.5 Other glycosyl transferases involved in the synthesis of β-glucans............................ 87
6.6 Synthesis of β-1,6-glucans................................................................................................... 89
6.7 Synthesis of α-1,3-glucans................................................................................................... 91
References....................................................................................................................................... 95
Chapter 7 Proteins...................................................................................................................103
7.1 Introduction........................................................................................................................ 103
7.2 Chemical structure of glycoproteins.............................................................................. 104
7.3 Different classes of covalently bound glycoproteins.................................................... 106
7.3.1 GPI-proteins.......................................................................................................... 107
7.3.2 Proteins with internal repeats........................................................................... 107
7.3.3 Proteins associated with the wall through disulfide bonds......................... 108
7.4 Biosynthesis of glycoproteins.......................................................................................... 108
7.4.1 Synthesis of the inner core, or the “dolichol pathway”.................................. 109
7.4.2 Transfer of the inner core to proteins............................................................... 111
7.4.3 Processing and maturation of the N-linked oligosaccharides...................... 111
7.4.4 Further glycosylation of the inner core to form the outer chain of
glycoproteins........................................................................................................ 112
7.4.5 O-glycosylation of mannoproteins................................................................... 113
7.5 Synthesis of GPI-proteins................................................................................................. 114
7.6 Nonclassical protein secretion......................................................................................... 116
References..................................................................................................................................... 117
Contents ix
Chapter 8 Cytological aspects of cell wall synthesis.......................................................123

8.1 Introduction........................................................................................................................ 123
8.2 Synthesis of secretory and membrane-bound proteins............................................... 123
8.3 Translocation of proteins to the endoplasmic reticulum............................................. 125
8.4 The importance of protein glycosylation in translocation.......................................... 127
8.5 Transfer of proteins along the endomembrane system................................................ 129
8.5.1 From ER to Golgi................................................................................................. 131
8.5.2 Out from Golgi..................................................................................................... 134
8.6 The secretion mechanism in filamentous fungi........................................................... 135
8.7 The Spitzenkörper............................................................................................................. 135
8.8 Vesicles and chitosomes in fungal cell wall growth.................................................... 136
8.9 The porosome..................................................................................................................... 142
References..................................................................................................................................... 143
Chapter 9 Cell wall growth and expansion........................................................................ 149

9.1 Introduction........................................................................................................................ 149
9.2 Expansion of the cell wall................................................................................................. 150
9.2.1 Introduction.......................................................................................................... 150
9.2.2 The physical stages of nascent and mature wall and their relation to
cell wall expansion and fungal growth............................................................ 150
9.2.3 Intermittent growth of the cell wall.................................................................. 152
9.2.4 Mobilization of microvesicles and vesicles involved in wall growth.......... 153
9.2.5 Reinitiation of wall growth................................................................................ 156
9.3 Polarization of cell wall synthesis and apical growth.................................................. 162
9.3.1 Introduction.......................................................................................................... 162
9.3.2 Establishment of polarity................................................................................... 162
9.3.3 Development of the hyphal shape..................................................................... 165
References..................................................................................................................................... 168
Preface
Twenty years ago, I wrote the first edition of this book on the fungal cell wall struc-
ture and synthesis, a topic in which I have been interested for many years. As occurs in
Twenty Years After, second in the saga of The Three Musketeers, by Alexander Dumas, many
changes have occurred during this 20-year time period, beginning with those occurring
in my own person. The field of cell wall research is no exception, and the main topics
of investigation have oscillated from those mainly centered on the determination of the
chemical composition of the wall, and the biochemical and physiological aspects of cell
wall growth and expansion, to a field dominated, in great part, by molecular biology
and its ramifications. This change is obvious, since it is the wave in biology in general. It
is positive because it has brought light to many aspects of the behavior of the cell wall,
the synthesis of its main components, and the enzymes involved and their regulation.
However, it is negative because many important aspects have been left out because they
are not fashionable.
With this background, I have dared to take the responsibility of writing this second
edition, maybe without taking into consideration that I am probably old-fashioned and
perhaps without the necessary background to tackle the new ideas on the subject. But,
with the courage to engage in an attractive enterprise and my long interest in the fungal
cell wall, I expect to cover these deficiencies. Only the readers will judge if I have suc-
ceeded or not.
The cell wall is an attractive subject of study from many points of view, mainly because
it is the structure responsible for the way of life of a large number of phyla; bacteria, fungi,
algae, and plants, that is completely different from the animals and related wall-less organ-
isms, with regard to mechanisms of nutrition, mobility, the capacity to colonize distinct
ecological niches, and so forth. The appearance of the eukaryotic wall, completely differ-
ent from the prokaryotic one, was a giant step in evolution that also marked the departure
from wall-less organisms. This process was followed by the acquisition of walls with dif-
ferent composition by fungi, a process that separated them from plants and algae, and
later, by the specialization of the fungal enzymes involved in the synthesis of the different
components of the cell wall from the several groups of these organisms. These processes
can now be analyzed using the modern phylogenetic, genetic, and in silico tools. The chem-
ical characteristics of the different components of the cell wall can also be approached now
with modern analytical procedures of extremely high sensitivity. The knowledge of the
mechanisms of action and interaction of the enzymes involved in the synthesis of the wall
components is now within our reach with the advent of techniques for gene isolation and
mutation, and the concepts introduced by synthetic biology. Modern cytological proce-
dures, the use of fluorescent probes, and new microscopic techniques have added excellent
results to our knowledge of the location, displacement, and deployment of enzymes and
organelles involved in wall growth.
xi
xii Preface
All these concepts have made a great impact on the applied aspects of mycology; our
knowledge of the bases of fungal pathogenicity to both man and animals and plants,
where the wall plays an important role, the ecological importance of fungi, and the use
of fungi in the production of a great number of compounds with important applications.
With all this acquired knowledge, it is surprising that many aspects related to the
fungal wall remain either unknown or untouched: isolation and structural analyses of
chitin and β−glucan synthases; mechanism of β-1,6-glucan biosynthesis; detailed physical
structure of the cell wall; mechanical properties of the cell wall and their relation to wall
growth and expansion; mechanisms of association of moonlight proteins into the wall; the
development of specific antimycotics based on chitin inhibition, and so forth.
This book has been divided into chapters with almost the same topics as the first
edition to try and allow those readers interested in only some selected aspects of the gen-
eral subject to read them as separate reviews. In the first chapters, I present a review of
the chemical composition and structure of the fungal cell wall, a matter almost closed to
modern research. These chapters are followed by chapters describing the structure and
synthesis of the most important components of the fungal cell wall. The last chapters are
devoted to the analysis of the mechanisms of cell wall expansion and growth. For the sake
of completeness, I have provided brief introductions on the basic concepts from which our
modern ideas originated, citing older publications where necessary.
José Ruiz-Herrera
Irapuato, Mexico
Acknowledgments
I want to thank all my collaborators and students for their work which helped to obtain the
results from my lab that are included in this book, the Centro de Investigación y Estudios
of the National Polytechnic Institute for the facilities received, and the Consejo Nacional
de Ciencia y Tecnología (CONACYT), Mexico for the partial support of my research.
xiii
The author
José Ruiz-Herrera, Ph.D., is Professor Emeritus in the Department of Genetic Engineering
at the Center for Research and Advanced Studies of the National Polytechnic Institute,
Mexico (Irapuato Unit).
He was born in Mexico City, and received his professional degree in microbiology at
the National Polytechnic Institute in Mexico City, and his Ph.D. in microbiology at Rutgers
(New Jersey).
Dr. Ruiz-Herrera was Professor and Chairman of the Department of Microbiology at
the School of Biological Sciences at the National Polytechnic Institute, and the Department
of Genetics and Molecular Biology at the Center for Research and Advanced Studies in
Mexico City. He was creator and director of the Institute of Experimental Biology of the
University of Guanajuato in Mexico, and has been visiting professor and/or visiting inves-
tigator at the University of California, San Diego and Riverside, and at the Universities of
Valencia, Salamanca, Sevilla, and Extremadura in Spain.
He has been the recipient of important awards, among which the following may be
cited: Award of the Academy of Scientific Research, Mexico; Miguel Hidalgo y Costilla
Award from the Congress of the State of Guanajuato, Mexico; Elsevier/Scopus/Science
and Technology Council of the State of Guanajuato Award; the Ruth Allen Award of
the American Phytopathological Society; and the National Award in Sciences from the
Mexican Government.
He is a member of several scientific societies, having served as president of the Mexican
Society for Microbiology and the Mexican Society for Biochemistry, and has served on
many scientific committees. He is reviewer for several prestigious journals and serves or
has served as associate editor for some of them.
Dr. Ruiz-Herrera has published more than 220 articles in international journals and
presented over 230 lectures. He has directed 53 postgraduate theses: 24 M.Sc. theses and
29 Ph.D. theses.
His current research interests include the structure and synthesis of the fungal
cell wall; synthesis of chitin and glucans; molecular bases of fungal differentiation and
morphogenesis; dimorphic transition in fungi; and, the role of polyamine metabolism
in fungal differentiation.
xv
chapter one
Introduction
There is a phrase from Winston Churchill that was applied within a political or socio-
logical context on how we end up being influenced by our own creations: “We shape our
houses, and later they shape us.” I use this phrase in relation to organisms such as bacte-
ria, archea, algae, plants, and fungi that possess cell walls. The presence of this cell wall
structure creates the apparent vicious circle between the morphology of these cells and the
shape of their walls. Which is due to which? I think that it is important to break the circle,
and clearly state that these cells owe their shape to the cell wall, but that the architecture
of the wall is the product of the vectorial mechanisms of wall assembly imposed by the
cell. Of course shape is not the only important function of the cell wall, rigidity is another
one. Without cell walls, sequoias would be no more than shapeless masses of cytoplasm,
the same as a vertebrate that has lost its skeleton.
That shape of these organisms depends on the form of the cell walls can be easily
demonstrated. If walled cells were subjected to the action of lytic enzymes that destroyed
their walls, they would be converted into protoplasts retaining their integrity only if
maintained in a hypertonic medium that equilibrated the intracellular pressure, which
is normally higher than that from the tire tube of a racing bicycle. But they would adopt
a spherical shape, since spheres have the minimal surface/volume ratio. If placed in a
hypotonic medium, the absence of the protective structure would make the cells blow out,
because of the difference in internal versus external pressure (see Figure 1.1). The opposite
experiment would be to isolate the cell walls such that, free of internal components, they
would still retain the original shape of the organism, although deflated, of course.
According to these simple concepts, it may be concluded that all differentiation pro-
cesses that bring about a morphogenetic alteration in these organisms, have the cell wall
as their final target. All changes in morphology will be due to alterations in the shape of
the cell wall. Therefore, this is the importance of analyzing the mechanisms involved in
the synthesis and organization of this cellular structure.
Acquisition of a wall by prokaryotic cells was an early event in evolution, a process
that occurred about 3.5 billion years ago, and by eukaryotes probably at the appearance of
the precursors of the primitive algae, plants, and fungi more than 1 billion years ago. As
indicated above, in either type of cells, the most important function of the original wall
was to preserve cell integrity by providing support to the cell membrane to withstand the
difference in osmotic pressure between the cytoplasm and the external medium, permit-
ting its growth in extremely hypotonic media.
Besides this crucial role, when cellular functions became more sophisticated, other
important functions of the wall were established, among others: (i) to protect the cell from
the action of enzymes and different deleterious compounds, (ii) as indicated above, to
provide all the extreme variety in cellular shapes necessary for different functions such
as mating, dispersal, colonization, and so forth, (iii) to bear molecules responsible for
the recognition of predators or hosts, (iv) to accumulate enzymes which digest different
impermeable compounds such as polysaccharides or proteins into small products that can
penetrate the permeability barrier of the cell in order to be further metabolized, and (v) to
1
2 Fungal cell wall: Structure, synthesis, and assembly
Figure 1.1 Cell wall digestion and protoplast lysis. (A) Germlings of Mucor rouxii grown in complex
media. (B) Protoplasts obtained from germlings by treatment with a lytic complex maintained in a
hypertonic medium. (C) Lysis of protoplasts by transfer to hypotonic medium.
convey information to the nucleus in regards to the environment, allowing the cell to con-
trol secretion of specific molecules, rate of growth, and other specific reactions in response
to different types of stimuli. Besides, it must be recalled that in mushrooms, fruit bodies,
large brown algae, and plants, the wall constitutes the skeletal structure that provides the
rigidity necessary to sustain the full organism (see Table 1.1).
If the functions are similar, the structure and chemical composition of the cell walls
of prokaryotes and eukaryotes are basically different. The origin for this difference is evo-
lutionary. Prevalent ideas sustain the concept that all eukaryotic organisms descend from
an ancestral unicilliate eukaryote (Cavalier-Smith 2002, 2004; Steenkamp, Wright, and
Chapter one: Introduction 3
Table 1.1 Main Functions of the Cell Walls

1. Resistance to the difference in internal and external osmotic pressure
2. Resistance to harmful physical, chemical, and biological aggression
3. Provision for the shape of the cell
4. Recognition of external surfaces: inert or biological
5. Reception of external stimuli
6. Selective permeability to large molecules
7. Accumulation of molecules important to the physiology of the cell, including nutrition
Baldauf 2006). Accordingly, at the time of the appearance of cell-walled eukaryotic organ-
isms, the prokaryotic cell wall had to be substituted by a completely different structure,
more adequate to fit the necessities of larger organisms. The structure of the wall in most
prokaryotic organisms is a giant tridimensional net, the sacculus, made of short chains of
a repeating disaccharide made of N-acetylglucosamine and N-acetylmuramic acid in bac-
teria or N-acetylalosaminouronic acid in archea, covalently bound through polypeptidic
bridges made of a few amino acids (the peptidoglycan in bacteria, and the pseudopeptido-
glycan in archeae). Characteristic of these structures is the presence of rare and D-amino
acids, some joined through nonpeptidic bonds.
On the other hand, the structure of the cell walls from eukaryotic organisms is based
on the presence of microfibrils made by the association through hydrogen bonding of
long chains of linear polysaccharides. Characteristically, microfibrillar polysaccharides
are polymers of a single type of sugar bound through glycosidic β-linkages. Repeatedly,
the structure of the eukaryotic cell wall has been compared to synthetic composites made
of a structural component responsible for the resistance to tensions, and an amorphous
component that confers resistance to pressure and protects the structural component from
fracture and from the action of deleterious components of the environment. The main
structural microfibrillar components, depending on the type of organism, are cellulose
and chitin. Both of them are linear polysaccharides made of a large number (normally
more than 2000 units) of either glucose for cellulose or N-acetylglucosamine (2-acetamido-
2-deoxy-D-glucose, GlcNAc) for chitin, joined by β-1,4-linkages. Because of the crystalline
arrangement of both polysaccharides that eliminates water from their structure, they are
extremely insoluble, and with a high-tensile strength, higher for chitin than for man-made
fibers (steel, carbon, or boron fibers) (reviewed by Ruiz-Herrera and Ruiz-Medrano 2004)
(see Table 1.2). The chemical components that constitute the amorphous part of the cell
wall are different nonmicrofibrillar polysaccharides, proteins, and lipids, plus salts and
pigments in small amounts (see Sentandreu et al. 2004 for review). Recently, and due to
their important roles in the cell wall, great interest has been placed on fungal wall pro-
teins, recognizing, in general, two different classes according to the way they associate
with other wall components: noncovalently bound and covalently bound proteins.
Table 1.2 Structural Components of the Cell Wall from

Different Organisms
Prokaryotes (Nonfibrillar) Archea Pseudopeptidoglycan
Bacteria Peptidoglycan
Fungi Chitin
Eukaryotes (Fibrillar) Algae Cellulose
Plants Cellulose
The importance of the cell wall can be measured by the fact that in Saccharomyces cere-
visiae, about 20% of its genetic information is related to the composition and structure of
the wall (De Groot et al. 2001), and that the cell wall in fungi makes up close to 30% of the
cell dry weight (Valentin et al. 1987; Fleet 1991), 80% to 90% of which is made by polysac-
charides (Bartnicki-García 1968).
The wall is far from being a static structure; it must suffer physical and chemical
changes, some of them very rapid, in order to fulfill its different functions. The nascent
eukaryotic cell wall is in a deformable state called viscoelastic, but as it matures it becomes
rigid and resistant to enzymatic attack, a prerequisite necessary to protect the protoplast
from the harming extracellular conditions. For example, fungi can grow in extremely
hypotonic media, which subject them to large differences in osmotic pressure, and hyphae
can traverse long distances under aggressive environments during colonization, search for
food or for sensitive hosts, and disseminate spores. In the same sense, trees have to sustain
an amazing amount of weight. Were it not for the cell wall, these organisms would be
unable to perform those tasks or survive in hostile environments. The process of transfor-
mation of the viscoleastic wall into a rigid one involves the organization of all the compo-
nents that constitute the whole structure, and the establishment of covalent bonds among
them, probably by the action of enzymes present in the wall itself. But some other func-
tions of the cells or the organisms, such as expansion, branching, mating, and so forth,
require changes in the structure of the wall; accordingly, the organisms must (and do) pos-
sess mechanisms to transform the wall from rigid into viscoelastic again. These involve
hydrolytic activities or changes in pH or ion binding capacity that produce a controlled
alteration in the mechanical properties of the wall that allow its deformation under the
high turgor pressure of the cell.
In the specific case of fungi, changes in cell wall composition and structure also occur
during sporulation and the formation of resistance bodies that help the organism to be dis-
persed, and to withstand harmful conditions for large periods of time. Some plant patho-
gens contain special structures that help them to cross the host barriers during invasion.
Additionally, through the production of large amounts of certain wall-bound polysaccha-
rides, fungi can escape from the host immunological defenses or unchain allergic reactions.
Study of the fungal cell wall, as occurs with any specific scientific matter, has gone
through different stages. The original problem with its study was the necessity to develop
methods for its isolation without alterations in its chemical composition. This required the
elimination of the originally used harsh chemical procedures, and its substitution by mild
physical ones. Once this problem was solved, a frantic stage of chemical analysis of the cell
wall from organisms belonging to almost all taxonomic groups followed. This stage has
practically come to an end, although some minor groups remain almost untouched. The
following stage in the study of the cell walls was the analysis of the mechanisms involved
in the synthesis of the most important wall components. This process almost coincided
with the development of the methods for gene cloning that greatly facilitated the task.
Most of the studies that are now in progress refer to the mechanisms that regulate cell wall
organization, biosynthesis and turn over, and the cellular bases of cell wall growth and
polarization. The advent and evolution of molecular biology techniques, and cell biology
methods such as the selective labeling of cell organelles has attracted a large number of
scientists to these fields. This is fortunate, but has made other important aspects of these
fields remain completely or almost completely forgotten; for example, the mechanisms of
the physical bases of growth and cell expansion that have remained almost untouched
for many years; the mechanisms of the reactions that convey information from the envi-
ronmental cues to alter the structure and expansion of the cell wall; and finally, critical
Chapter one: Introduction 5
analysis of the prevalent idea that the cell walls from all fungal species have the same basic
structure, organization, and behavior as the most popular models of study, a generaliza-
tion that may be far from being true.
Almost 20 years ago, I wrote a book that reviewed all the different aspects of study
of the cell walls (Ruiz-Herrera 1992). Since this time, and as occurs in all fields of science,
great advances have taken place in our knowledge of the fungal cell wall. This has moved
me to write a second edition trying to bring to scholars and students interested on this
subject, not a revised version of the former text, but a completely new vision of what we
now know of the fungal cell wall. For the sake of completeness, I have included data on
subjects that have remained almost unchanged or closed during this time, and the reader
may find them similar to their description in the former edition. Also, thinking of those
readers who may be interested only in specific aspects of the fungal cell walls, I have tried
to contain in each chapter all the material relevant and necessary for the comprehension
of the subject indicated in that chapter title; a necessity that has obliged me to repeat some
ideas and concepts in different chapters. I hope the readers do not find this repetitious.
Accordingly, if necessary, each chapter may be read independently of the rest, but it is
my expectation that most readers will decide to go though all of them. If this objective is
reached, I will be more than satisfied with the work done.
References
Bartnicki-García, S. 1968. Cell wall chemistry, morphogenesis and taxonomy of fungi. Annu. Rev.
Microbiol. 22:87–108.
Cavalier-Smith, S. 2002. The phagotrophic origin of eukaryotes and phylogenetic classification of
Protozoa. Int. J. Syst. Evol. Microorg. 52: 297–354.
Cavalier-Smith, S. 2004. Only six kingdoms of life. Proc. R. Soc. Lond. B. 271:1251–1262.
De Groot, P. W. J., Ruiz, C., Vazquez de Aldana, C. R., Andel, A., Caubin, J., Arroyo, J., Garcia, J. C.,
Gil, C., Molina, M., Garcia, L. J., Nombela, C., and Klis, F. M. 2001. A genomic approach for the
identification and classification of genes involved in cell wall formation and its regulation in
Saccharomyces cerevisiae. Comp. Funct. Genomics. 2:124–142.
Fleet, G. H. 1991. “Cell Walls.” In The Yeasts, Vol. 4. Edited by A. H. Rose and J. S. Harrison, 199–277.
New York: Academic Press.
Ruiz-Herrera, J. 1992. Fungal Cell Wall: Structure, Synthesis, and Assembly. Boca Raton, FL: CRC Press.
Ruiz-Herrera, J. and Ruiz-Medrano, R. 2004. “Chitin Biosynthesis in Fungi.” In Handbook of Fungal
Biotechnology, 2nd ed. Edited by P. K. Arora, 315–330. New York: Marcel Dekker.
Sentandreu, R., Elorza, M. V., Valentin, E., and Ruiz-Herrera, J. 2004. “The Structure and Composition
of the Fungal Cell Wall.” In Pathogenic Fungi: Structural Biology and Taxonomy. Edited by G. San-
Blas and R. Calderone, 3–39. Norfolk, UK: Caister Academic Press.
Steenkamp, E. T., Wright J., and Baldauf, S. L. 2006. The protistan origins of animals and fungi. Mol.
Biol. Evol. 23:93–106.
Valentin, E., Herrero, E., Rico, H., Miragall, F., and Sentandreu, R. 1987. Cell wall mannoproteins dur-
ing the population growth phases in Saccharomyces cerevisiae. Arch. Microbiol. 148:88–94.
chapter two
Cell wall composition

2.1 Introduction
The initial studies on the fungal cell wall had as their main objective to determine its
chemical composition compared to the cell wall from plants. These studies originally
involved the isolation of the cell wall using harsh chemical methods that respected the
integrity of the most resistant compounds only. The first example of these types of studies
was the isolation of chitin (named at that time fungine) from Agaricus volvaceus by Henry
Braconnot (Braconnot 1811). As the studies of the structure and chemical composition of
the cell wall of fungi became more refined, it became more obvious that chemical methods,
even when using lower concentrations of acid or alkali, were deleterious for important
components of the cell wall. Accordingly, authors turned to the use of mechanical proce-
dures to break the cells, and to separate the walls by centrifugation, taking advantage of
the fact that they are the heaviest components of the fungal cells. Chemical methods were
left only for experiments designed to measure polarization of the synthesis of the most
resistant polysaccharides: chitin, β-glucans, or chitosan (for example, Bartnicki-García and
Lippman 1969, 1977; Sentandreu and Ruiz-Herrera 1978), and for analysis of the material by
physical techniques, X-ray diffraction, electron diffraction, and electron microscopy (for
example, Aronson and Preston 1960).
It is not an exaggeration to state that now the most common method of cell breakage
for the isolation of intact walls, is to shake them in the presence of glass beads of a diam-
eter around 1 mm. The types of instruments used depends on the size of the sample, but
may go from the most sophisticated ones, to the use of simple centrifuge tubes agitated
in a vortex-type mixer. Taking into consideration that the cell wall contains proteins, and
that proteases are liberated during the breakage procedure, it is common to add prote-
ase inhibitors to preserve them. Depending on the future analysis to be made, the walls
are washed with buffer and saline solutions, and if necessary, with detergents or organic
solvents. Purity of the isolated walls is then normally confirmed by microscopic analysis
(either light or electronic, as required). A thorough review of all these procedures was
described in the first edition of the book (Ruiz-Herrera 1992).
These studies have demonstrated that the fungal cell wall has an extremely com-
plex chemical composition that may be different in the several taxonomic groups, from
the phylum to the genus, and even among the species. This idea, originally stated by
Bartnicki-García (1968), pointed out the chemotaxonomic value of these analyses, and it
was followed by several authors who have used different wall components and synthases
involved in their synthesis as taxonomic tools, or to analyze the evolution of fungal taxa
(see Table 2.1). Among these studies we may cite Bernabé et al. (2002), whose study used
the presence of certain types of polysaccharides from the cell wall as a taxonomic deter-
minant for some Ascomycota species; Coronado et al. (2007), who analyzed the presence of
discrete motifs in the cell wall proteins of yeasts as evolutionary markers; and our analy-
sis of the evolution of the fungal cell wall by analyses of the covalently bound wall pro-
teins and the enzymes involved in the synthesis of wall polysaccharide (Ruiz-Herrera and
Ortiz-Castellanos 2010). Also, the identification of specific components of the cell wall in
7
Table 2.1 Classification of Chemotypes of Fungal Cell Walls

Original class Cell wall components Taxonomic groups*
IV Chitosan, Chitin Zygomycota
V Chitin, Glucans Chitridiomycota
Euascomycota
Homobasidiomycota
Deuteromycota
VI Mannan, Glucan Hemiascomycota
VII Mannan, Chitin Heterobasidiomycota
Source: Modified from Bartnicki-García, S., 1968, Cell Wall Chemistry,
Morphogenesis and Taxonomy of Fungi, Annu. Rev. Microbiol., 22:87–109.
* Excluding taxonomic groups not considered fungi any longer.
different species has revealed their value in taxonomic and ecological studies. When these
specific components are antigenic, they have been used for the development of diagnostic
procedures in the medical field.
These analyses have also revealed that the chemical composition of the wall from a
fungal species may change depending on the age of the culture, the environmental condi-
tions of growth, the use of solid or liquid media, the carbon and nitrogen sources, ion con-
centration, temperature, pH, illumination, addition of different compounds, and so forth.
These data reveal a complex regulation of the cell wall synthesis that we are only starting
to understand.
Chemical analyses of the fungal cell wall have revealed the presence of polysaccha-
rides, proteins, lipids, and other components in smaller proportion: pigments, and inor-
ganic salts. Of these, polysaccharides are the most abundant components, comprising
about 80% of the dry weight, followed by proteins ranging from 3% to 20%, depending on
the organism and growth conditions. Some early studies reported the presence of nucleic
acids in the cell walls of several fungi, but modern concepts led to the consideration that
they were really contaminant material liberated during cell breakage that bound to the
wall by ionic attractions. According to the roles played by the different wall components,
they can be divided into structural and amorphous components (see Table 2.2).
Hydrolysis, usually acid, of the cell walls from fungi representing the different taxa
have revealed that the most abundant sugars are hexoses, in the following order of abun-
dance and distribution: glucose, glucosamine (a hydrolytic product from chitin and chi-
tosan), mannose, galactose, and galactosamine. Uronic acids, mainly glucuronic acid,
pentoses (mostly arabinose and ribose), and deoxyhexoses (mainly fucose) have been found
also (see Table 2.3).
2.2 Cell wall composition

2.2.1 Cell wall polysaccharides
As expected from the results obtained by analysis of the sugars released by wall hydro-
lysis, glucans are the most abundant polysaccharides present in the fungal wall, but their
distribution may be taxonomically restricted. These polysaccharides have been classified
according to the type of bond linking the glucose units. Thus we recognize α-glucans,
where sugar units are linked through an α-1,3-bond, and β-glucans of two types of link-
ages: β-1,3-glucans, and β-1,6-glucans. α-1,3-glucans have been identified in a large number
Chapter two: Cell wall composition 9
Table 2.2 Components of the Fungal Cell Walls

Class Cellular function Chemical nature Examples
Structural (Fibrillar) Rigidity Polysaccharides Chitin
Shape — β-1,3-glucans
Amorphous Cementing Polysaccharides, Mannogalactan
Enzymatic Glycoproteins Invertase
Recognition — Aglutinins
Table 2.3 Main Sugars Identified in Hydrolyzates of Fungal Cell Walls

Taxonomic Groupa Glc Gal GlcN Mann GalN Glucur Rham Fuc Xyl Ara
Chitridiomycota ++ + +++ tr tr 0 tr +/– +/– +/–
Zygomycota + + +++ + 0 ++ tr + 0 0
Hemiascomycota +++ + + +++ 0 0 0 0 0 0
Euascomycota +++ ++ ++ + + + +/– 0 tr tr
Heterobasidiomycota +++ tr + ++ 0 + 0 tr tr tr
Homobasidiomycota +++ + ++ + tr 0 + + + +
Source: Modified from Ruiz-Herrera, J., 1992, Fungal Cell Wall: Structure, Synthesis, and Assembly, Boca Raton,
FL: CRC Press. With permission from Taylor & Francis.
Note: tr = traces; +/– = less than 1%; + = 1.1–5%; ++ = 6–20%; +++ = more than 21% of the total sugars present
in the wall.
a Glc = glucose; Gal = galactose; GlcN = glucosamine (a product of polysaccharides containing glucosamine or
N-acetylglucosamine); Mann = mannose; GalN = galactosamine (a product of polysaccharides containing

galactosamine or N-acetylgalactosamine); Glucur = glucuronic acid; Rham = rhamnose; Fuc = fucose; Xyl =
xylose; Ara = arabinose.
of Ascomycota, and in a more restricted number of Basidiomycota species, some of them

human pathogens (Bacon et al. 1968; Kanetsuna and Carbonell 1971; Niederprum and
Wessels 1969), where they may be involved in virulence (San-Blas, San-Blas, and Serrano
1977). The recent studies on fungal taxonomy and phylogeny that took out groups of fun-
gal-like organisms such as Oomycetes from the kingdom Fungi, also led to the concept
that cellulose is absent in this kingdom, although some minor polysaccharides may have
β-1,4-glucans bonds (see below). β-glucans are present in a larger numbers of fungi; β-1,3-
glucans are present in Zygomycota, Basidiomycota, and Ascomycota phyla, whereas β-1,6-
glucans are apparently present only in Ascomycota and Basidiomycota (see Ruiz-Herrera
and Ortiz-Castellanos, 2010). One interesting characteristic of β-1,3-glucans is that they
may have cell locations other than the wall itself; accordingly, they may be intracellular
or be secreted to the medium; also their structure and molecular size is extremely vari-
able, being linear or branched (Ruiz-Herrera, 1992). The most characteristic β-1,3-glucans
that contribute to the structural rigidity of the cell walls are the large linear molecules,
associated in the form of microfibrils (Kopecká and Krieger 1986; Larriba, Morales, and
Ruiz-Herrera 1981). Similar to β-1,3-glucans, β-1,6-glucans constitute a large group of poly-
saccharides with different molecular size, structure, chemical characteristics, and cellular
location (see Ruiz-Herrera 1992 for review). The most widely studied β-1,6-glucan is the
one present in the S. cerevisiae cell wall, a highly branched glucose polysaccharide made of
ca. 350 glucosyl units, covalently linked to β-1,3-glucan representing about 10% of the total
glucans from the wall of the organism (Shahinian and Bussey 2000).
A different and interesting type of β-glucan was originally described in the cell wall
of Aspergillus fumigatus (Bernard and Latgé 2001). This is a highly insoluble polysaccharide
where glucosyl units are linked through alternating β-1,3, and β-1,4 bonds, in a similar
fashion to the case of the α-bound polysaccharide nigeran (see below). This polysaccharide
is covalently bound to the common β-1,3-glucan.
Chitin is normally present in lower amounts than β-glucans, but its presence is univer-
sal in the fungal kingdom, thus being the basic structural polysaccharide in the cell walls
of fungi. Chitin is a linear and large polymer made of residues of N-acetylglucosamine
linked through β−1,4-glycosidic bonds. Chains of the polysaccharide associate by hydro-
gen bonds to form microfibrils. Depending on the arrangements of the chains in the
microfibrils, three different forms have been shown to exist in nature: β-chitin , with par-
allel chains [i.e., all chains in the same orientation (polarity)], α-chitin, where chains are
antiparallel (polarity of the chains alternates), and γ-chitin, in which two parallel chains
are adjacent to one antiparallel chain. The form present in the fungal cell walls is only
α-chitin (reviewed in Ruiz-Herrera and Ruiz-Medrano 2004).
Chitosan is a polysaccharide of basic nature made mostly of glucosamine and a vari-
able number of dicetyl chitobiose residues all bound through β−1,4-linkages. Chitosan is
a characteristic component of the cell wall from Zygomycota species, originally identified
in a member of this taxon, Phycomyces blakesleeanus (Kreger 1954). This characteristic led
to the belief that it was specific to this fungal group. Nevertheless, more recent studies
have demonstrated that chitosan is present in the cell walls of different Ascomycota and
Basidiomycota species (see Briza et al. 1988; Matsuo 2005; Banks 2005). Chitosan is synthe-
sized from chitin through the action of specific deacetylases that eliminate the acetyl group
of GlcNAc (Davis and Bartnicki-García 1984a, 1984b; Calvo-Mendez and Ruiz-Herrera
1987). Accordingly, the discovery of genes encoding chitin deacetylases in the genomes of
a large number of fungal species can be taken as evidence of the wide distribution of this
polysaccharide in the fungal kingdom.
Another glucose-containing polysaccharide is nigeran. Nigeran is not a common or
constant component of the fungal cell wall. It has been described only in a few members
of a specific group of Ascomycota: Aspergillus and Penicillium species (Johnston 1965; Tung
and Nordin 1967), but only under some conditions of growth. Nigeran is a peculiar amor-
phous polysaccharide made of glucosyl units linked by alternating α-1,3- and α-1,4-bonds.
Mannose polymers in the cell fungal cell wall are mostly in the form of the carbohy-
drate moiety of glycoproteins, where they are commonly described as mannans, although
the presence of mannose in some wall heteropolysaccharides has also been described (for
example, see Bush et al. 1974; Ruiz-Herrera 1967; Horikoshi and Iida 1964; Nakajima et al.
1970). Mannan polysaccharides are present in all species of fungi. Two types of mannan
molecules exist associated with the wall proteins: short chains made of at most five or
six mannosyl units bound through α-1,3- and α-1,2-bonds to serine or threonine residues
of the protein by a so-called O-glycosidic linkage. The other type are high Mr branched
polysaccharides made of dozens and even hundreds of mannosyl units, depending on the
glycoprotein and the fungal species, bound through α-1,2-, α-1,3-, and α-1,6-bonds, some of
them phosphorylated. Interestingly, it was described that the wall phosphomannan resi-
due in the fungal cell wall was responsible for osmotin binding (Ibeas et al. 2000) (osmotin
is a defense plant PR-5 protein toxic for a wide variety of fungal species). These phospho-
mannan molecules are bound (N-linked) to asparagine residues of the protein through a
disaccharide made of GlcNAc, diacetyl chitobiose.
Heteropolysaccharides made of different hexoses, or hexoses and deoxyhexoses, or
pentoses are common in the cell walls of different fungal species, constituting an impor-
tant component of the so-called matrix or cementing part of the cell wall. These poly-
saccharides may be antigenic determinants of specific fungi. Representative examples of
this property are the polysaccharide fractions from the cell wall of two chemotypes of
Histoplasma capsulatum released by enzymatic hydrolysis (Reiss, Miller, and Kaplan 1977). It
was observed that an antigenic fraction different to histoplasmin released by several poly-
saccharidases (chem.1, bG1) contained different sugars in the following order of abundance:
glucose > mannose > glucosamine > galactose
Important heteropolysaccharides from fungal cell walls are galactomannans, described

in a number of Ascomycota species (Sakaguchi, Yokota, and Suzuki 1969). The structure
of a polysaccharide extracted with alkali from the wall of Aspergillus niger was described
by Bardalaye and Nordin (1977). The polysaccharide was found to contain almost equi-
molecular amounts of galactose and glucose, arranged in the form of a backbone made of
mannose units bound by 1,2-linkages, with branches of 1,6-bound short oligosaccharides
made of galactose joined by 1,4-linkages. In the important fungus pathogenic for man,
Aspergillus fumigatus, different noncovalently bound wall proteins are antigenic and the
most important antigenic component used for diagnosis is the galactofuran side chain of
the wall galactomannans for which a commercial kit exists (Bernard and Latgé 2001). In a
different study, a monoclonal antibody was raised against an epitope from the cell wall of
Penicillium frequentans that recognized different species of Penicillium and Aspergillus, and
that was identified also as a galactomannan (Fuhrmann et al. 1992); and in Candida albicans,
a wall antigen not completely characterized yet (3H8) has been used for the clinical identi-
fication of the fungus (R. Sentandreu, personal communication).
Glucuronic acid is present in polysaccharides of the cell wall of a number of fungi.
Originally discovered in the cell walls of Ascomycota species (Gancedo, Gancedo, and
Asensio 1966), glucoronic acid was later found to be constituents of some heteropolysac-
charides, for example, polysaccharides containing mannose, galactose, and either glucose
or fucose, from Verticillium albo-atrum (Wang and Bartnicki-García 1970) and Aureobasidium
pullulans (Brown and Lindberg 1964), respectively. Further examples of glucuronic acid–
containing polysaccharides were later described in Zygomycota (Mucor) species (Bartnicki-
García and Reyes 1968; Datema, Van Den, and Wessels 1977).
2.2.2 Cell wall proteins

From a quantitative point of view, proteins are less important than polysaccharides in the
cell wall, constituting only about 3% to 20% of the cell wall dry weight. Nevertheless, the
roles of proteins are extremely important for the cell wall structure, physiological activi-
ties, and organization. It is a paradox that proteins were for a long time considered to be
contaminants of the cell wall and in early studies it was common to treat the walls with
hot alkali, proteases, or detergents to eliminate the proteins from the wall preparations,
prior to their analyses, whereas in recent times they are probably the most widely studied
component of the cell wall.
Historically, use of radio-labeled cytoplasmic proteins added to cell wall preparations
to detect whether they might become associated as contaminants led to the conclusion that
the proteins present in wall preparations were indeed bona fide components of them (Dyke
1964; Martínez et al. 1991). But perhaps more important were some early results describ-
ing the existence of peptides bound to glucans and mannans (Kessler and Nickerson 1959;
Korn and Northcote 1960), and especially studies from Sentandreu and collaborators that
showed the then surprising result that the fungal cell wall contained covalently linked
proteins (see Sentandreu et al. 2004, for review).
All bona fide wall proteins are glycosylated, therefore, their denomination as glyco-
proteins. As mentioned above, two main types of binding of the carbohydrate (mannan)
moieties to the proteins are recognized: O-glycosylation where a short chain of sugars
binds to serine or threonine residues, and N-glycosylation in which the sugar chains bind
to asparagine. The relative proportion of the carbohydrate moiety may vary from less than
10% to more than 95% of the whole glycoprotein Mr. Wall glycoproteins can be divided into
two large groups: proteins bound through covalent or noncovalent bonds. In Ascomycota,
which are the best studied examples, three types of covalently bound proteins have been
described: (i) GPI proteins, that are characterized by the presence of a remnant of a gly-
cosylphosphatidylinositol group (GPI), and are bound to β-1,6-glucans by a glycosidic
linkage; (ii) Pir-proteins, that bind to β-1,3-glucans by an alkali-sensitive linkage and are
characterized by the presence of internal repeats, and (iii) proteins bound to other wall
proteins through disulfide bonds. It is important to indicate that Pir-proteins exist only
in Ascomycota, and that the presence of GPI proteins linked to β-1,6-glucans be restricted
to the fungal groups containing these polysaccharides, that is, members of the Dikarya
clade, although their presence in Basidiomycota is still undecided (see Ruiz-Herrera and
Oriz-Castellanos 2010). Some noncovalently bound proteins may be those that are retained
in the wall during their transit to the medium, but others are tightly retained and present
only in the wall itself.
Proteins have extremely important roles in the fungal cell wall. Some wall proteins
have hydrolase activities degrading macromolecules into their products to make them
accessible to be used as nutrients, or are involved in the controlled degradation of wall
polymers during growth (see Adams 2004). Others are important in the interaction of
fungi with the environment, or participate in the architecture of the cell wall, and, there-
fore, in its morphology (Sentandreu et al. 1993). Other glycoproteins may be important
from a structural point of view, whereas another special group of proteins collectively
known as hydrophobins are responsible for the hydrophobic properties of the wall surface
of a large number of fungal species (Wessels 1997). Also, due to their hydrophobic nature,
hydrophobins lower the surface tension thus allowing the growth of aerial hyphae, act as
adhesive agents, and facilitate attachment of fungi to surfaces (Paananen et al. 2003).
2.2.3 Lipids from the cell walls

Lipids comprise the almost forgotten chapter of cell wall studies. As occurred with pro-
teins, for a long time they were considered to be contaminants of the fungal cell wall,
probably arising from the cell membranes. The first evidence that lipids were probably
bona fide components of the walls came from Dyke studies with the yeast Nadsonia elon-
gata (Dyke 1964). This author found that the most abundant fatty acid in the cell lipids,
palmitoleic acid, was absent in the lipids extracted from the cell wall. Later, Domer and
Harmon (1972) concluded from comparative studies of the lipid composition of cells and
walls of Sepedonium species, that the lipid composition of the wall was more constant
than the former. With few exceptions, lipids are present in the fungal cell walls in minor
amounts, from 1% to 10%. Some lipids may be readily extractable with solvents, whereas
others require hydrolysis prior to extraction, suggesting that they are bound to other wall
components by means of covalent linkages. Different authors identified free fatty acids; tri-
glycerides, sterol, sterol esters, and phospholipids in the walls of different fungi. The most
abundant fatty acids in the wall are C16 and C18. In spores from several fungi: Rhizopus sto-
lonifer, Alternaria tenuis, Botrytis fabae, and Neurospora crassa, differences in the composition
of the lipids from whole spores and those present in the cell wall were observed (Fisher,
Brown, and Holloway 1978). The role of the lipids in the cell wall is obscure, but apparently
the lipids may be involved in providing hydrophobic character and protection from desic-
cation (reviewed by Ruiz-Herrera 1992).
Exceptions to the simple and passive role indicated above are complex lipids, for
which important roles in growth, signaling, and development have recently been sug-
gested (see below). For example a C. albicans glycolipid that reacts with antibodies specific
for β-1,2-oligomannosides has been described (Mille et al. 2004). The lipid contained an
oligomannoside chain bound to a phosphomannoinositide residue and it was suggested
to be involved in adhesion, protection, and signaling (Mille et al. 2004).
Another group of complex lipids that have recently gained the interest of researchers
are cerebrosides. Cerebrosides, ceramide monohexosides (CMH), are glycosphingolipids
composed of a hydrophobic ceramide linked to one sugar unit. In fungi, their structure
is conserved and contains one residue of glucose or galactose, although the presence of a
disaccharide has been also described, suggesting an alternative biosynthetic mechanism.
Evidence for the localization of these lipids in the cell wall was obtained by immunological
studies performed with Cryptococcus neoformans (Rodrigues et al. 2000). These authors sug-
gested that the lipid arrived at the wall with exocytic vesicles. Recent evidence suggests an
important role of these lipids in growth and differentiation and signaling, making them
an important target for antifungal drugs (for a thorough reviews on this important group
of lipids see Barreto-Bergter, Pinto, and Rodrigues 2004; Rhome and Del Poeta 2009, and
references therein). Some CMHs have been found to be antigenic, for example, the one
from C. neoformans is recognized by sera from cryptococcosis patients. The observation
that antibodies directed against the lipid were inhibitory to the fungus suggests that it is
involved in growth (Rodrigues et al. 2000). A role of these lipids in cell differentiation was
obtained when it was observed that antibodies raised against CMH from Pseudallescheria
boydii inhibited mycelium formation of this and C. albicans (Pinto et al. 2002), and by the
observation that inhibitors of their synthesis blocked sporulation and hyphal growth in
Aspergillus nidulans and A. fumigatus (Levery et al. 2002). The possibility that the role of
these lipids is to bind to proteins involved in the synthesis or organization of the cell wall
has been entertained (Barreto-Bergter et al. 2004). And although not strictly having a wall
location, an important role of lipid rafts has been recently described in cell wall polariza-
tion (see Chapter 9).
2.2.4 Pigments
The cell walls from different fungi become pigmented at different stages. This is particu-
larly noticeable in the spores. Among the pigment present in the wall, melanins are prob-
ably the most amply distributed, and may be present in the cell walls of some organisms
in high amounts, as much as 16% to 21% of the cell wall dry weight (Benitez, Villa, and
García-Acha 1976; Bull 1970). Melanins are dark pigments derived from the enzymatic oxi-
dation of tyrosine (indole melanins, divided in eumelanins and pheomelanins), or from
other phenolic compounds (nonindole melanins) (see Nicolaus 1969 for review). In the cell
walls, they occur in the form of small electron-dense round bodies (for example, Joppien,
Burger, and Reisenner 1972; Ellis and Griffiths 1975; Hegnauer, Nyhlen, and Rast 1985). The
role of melanins is probably to protect the cell from light, both visible and ultraviolet. Also,
the role of melanins in virulence of pathogenic fungi has been demonstrated (see Jacobson
2000; Gomez and Nosanchuk 2003, for reviews). In the case of Exophiala (Wangiella) dermatiti-
dis, it was observed using mutants deficient in melanin biosynthesis compared to the wild-
type strain, that melanin protected the fungus from being killed within phagolysosomes
of neutrophils (Schnitzler et al. 1999). The observation that antibodies directed to melanins
reduced lethality of C. neoformans to mice confirms this hypothesis (Rosas, Nosanchuk,
and Casadevall 2001). Interestingly, a correlation between sphingolipids (see above) and
melanins in virulence was described when it was observed that phosphoceramide syn-
thase is involved in the regulation of melanin synthesis in C. neoformans (see Rhome and
Del Poeta 2009 for review). Melanins produced by different pathogenic fungi such as C.
neoformans and H. capsulatum, can bind antifungals, reducing their inhibitory activity; while
in others, such as A. niger, melanin may affect the immune response, and even protect the
fungus from toxic metals. An interesting positive effect of melanins on fungal growth
was described, especially when melanized fungi were subjected to ionizing radiation that
changed the structure of the pigments (Dadachova et al. 2007).
It has been also shown that melanin may be an important virulence factor for some
phytoplathogenic fungi in invasion and in resistance to stress conditions in the host.
Another role is to provide resistance to the aggressive conditions of the environment (see
Henson, Butler, and Day 1999 for review). Melanins also have an effect on changing the
structure of the wall of appresoria to support the high turgor pressure necessary for pen-
etration of phytopathogenic fungi into their hosts (Chumley and Valent 1990; Howard and
Ferrari 1989).
Carotenoids are a class of hydrocarbons (carotenes) and their oxygenated derivatives
(xanthophylls) that consist of eight isoprenoid units. These pigments are abundant in dif-
ferent fungal species, and carotenoid pigments have been located in the cell walls. Studies
on a number of fungi, including N. crassa, Blakeslea trispora, Mucor hiemalis, and, particu-
larly, Phycomyces blakesleeanus, have figured prominently in the literature of carotenoid bio-
synthesis. Examples of these are their location in the cell wall of Dacryopinax spathularia
as demonstrated by use of cytochemical techniques and analysis of the isolated cell walls
(Vail and Lilly 1968), and in the perithecial wall of N. crassa, where its acccumulation was
induced by light (Perkins 1988).
By use of in situ Raman microspectroscopy, carotenoids were located in the cell wall
of Arthrobotrys ferox, a fungus isolated from moss samples from continental Antarctica
(Arcangeli and Cannistraro 2000. Exophiala (Wangiella) dermatitidis contains melanin and
the carotenoids torulene and torularhodin in the cell wall, and it has been shown that the
carotenoids protect the cell from irradiation (Geis and Szaniszlo 1984), but apparently are
not involved in protection of the cells from killing after phagocytosis (Schnitzler et al. 1999).
Sporopollenins are extremely resistant polymers derived from carotenoids that are
present in the walls of the spores from different fungi, for example, the zygospores of
mucoraceous fungi (Gooday et al. 1973; Furch and Gooday 1978). According to their chemi-
cal and physical properties, sporopollenins have been suggested to protect the wall from
chemical or enzymatic insults (see Gooday 1981 for review).
A different kind of pigment synthesized by many fungi are polyketide derivatives.
These may be toxic or harmless, and have been suggested as a source for natural pigments
with diverse uses. Among the species producing these types of pigments, mainly in the
spores are those belonging to the genera Monascus, Aspergillus, and Penicillium.
2.2.5 Inorganic salts in the walls

Analyses of the fungal cell walls have revealed the presence of inorganic salts left after
ignition at high temperatures in variable amounts. Their amounts vary from 1% to 5% of
the wall dry weight, but amounts as high as 29.5% were reported for Penicillium digitatum
(Grisaro, Sharon, and Barkai-Golan 1968). Ca++, K+, Fe+++ were the most abundant cations
identified, while Cl– and especially phosphate were the most common anions. It is most
likely that these salts are retained by ionic components both negative and positive pres-
ent in the cell wall (polyuronides, chitosan, proteins, etc.), but other salts are present in
the form of coherent structures. Thus, calcium oxalate polyhydrate (weddelite) was pres-
ent in the form of spine-like structures or crystals on the surface of the wall of different
Mucorales (Jones, McHardy, and Wilson 1976; Urbanus, Van Den Ende, and Koch 1978). A
role of fungi in calcium oxalate biomineralization in calcretes through the transformation
of calcium oxalate polyhydrate into oxalate monohydrate (whewellite), and then into cal-
cium carbonate has been proposed (Verrecchia, Dumont, and Verrecchia 1993).
2.3 Differentiation of the cell wall chemical composition

As might be expected, the data for the composition of the cell wall from specific fun-
gal species described above were obtained after cell growth under arbitrary conditions
selected by the investigators (medium composition, temperature, pH, time of incubation,
etc.). But is the composition of the cell wall of a specific fungus constant under all growth
conditions? We can anticipate that this would hardly be possible considering the capacity
of all living organisms to adapt themselves to cope with the different stimuli they receive
from the environment.
Early experiments using isotopic dilution methods demonstrated that, maintained
under constant conditions, cell wall polysaccharides were rather stable (Corina and
Munday 1971; Villa, Notario, and Villanueva 1980). But in contrast to this stability, the
composition and structure of the whole cell wall of a fungus is far from being stable when
the organism is grown under variable conditions, or in its different developmental forms,
for example, yeast, mycelium, reproductive structures, spores, and so forth. This apparent
contradiction of stability on one side, and capacity to change on the other can be explained
as follows: cell wall components, mainly polysaccharides (but also the bulk of proteins)
(see Ruiz-Herrera, Martinez, and Sentandreu 2002) have a very low turn over, but de novo
synthesis of the cell wall during expansion, branching, mating, formation of reproductive
structure, and with all kinds of spores, and so forth, may introduce substantial changes
in the cell wall structure and composition. As expected, some of these processes are pre-
ceded by a stage of controlled hydrolysis of selective regions of the cell wall that permit the
synthesis of a new wall with similar or different characteristics. These processes are sub-
jected to delicate regulatory mechanisms, not entirely known yet, that will be discussed
in Chapter 9.
Minor and mainly quantitative changes in cell wall composition may occur during
aging of fungal cultures, as observed in the content of different polysaccharides along the
growth curve of Penicillium notatum (Martin, Nicolas, and Villanueva 1973), Trichoderma
viride (Benitez, Villa, and García-Acha 1976), Colletotrichum langenarum (Auriol 1974), and S.
cerevisiae (Masschelein 1957), among many other fungi.
Examples of alterations in cell wall composition by changes in the environmental
conditions are numerous, and a few examples follow: notorious changes in wall polysac-
charides took place in a Paecylomyces persicinus strain during the production of cephalo-
sporin C (Malowitz and Pisano 1982). Growth of Penicillium janczewskii with inulin as the
carbon source drastically changed the structure, and the relative amounts of glucose– and
galactose–containing polysaccharides, as compared to mycelium grown in a medium con-
taining sucrose (Pessoni et al. 2005). Variations in the relative amounts of chitin in the
cell walls were described for Neolentinus lepideus and Phialophora sp., depending on the
nutrient content of the medium and the growth temperature. These data were useful in
applying chitin determination as an indicator of the biomass of wood-decay fungi during

the process of wood degradation (Nilsson and Bjurman 1998). Incubation of a strain of N.
crassa in a medium with acetate as the carbon source reduced the amount of chitin and
wall proteins, as compared with a sucrose-grown mycelium (Coniordos and Turian 1968).
Growth under illumination may also induce changes in the chemical composition of the
cell wall as compared to cultures maintained in the dark as demonstrated by changes
in the amounts of chitin, but not of proteins, in the wall of a mutant of Aspergillus gigan-
teus (Fiema 1983a; 1983b). In P. blakesleeanus, illumination with white (but not red) light
increased the synthesis of chitin (Herrera-Estrella and Ruiz-Herrera 1983). Changes in
minor, but important cell wall components have been also reported to occur when fungi
are incubated under different conditions, for example, changes in melanin composition
were observed when Cladosporium sphaerospermum was grown in different culture media
(Dadachova et al. 2007).
The most relevant changes in cell wall composition, both quantitative and qualitative,
have been recorded during differentiation of fungi, including the dimorphic transition.
Among these, the developmental changes suffered during the encystment of fungal (and
also oomycete) zoospores is probably the most dramatic alteration during development
from the points of view of structure, function, and for the aim of the point analyzed here,
of the cell wall. During these processes, a flagellated swimming cell that lacks a wall,
after receiving a signal, which is mainly specific changes in the environment, binds to a
solid substrate, loses the flagellum by an endocytic process, becomes sessile, and makes a
complete cell wall in a process as short as a few minutes. At later stages, the cyst develops
into a mature plant, forms rhizoids for fixation to the substrate, and some species may
grow in a mycelial stage. Well-known examples of this process are Chitridiomycota mem-
bers of the genera Blastocladiella (Lovett 1975) and Allomyces (Barstow and Pommerville
1980). During encystment these fungi rapidly synthesize chitin microfibrils that cover the
cell providing a rigid structure resistant to osmotic shock that serves as receptor for the
deposition of proteins and polysaccharides that form a complete wall. In Allomyces mac-
rogynus for example, the nascent wall contains polysaccharides made of glucose, galac-
tose, mannose, fucose, rhamnose, xylose, and arabinose (Kroh et al. 1977). It is important
to indicate that chitin is not accumulated in the zoospores, and that the inability of these
zoospores to synthesize the polysaccharide is not due to absence of the enzyme that
synthesizes it (chitin synthase), as clearly shown by Cantino and collaborators. These
authors demonstrated that this was accumulated in γ particles, specific organelles of the
zoospores of Blastocladiella emersonii (Cantino and Mills 1983). The same particles, play-
ing the same role have been also described in A. macrogynus (Rone and Boye 1977; Kim,
Youn, and Cho 1996). Inability to synthesize chitin by the zoospores in B. emersonii (and
A. macrogynus) was explained by the different location of chitin synthase on one side
(γ particles) and the enzymes involved in the synthesis of the substrate of chitin syn-
thase (uridine-diphospho-N-acetylglucosamine [UDPGlcNAc]) on the other. Only during
encystment does there occur a liberation of chitin synthase by disorganization of the γ
particles with liberation of microvesicles (chitosomes) carrying the enzyme that move to
the surface and start making the polysaccharide (Cantino and Mills 1983; Kim et al. 1996).
Additionally, evidence was obtained of the existence of a complex regulation mechanism
due to the sensitivity of the first enzyme in the pathway leading to UDPGlcNAc for-
mation: 2-amino-2-amido-deoxy-D-glucose-6-phosphate ketol-isomerase (amido trans-
ferase) (Selitrennikoff, Allin, and Sonneborn 1976; Costa-Maia 1994). Accordingly, in the
zoospore, the high levels of UDPGlcNAc would inhibit its own synthesis, acting as an
inhibitor of amido transferase, but during encystment, UDPGlcNAc levels would rapidly
decay being utilized for the initial chitin synthesis, thus relieving the inhibition of amido
transferase, and leading to its rapid synthesis.
As mentioned above, during the differentiation stages of fungi, the cell wall of fungi
may suffer changes in its chemical composition, structure, or both. Accordingly, during
the further development of the cysts of Chitridiomycota species, changes in the chemical
composition of their corresponding cell walls take place. An example is the wall compo-
sition of the species Allomyces arbuscula where a comparison of the cell wall of cysts, rhi-
zoids, and a mixture of rhizoid and hyphae revealed minor differences in chitin content
among the three stages; an increase in glucose concentration during cyst germination, con-
trasting with a decrease in the content of galactose, xylose, and fucose (Kroh et al. 1977).
Qualitative changes occurring at the surface of the cell wall of A. macrogynus were found
to occur as revealed by the use of an antiserum prepared against mycelium adsorbed with
cell walls (Fultz and Sussman 1966).
Possibly, the most thorough chemical analyses of the wall during differentiation are
the ones corresponding to the Zygomycota species Mucor rouxii and P. blakesleeanus. In the
case of the dimorphic fungus M. rouxii (see below), striking differences in composition at
its different stages of development were recorded (Bartnicki-García 1968). The most sig-
nificant difference was found for β-1,3-glucans which were present mostly in the spore
wall, while they were absent in the wall of the vegetative forms, either mycelium or yeasts,
and were present in very low amounts in the sporangiophores. On the other hand, fucose
and galactose appeared to be absent only in the spore wall. The spore wall contained very
small amounts of glucuronic acid, in contrast with its abundance in the cell wall of myce-
lium, yeast, and sporangiophores. These results were similar to those reported for the cell
walls of the sporangiophores (Cansino and Ruiz-Herrera 1979) and sporangiospores of P.
blakesleaanus (Van Laere and Van Assche 1987). A further difference in the chemical com-
position of the cell wall from the spores and the vegetative forms of P. blakesleeanus was its
significant content of sporopollenins. As indicated above, these are oxygenated polymers
of carotenoids, extremely resistant to physical and chemical attack, which probably play a
protective role for the dormant spores.
Differentiation of Sphaerostilbe repens hyphae into aggregated mycelium and rhizo-
morphs brought about differences in composition of the cell wall, where the amounts of
glucose, protein, and chitin increased during the formation of aggregated mycelium and
rhizomorphs. This is in contrast to uronic acids, lipids, and especially minerals, mainly
calcium and phosphate, which decreased when compared to the cell walls of the vegeta-
tive mycelium (Bolton and Bonaly 1982).
Another example of cell wall changes during development occurred in Schizophyllum
commune, where carbon source depletion induces pileus formation in some fruit body pri-
mordia, a process involving breakdown of polysaccharides from the cell wall where both
alkali soluble and insoluble β-1,3-glucans decreased noticeably, in contrast to an increase
in α-1,3-glucans (Kanetsuna and Carbonell 1971). The positive correlation existing between
the stipe elongation and polysaccharide autolysis suggested the role of this process in dif-
ferentiation (Niederpruem and Wessels 1969; Wessels and Koltin 1972). Similar conclusions
were reached for Coprinus macrorhizus (Kamada, Hamada, and Takemaru 1982), C. cinereus
(Kamada and Takemaru 1983), and Agaricus bisporus (García-Mendoza et al. 1987) where
analysis of the cell walls obtained at the various stages of the fruiting processes revealed
a positive correlation between the rate of stipe elongation and the rate of glucan autolysis.
Structural and chemical changes in the cell wall that occur during germination of
fungal spores have been observed in many species. For example, during the germina-
tion of Zygomycota sporangiospores, a new wall is synthesized below the spore wall
(Bartnicki-García 1981). In M. rouxii, it was observed that the new cell wall was structur-
ally and chemically different to the spore wall (Bartnicki-García 1981; Obregon et al. 1990).
In the related species P. blakesleeanus, changes in the wall composition also occurred along
the germination process: the proportion of aminosugar–containing polysaccharides (chi-
tin and chitosan) increased, as well as uronic acids, and galactans, but glucans showed a
slight decrease only (Van Laere, Van Assche, and Furch 1987). In a different Zygomyota
species, Conidiobolus obscurus, belonging to the group of the Entomophtorales, opposite
changes in protein and polysaccharide content took place, whereas the cell wall of spores
contained 20.1% protein, 20.5% β-1,3-glucan, and 52.3% chitin, the mycelial wall contained
only 4% protein and 15% chitin, but 56.3% β-1,3-glucan (Latgé et al. 1984). Other notorious
examples of the changes in cell wall composition occurring during the various differen-
tiation processes of several fungal species that have been reported in the literature were
previously described in the first edition of the book (Ruiz-Herrera 1992). Some of these are
described below.
During germination of P. notatum spores, a continuous increase in the cell wall content
of glucosamine, galactosamine, and glucose occurred from the stage of resting spores to
swollen spores, to germlings, and to grown mycelium, whereas galactose decreased when
spores reached the swollen stage (Martin, Nicolas, and Villanueva 1973). Another fungus
where important differences in wall composition during germination were reported was
Colletotrichum lagenarium conidia where the content of mannose in the cell wall decreased,
and xylose and rhamnose disappeared in the mycelial wall (Auriol 1974). In T. viride it
was observed unexpectedly that the cell wall from conidia contained no chitin at all,
in contrast to grown mycelium (Benitez, Villa, and García-Acha 1976). A further change
occurring during spore germination was the increase in the ratio between β-1,3- and
β-1,6-glucans that shifted from 0.3 in spores to about 3.0 in the young mycelium (Benitez,
Villa, and García-Acha 1976). Less drastic changes occurred during spore germination
from Puccinia graminis var. tritici where the amounts of neutral sugars in the cell walls
decreased, but in contrast, the content of chitin and protein increased in the mycelial wall
(Ellis and Griffiths 1974).
As would be expected from the above examples, changes in the chemical composi-
tion of the cell wall occurring during the formation of different fungal spores have been
recorded also. An interesting qualitative change in the cell wall composition in the asco-
spores as compared with the yeast vegetative form from S. cerevisiae was revealed when
the existence of a layer made of chitosan was identified in the wall of the ascospore (Briza
et al. 1988). As mentioned above, previously it had been considered that chitosan was a
specific wall component of Zygomycota species. An ecological role of this layer of chito-
san was suggested, when it was observed that ascospores from S. cerevisiae are resistant
to digestion in the gut of Drosophila spp. that has been fed on yeasts (Coluccio et al. 2008).
Using an ingenious quantitative assay, the authors found that, whereas yeast cells were
digested, most of the fed ascospores were recovered in the flies feces, a resistance ascribed
to the chitosan layer, since ascospores from mutants unable to synthesize chitosan were as
sensitive as yeasts.
Quantitative differences in cell wall components of different kinds of spores or the
vegetative forms of different fungi are common. Thus, it was reported that whereas man-
nose content of the yeast wall of A. pullulans was almost twice the amount present in the
wall from the chlamidospores, the cell wall of these chlamidospores contained three
times more glucose. Similarly, differences in the composition of the cell walls from conidia
and chlamydospores of Fusarium sulphureum were observed; the conidial wall being pro-
portionally richer in glucans when compared to chlamydospore walls, whereas these
contained higher amounts of chitin and protein, and a reduced proportion of glucuronic
acid (Schneider et al. 1977).
Changes in cell wall chemical composition and structure also occur during mating of
different fungi. Reported examples are the changes in the wall of the reproductive struc-
tures called shmoos from S. cerevisiae, as compared to vegetative yeasts. It was reported
that the wall of these structures contained higher amounts of chitin, which in contrast to
yeasts that accumulate the polysaccharide in budding scars, appeared homogeneously dis-
tributed in all the cell surface (Díaz, Zinker, and Ruiz-Herrera 1984); additionally, a change
in glycoprotein synthesis took place. In the Basidiomycota species Tremella mesenterica, the
conjugation tubes formed in response to the sexual pheromone from sexually compatible
strains contained higher amounts of β-1,3 and β-1,6-glucans, xylose, mannose, and gluc-
uronic acid, and a lower content of α-1,3-glucans, chitin and proteins, as compared to the
walls of nonmating cells (Reid and Bartnicki-García 1976).
A completely different phenomenon; the synthesis of a new and chemically different
cell wall below the original one was reported for the pathogenic Basidiomycota species C.
neoformans. When a culture of the organism reaches the stationary phase in an unbuffered
medium, there occurs a drastic decrease in pH of the cultures, and wall-lytic enzymes
accumulate leading to cell autolysis. However, a high proportion of cells start to synthesize
a new wall behind the original one that allows their survival and capacity to grow when
transferred to fresh media. It appears that under these conditions, a signal transduction
pathway becomes activated leading not to a reinforcement of the damaged cell wall, but to
the complete synthesis of a new and chemically different cell wall, that provides resistance
to the lytic enzymes (Farkas et al. 2009).
Other effectors may affect the chemical composition of the fungal cell wall or its struc-
ture. Among these, we may cite the composition of the medium; temperature of growth;
illumination; addition of different compounds; addition of different concentrations of
growth factors to auxotrophic mutants; and, the presence of inhibitors. These inhibitors
are not only specific for the synthesis of cell wall components, but also compounds that
physically affect the association of wall components or microfibril assembly of structural
polysaccharide or inhibitors of cell growth. Representative examples were discussed in the
first edition of this book (Ruiz-Herrera 1992) (see Figure 2.1).
Dimorphism is the capacity of different fungal species to grow in the forms of myce-
lium or yeast, depending on the environmental conditions. Dimorphic capacity is not
restricted to a specific taxonomic group, but is distributed within at least Zygomycota,
Basidiomycota, and Ascomycota species; that is, it is not a taxonomic determinant (see
Szaniszlo 1983; Vanden Bosche, Odds, and Kerridge 1993, for reviews and references
therein). The first thorough analyses of the cell walls from the yeast and the mycelium
forms of a dimorphic fungus were made by Bartnicki-García and Nickerson in M. rouxii
(see references above and Bartnicki-García 1968; Bartnicki-García and Nickerson 1962). As
already shown, significant chemical differences exist in the cell wall of both forms, the
main ones corresponding to mannose and proteins; that is, in glycoproteins.
Interestingly, a significant number of dimorphic fungi are human pathogens, and dur-
ing the dimorphic transition, not only morphology changes, but also the structure and
chemical composition of the cell wall suffers important variations, although no general
pattern exists in the different species analyzed. An example of these differences is the con-
tent of α-glucans, which is higher in the yeast (virulent) than in the mycelial (saprophytic)
form of different human pathogenic species (Kanetsuna et al. 1969; San-Blas and Carbonell
1974). Evidence of a correlation between these two factors: virulence and the content of
α-glucan in the cell wall was provided by the observation that a mutant of Paracoccidioides
Figure 2.1 Alterations in the structure of the cell wall by two compounds that inhibit cell differ-
entiation. (A) Appearance of the cell wall in normal germlings of Mucor rouxii. (B) Structure of the
wall of germlings incubated with hydroxyurea, an inhibitor of DNA synthesis. (C) Structure of
the wall of germlings of the fungus incubated with diaminobutanone, an inhibitor of ornithine
decarboxylase that inhibits polyamine biosynthesis and blocks cell differentiation. Magnification
bar: 1 μm. (Modified from Obregon, A., Monzalvo, S., Calvo-Mendez, C., and Ruiz-Herrera, J., 1990,
Ultrastructural and Chemical Alterations in Germinating Spores of Mucor rouxii (Zygomycetes),
Induced by Two Compounds Which Inhibit Their Developmental Pattern, Crypt. Bot., 1:323-331.)
brasilensis unable to synthesize this polysaccharide was avirulent (San-Blas, San-Blas, and
Cova 1976).
Other quantitative differences in the content of chitin of the yeast and mycelium walls
of dimorphic fungi are known. Considering the structural role of chitin, these differences
have been considered of particular importance. Characteristically, the mycelial forms of
Yarrowia lipolytica (Vega and Dominguez 1986) and C. albicans (Chattaway, Holmes, and
Barlow 1968) contain larger amounts of chitin in their walls. Interestingly, it was observed
that cells of C. albicans yeast monomorphic mutants grown under conditions that induce
mycelial growth, displayed a chemical composition in their cell walls almost equal to the
yeast form of the wild-type strains, with a low proportion of chitin (Elorza, Sentandreu,
and Ruiz-Herrera 1994). Nevertheless, it must be noticed that in not all species analyzed,
the cell wall from the mycelial form contains higher amounts of chitin. In other species,
it has been shown that it is the yeast form that contains higher amounts of this polysac-
charide in the cell wall, for example, Blastomyces dermatitis (Kanetsuna and Carbonell
1971) and P. brasiliensis (Kanetsuna et al. 1969); or else there does not exist significant
differences in the amount of chitin in the cell wall of both forms, as occurs in the plant
pathogenic fungus Ustilago maydis (Ruiz-Herrera et al. 1996).
As would be expected, quantitative as well as qualitative differences in other polysac-
charides between the cell walls of the yeast and mycelial forms have been reported for dif-
ferent fungal species. Among them we may cite the described differences in the relative
amounts of β-1,3-glucans in Histoplasma farciminosum (San-Blas and Carbonell 1974) and
B. dermatidis (Kanetsuna and Carbonell 1971), of β-1,6-glucans in Saccharomyces guttulata
Table 2.4 Main Chemical Differences of the Cell Wall of the Mycelial and Yeast-Like Forms of
Some Dimorphic Fungi
Organism Main differences
Histoplasma capsulatum More β-1,3-glucan, galactosamine, and chitin in the mycelial form
Trigonopsis variablilis Mannose only and more lipids in the triangular form
Blastomyces dermatitidis Mannose and galactose only, and more chitin and β-glucan in the
yeast form
Candida albicans Less neutral sugar and more chitin in the mycelial form
Aureobasidium pullulans Uronic acid only and more mannose in the yeast form
Histoplasma capsulatum More glucose and mannose, and less glucosamine in the mycelial form
Mucor rouxii More mannose and protein in the yeast form
Saccharomyces gutulata More chitin and β-1,6-glucan in the mycelial form
Yarrowia lipolytica More chitin, and less protein and glucans in the mycelial form
Source: Slightly modified from Ruiz-Herrera, J., 1992, Fungal Cell Wall: Structure, Synthesis, and Assembly, Boca
Raton, FL: CRC Press. With permission from Taylor & Francis.
(Buecher and Phaff 1968) and mannans in Trigonopsis variabilis (Sentheshanmuganathan

and Nickerson 1962). Qualitative differences in rhamnomannans and galactomannans
were reported for the cell wall from the yeast and mycelial forms of Sporothrix schenkii
(Mendonca et al. 1976), whereas the cell wall from U. maydis mycelium was found to con-
tain polysaccharides made of glucose, galactose, mannose, and xylose, the yeast wall was
found to contain in addition to these, four sugars, ribose, arabinose, and fucose (Ruiz-
Herrera et al. 1996) (see Table 2.4).
I hope that the description of these few examples of qualitative and quantitative
changes in the chemical composition of the cell wall of different fungal species, which
take place depending on changes in the environmental conditions (during growth, when
morphological transitions occur, or along the life cycle), will convince the reader of the
extreme plasticity of the fungal cell wall. These alterations, which respond to complex
regulatory mechanisms described in Chapter 9, permit fungi to adapt to the alteration of
their ecological niches, or better fulfill their different roles necessary for their survival,
growth, or dispersion.
References
Adams, D. J. 2004. Fungal cell wall chitinases and glucanases. Microbiology 150:2029–2035.
Arcangeli, C. and Cannistraro, S. 2000. In situ Raman microspectroscopic identification and localiza-
tion of carotenoids: Approach to monitoring of UV-B irradiation stress on antarctic fungus.
Biopolymers (Biospectroscopy) 57:179–186.
Aronson, J. M. and Preston, R. D. 1960. The microfibrillar structure of the cell walls of the filamentous
fungus Allomyces. J. Biophys. Biochem. Cytol. 8: 247–256.
Auriol, P. 1974. Sur les polyosides des parois cellulaires de conidies de mycelium de Colletotrichum
lagenarium (Pass.) Ell. et Halst., Compt. Rend. Acad. Sci. Paris 279 (Serie D):1867–1869.
Bacon, J. S. D., Jones, D., Farmer, V. C., and Webley, D. M. 1968. The occurrence of α (1-3)-glucan in
Cryptococcus, Schizosaccharomyces, and Polyporus species and its hydrolysis by a Streptomyces
culture filtrate lysing cell walls of Cryptococcus. Biochim. Biophys. Acta. 158:313–315.
Banks, I. R., Specht, C. A., Donlin, M. J., Gerik, K. J., Levitz, S. M., and Lodge, J. K. 2005. A chitin
synthase and its regulator protein are critical for chitosan production and growth of the fungal
pathogen Cryptococcus neoformans. Eukaryot. Cell 4:1902–1912.
Bardalaye, P. C. and Nordin, J. H. 1977. Chemical structure of the galactomannan from the cell wall
of Aspergillus niger. J. Biol. Chem. 252:2584–2591.
Barreto-Bergter, E., Pinto, M. R., and Rodrigues, M. L. 2004. Structure and biological functions of
fungal cerebrosides. An. Acad. Bras. Cienc. 76:67–84.
Barstow, W. E. and Pommerville, J. 1980. The ultrastructure of cell wall formation and of gamma
particles during encystment of Allomyces macrogynus zoospores. Arch. Microbiol. 128:178–189.
Bartnicki-García, S. 1981. “Cell Wall Construction during Spore Germination in Phycomycetes.” In
The Fungal Spore: Morphogenetic Controls. Edited by G. Turian and H. R. Hohl, 533–556. London:
Academic Press.
Bartnicki-García, S. and Lippman, E. 1969. Fungal morphogenesis: Cell wall construction in Mucor
rouxii. Science 165:302–304.
Bartnicki-García, S. and Lippman, E. 1977. Polarization of cell wall synthesis during spore germina-
tion of Mucor rouxii. Exp. Mycol. 1:230–240.
Bartnicki-García, S. and Nickerson, W. J. 1962. Isolation, composition and structure of cell walls of
filamentous and yeast-like forms of Mucor rouxii. Biochim. Biophys. Acta 58:102–119.
Bartnicki-García, S. and Reyes, E. 1968. Chemical composition of sporangiophore walls of Mucor
rouxii. Biochim. Biophys. Acta 165:32–42.
Benitez, T., Villa, T. G., and García-Acha, I. 1976. Some chemical and structural features of the conid-
ial wall of Trichoderma viride. Can. J. Microbiol. 22:318–321.
Bernabé, M., Ahrazem, O., Prieto, J., and Leal, A. 2002. Evolution of fungal polysaccharides f1ss and pro-
posal of their utilization as antigens for rapid detection of fungal contaminants. EJEAFChe. 1:30–45.
Bernard, M. and Latgé, J. P. 2001. Aspergillus fumigatus cell wall: Composition and biosynthesis.
Medical Mycol. 39:9–17.
Bolton, R. and Bonaly, R. 1982. Cell wall composition of the ascomycete Sphaerostilbe repens at differ-
ent developmental stages. Arch. Microbiol. 131:291–297.
Braconnot, H. 1811. Ann. Chim. (Paris) 79:265. Cited in Foster, A. B. and Webber, J. M. Chitin. Adv.
Carb. Chem. 15:371–396.
Briza, P., Ellingerg, A., Winkler, G., and Breitenbach, M. 1988. Chemical composition of the yeast
ascospore wall. The second outer layer consists of chitosan. J. Biol. Chem. 263:11569–11574.
Brown, R. G. and Lindberg, B. 1964. Polysaccharides from cell walls of Aureobasidium (Pullularia)
Pullulans. Acta Chem. Scand. 21:2383–2389.
Buecher, E. J. and Phaff, H. J. 1970. Cell wall composition of Saccharomyces guttulata Schionning.
In Proceedings of the Second International Symposium of Yeast Protoplasts. Brno, Czechoslovakia,
August 20–23, 1968. Acta Facultatis Medicae Unversitatis Brunensis 37:165–170.
Bull, A. T. 1970. Chemical composition of wild-type and mutant Aspergillus nidulans cell walls. The
nature of polysaccharide and melanin constituents. J. Gen. Microbiol. 63:75–94.
Bush, D. A., Horisberger, M., Horman, I., and Wursch, P. 1974. The wall structure of Schizaccharomyces
pombe. J. Gen. Microbiol. 81:199–206.
Calvo-Mendez, C. and Ruiz-Herrera, J. 1987. Biosynthesis of chitosan in membrane fractions from
Mucor rouxii by the concerted action of chitin synthetase and a particulate deacetylase. Exp.
Mycol. 11:128–140.
Cansino, J. M. and Ruiz-Herrera, J. 1979. Aislamiento, purificación y composición química de la
pared celular de los esporangióforos de Phycomyces blakesleeanus. Bol. Soc. Mex. Micol. 13:39–45.
Cantino, E. C. and Mills, G. L. 1983. “The Blastocladielan γ Particle: Once Viral Endosymbiont, Now
‘Chitosome’ Progenitor.” In Fungal Differentiation. Edited by J. E. Smith, 175–210. New York/
Basel: Marcel Dekker.
Chattaway, F. W., Holmes, M. R., and Barlow, A. J. E. 1968. Cell wall composition of the mycelial and
blastospore forms of Candida albicans. J. Gen. Microbiol. 51:367–376.
Chumley, F. G. and Valent, B. 1990. Genetic analysis of melanin-deficient, nonpathogenic mutants of
Magnaporthe grisea. Mol. Plant. Microbe. Interact. 4:135–143.
Coluccio, A. E., Rodriguez, R. K., Kernan, J. M., and Neiman, A. M. 2008. The yeast spore wall enables
spores to survive passage through the digestive tract of Drosophila. PLoS ONE 3:e2873.
Coniordos, N. and Turian, G. 1968. Recherches sur la differentiation conidienne de Neurospora crassa.
IV. Modifications chimio-structurales de la paroi chez le type sauvage et chez deux mutants
aconidiens. Ann. Microbiol. Inst. Pasteur 124A:5–28.
Corina, C. and Munday, A. 1971. The metabolic stability of carbohydrates in walls of hyphae of
Aspergillus clavatus. J. Gen. Microbiol. 65:253–257.
Coronado, J. E., Epstein, S. L., Qiu, W. G., and Lipke, P. N. 2007. Discovery of recurrent sequence
motifs in Saccharomyces cerevisiae cell wall proteins. MATCH Commun. Math. Comput. Chem.
58:281–299.
Costa-Maia, J. C. D. 1994. Hexosamine and cell wall biogenesis in the aquatic fungus Blastocladiella
emersonii. FASEB 8:848–853.
Dadachova, E., Bryan, R. A., Huang, X., Moadel, T., Schweitzer, A. D., Aisen, P., Nosanchuk, J. D.,
and Casadevall, A. 2007. Ionizing radiation changes the electronic properties of melanin and
enhances the growth of melanized fungi. PLosOne 2:e457.
Datema, R. H., Van Den, E., and Wessels, J. G. H. 1977. The hyphal wall of Mucor rouxii. 1. Polyanionic
polymers. Eur. J. Biochem. 80:611–619.
Davis, L. L. and Bartnicki-García, S. 1984a. Chitosan synthesis by the tandem action of chitin synthe-
tase and chitin deacetylase from Mucor rouxii. Biochemistry 23:1065–1073.
Davis, L. L. and Bartnicki-García, S. 1984b. The coordination of chitosan and chitin synthesis in Mucor
rouxii. J. Gen. Microbiol. 130:2095–2102.
Diaz, S., Zinker, S., and Ruiz-Herrera, J. 1984. “Influence of Sex Factor on the Biosynthesis of the Cell
Wall from Saccharomyces cerevisiae.” In Structure, Function and Biosynthesis of Plant Cell Walls.
Edited by W. M. Dugger and S. Bartnicki-García, 415–418. Baltimore: Waverly Press.
Domer, J. E. and Harmon, R. H. 1972. Lipid, manosaccharide and chitin content of cell walls of
Sepedonium. Sabouradia. 10:52–62.
Dyke, K. G. H. 1964. The chemical composition of the cell wall of the yeast Nadsonia elongata. Biochim.
Biophys. Acta 82:374–384.
Ellis, D. H. and Griffiths, D. A. 1975. Melanin deposition in the hyphae of a species of Phomopsis. Can.
J. Microbiol. 21:442–452.
Ellis, D. H. and Griffiths, D. A. 1974. The location and analysis of melanins in the cell walls of some
soil fungi. Can. J. Microbiol. 20:1379–1386.
Elorza, M. V., Sentandreu, R., and Ruiz-Herrera, J. 1994. Isolation and characterization of yeast mono-
morphic mutants of Candida albicans. J. Bacteriol. 176:2318–2325.
Farkas, F., Takeo, K., Maceková, D., Ohkusu, M., Yoshida, S., and Spiczki, M. 2009. Secondary cell
wall formation in Cryptococcus neoformans as a rescue mechanism against acid-induced autoly-
sis. FEMS Yeast Res. 9:311–320.
Fiema, J. 1983a. Some aspects of nitrogen metabolism in Aspergillus giganteus mut. alba. I. Chitin
content in the cell walls. Acta Physiol. Plant 5:113–121.
Fiema, J. 1983b. Some aspects of nitrogen metabolism in Aspergillus giganteus mut alba. II. Protein
content in cell walls. Acta Physiol. Plant 5:123–128.
Fisher, D. J., Brown, G. A., and Holloway, P. J. 1978. Influence of growth medium on surface and wall
lipid of fungal spores. Phytochemistry 17:85–89.
Fuhrmann, B., Lebreton, V., Van Hoegaerden, M., Kamphuis, H. J., and Strosberg, A. D. 1992. A mono-
clonal antibody specific for conidia and mycelium wall layer of Penicillium and Aspergillus.
Microbiol. Immunol. 36:1–12.
Fultz, S. A. and Sussman, A. S. 1966. Antigenic differences in the surface of hyphae and rhizoids in
Allomyces. Science 152:785–787.
Furch, B. and Gooday, G. 1978. Sporopollenin in Phycomyces blakesleeanus. Trans. Br. Mycol. Soc.
70:307–309.
Gancedo, J. M., Gancedo, C., and Asensio, C. 1966. Uronic acids in fungal cell walls. Biochem. Zeit.
346:328–332.
Garcia-Mendoza, C. G., Avellan, M. A., Sanchez, E., and Novaes-Ledieu, M. 1987. Differentiation and
wall chemisry of Agaricus bisporus vegetative and aggregated mycelia. Arch. Microbiol.148:68–71.
Geis, P. A. and Szaniszlo, P. 1984. Carotenoid pigments of the dematiaceous fungus Wangiella derma-
titidis. Mycologia 76:268–273.
Gomez, B. L. and Nosanchuk, J. 2003. Melanin and fungi. Curr. Opin. Infect. Dis. 16:91–99.
Gooday, W. G. 1981. “Biogenesis of Sporopollenins in Fungal Spore Walls.” In The Fungal Spore:
Morphogenetic Controls. Edited by G. Turian and H. R. Hohl, 487. London: Academic Press.
Gooday, G. W., Fawcet, P. Green, D., and Shaw, G. 1973. The formation of fungal sporopollenin in the
zygospore wall of Mucor mucedo: A role for the sexual carotenogenesis in the Mucorales. J. Gen.
Grisaro, V., Sharon, N., and Barkai-Golan, R. 1968. The chemical composition of the cell walls of
Penicillium digitatum Sacc. and Penicillium italicum Whem. J. Gen. Microbiol. 51:145–150.
Hegnauer, H., Nyhlen, L. E., and Rast, D. M. 1985. Ultrastructure of native and synthetic Agaricus
bisporus melanins. Implications as to the compartmentation of melanogenesis in fungi. Exp.
Mycol. 9:221–229.
Henson, J. M., Butler M. J., and Day, A. W. 1999. The dark side of the mycelium: Melanins of phyto-
pathogenic fungi. Annu. Rev. Phytopathol. 37:442–471.
Herrera-Estrella, L. and Ruiz-Herrera, J. 1983. Light response in Phycomyces blakesleeanus: Evidence
for roles of chitin synthesis and breakdown. Exp. Mycol. 7:362–369.
Horikoshi, K. and Iida, S. 1964. Studies of the spore coat of fungi. I. Isolation and composition of the
spore coats of Aspergillus oryzae. Biochim. Biophys. Acta 83:197–220.
Howard, R. J. and Ferrari, M. A. 1989. Role of melanin in appressorium function. Exp. Mycol. 13:403–418.
Ibeas, J. I., Lee, H., Damsz, B., Prasad, D. T., Pardo, J. M., Hasegawa, P. M., Bresan, R. A., and
Narasimhan, M. L. 2000. Fungal cell wall phosphomannans facilitate the toxic activity of a
plant PR-5 protein. Plant J. 23:375–383.
Jacobson, E. S. 2000. Pathogenic roles for fungal melanins. Clin. Microbiol. Rev. 13:708–717.
Johnston, I. R. 1965. The composition of the cell wall of Aspergillus niger. Biochem. J. 96:651–658.
Jones, D., McHardy, W. J., and Wilson, M. J. 1976. Ultrastructure and chemical composition of spines
in Mucorales. Trans. Brit. Mycol. Soc. 66:153–157.
Joppien, S., Burger, A., and Reisenner, H. J. 1972. Untersuchungen uber den chemischen Aufbau
von spores-und Keimschlauchwander der Uredosporen des Wizenrostes (Puccinia graminis var.
tritici). Arch. Mikrobiol. 82:337–352.
Kamada, T., Hamada, Y., and Takemaru, T. 1982. Autolysis in vitro of the stipe wall in Coprinus mac-
rorhizus. J. Gen. Microbiol. 128:1041–1046.
Kamada, T. and Takemaru, T. 1983. Modification of cell-wall polysaccharides during stipe elongation
in the basidiomycete Coprinus cinereus. J. Gen. Microbiol. 129:703–709.
Kanetsuna, F. and Carbonell, L. M. 1971. Cell wall composition of the yeast-like and mycelial forms
of Blastomyces dermatitidis. J. Bacteriol. 106:946–948.
Kanetsuna, F., Carbonell, L. M., Moreno, R. E., and Rodriguez, J. 1969. Cell wall composition of the
yeast and mycelial forms of Paracoccidioides brasiliensis. J. Bacteriol. 97:1036–1041.
Kessler, G. and Nickerson, W. J. 1959. Glucomannan-protein complexes from cell wall of yeast. J. Biol.
Chem. 234:2281–2285.
Kim, J. S., Youn, H. J., and Cho, C. W. 1996. Ultrastructural studes of encystment in Allomyces mac-
rogynus. J. Microbiol. 34:220–224.
Kopecká, M. and Krieger, D.R. 1986. Assembly of microfibrils in vivo and in vitro from (1→3)-β-D-
glucan synthesized by protoplasts of Saccharomyces cerevisiae. Arch. Microbiol. 143:387–395.
Korn, E. D. and Northcote, D. H. 1960. Physical and chemical properties of polysaccharides and gly-
coproteins of the yeast-cell wall. J. Biochem. 75:12–17.
Kreger, D. R. 1954. Observations on cell walls of yeast and some other fungi by X-ray diffraction and
solubility tests. Biochim. Biophys. Acta 13:1–9.
Kroh, M., Knuiman, B., Kirby, E. G., and Sassen. M. M. 1977. Cell wall formation in zoospores of
Allomyces arbuscula III. Carbohydrate composition of cell walls during development from meio-
spores to hyphae. Arch. Microbiol. 113:73–78.
Latgé, J. P., Fournet, B., Cole, G., Dubourdieu, D., and Tong, N. 1984. Composition chemique des parois
des corps hyphaux et des azygospores de Conidiobolus obscures. Can. J. Microbiol. 30:1507–1521.
Larriba, G., Morales, M., and Ruiz-Herrera, J. 1981. Biosynthesis of β-glucan microfibrils by cell-free
extracts from Saccharomyces cerevisiae. J. Gen. Microbiol. 124:375–383.
Levery, S. B., Momany, M., Lindsey, R., Toledo, M., Shayman, J., Fuller, M., Brooks, K., Doong, R.
L., Straus, A. H., and Takahashi, H. K. 2002. Disruption of the glucosylceramide biosynthetic
pathway in Aspergillus nidulans and Aspergillus fumigatus by inhibitors of UDP-Glc: Ceramide
glucosyltransferase strongly affects spore germination, cell and hyphal growth. FEBS Lett.
525:59–64.
Lovett, J. S. 1975. Growth and differentiation of the water mold Blastocladiella emersonii:
Cytodifferentiation and the role of ribonucleic acid and protein synthesis. Bacteriol. Rev.
39:345–404.
Malowitz, R. and Pisano, M. A. 1982. Changes in cell wall carbohydrate composition of Paecilomyces
persicinus P-10 Ml during growth and Cephalosporin C production. Appl. Environ. Microbiol.
43:916–923.
Martin, F. J., Nicolas, G., and Villanueva, J .R. 1973. Chemical changes in the cell wall of conidia of
Penicillium notatum during germination. Can. J. Microbiol. 19:789–796.
Martinez, J. P., Casanova, M., Gil, M. L., Sentandreu, R., and Ruiz-Herrera, J. 1991. Analysis of the
polypeptide composition of the cell walls of Neurospora crassa. Similarities with the protein-
aceous material secreted by the slime variant. Mycol. Res. 95:315–319.
Masschelein, C. A. 1957. Structure de la paroi cellulaire et mécanisme de la floculence des levures
basses. Rev. Ferment. Ind. Aliment. (Bruxelles). 14:59–112.
Matsuo, Y., Tanaka, K., Matsuda, H., and Kawamukai, M. 2005. CDA11, encoding chitin deacet-
ylase is required for proper spore formation in Schizosaccharomyces pombe. FEBS Lett.
579:2737–2743.
Mendonca, L., Gorin, P. A. J., Lloyd, K. O., and Travassos, R. 1976. Polymorphism of Sporothrix
schenkii surface polysaccharides as a function of morphological differentiation. Biochemistry
15:2423–2431.
Mille, C., Janbon, G., Delplace, F., Ibata-Ombetta, S., Gaillardin, C., Strecker, G., Jouault, T., Trinel, P.
A., and Poulain. D. 2004. Inactivation of CaMIT1 inhibits Candida albicans phospholipomannan
β-mannosylation, reduces virulence, and alters cell wall protein β-mannosylation. J. Biol. Chem.
279:47952–47960.
Nakajima, T., Tamer, K., Matsuda, K., Tanaka, H., and Ogasawara, N. 1970. Studies on the cell wall of
Aricularia oryzae. Part II. The chemical constituents of the cell wall. Agr. Biol. Chem. 34:553–560.
Nicolaus, R. A. 1969. Melanins. Paris: Herman.
Niederpruem, D. J. and Wessels, J. G. 1969. Cytodifferentiation and morphogenesis in Schizophyllum
commune. Bacteriol. Rev. 33:505–535.
Nilsson, K. and Bjurman, J. 1998. Chitin as an indicator of the biomass of two wood-decay fungi in
relation to temperature, incubation time, and media composition. Can. J. Microbiol. 44:575–581.
Obregon, A., Monzalvo, S., Calvo-Mendez, C., and Ruiz-Herrera, J. 1990. Ultrastructural and chem-
ical alterations in germinating spores of Mucor rouxii (Zygomycetes), induced by two com-
pounds which inhibit their developmental pattern. Crypt. Bot. 1:323–331.
Paananen, A., Vuorimaa, E., Torkkeli, M., Penttila, M., Kauranen, M., Ikkala, O., Lemmetyinen, H.,
Serimaa, R., and Linder, M. B. 2003. Structural hierarchy in molecular films of two class II
hydrophobins. Biochemistry 42:5253–5258.
Perkins, D. D. 1988. Photoinduced carotenoid synthesis in perithecial wall tissue of Neurospora crassa.
Fungal Genet. Newslett. 35:38–39.
Pessoni, R. A. B., Freshour, G., Figuereido-Ribero, R. C. L., and Braga, M. R. 2005. Cell-wall structure
and composition of Penicillium janczewskii as affected by inulin. Mycologia 97:304–311.
Pinto, M. R., Rodrigues, M. L., Travassos, L. R., Aaido, R. M. T., Wait, R., and Barreto-Bergter, E. 2002.
Characterization of glucosylceramides in Pseudallescheria boydii and their involvement in fungal
differentiation. Glycobiology 12:251–260.
Reid, I. D. and Bartnicki-García, S. 1976. Cell wall composition and structure of yeast cells and con-
jugation tubes of Tremella mesenterica. J. Gen. Microbiol. 96:35–50.
Reiss, E. Miller, S. E., Kaplan, W., and Kaufman, L. 1977. Antigenic, chemical, and structural proper-
ties of Histoplasma capsulatum yeast-form chemotypes 1 and 2 after serial enzymatic hydrolysis.
Infect. Immun. 16:690–700.
Rhome, R. and Del Poeta. 2009. Lipid signaling in pathogenic fungi. Annu. Rev. Microbiol. 63:119–131.
Rodrigues, M. L., Travassos, L. R., Miranda, K. R., Frazen, A. S., Rozental, S., De Souza, W.,
Alviano, C. S., and Barreto-Bergter E. 2000. Human antibodies against a purified gluco-
sylceramide from Cryptococcus neoformans inhibit cell budding and growth. Infect. Immun.
68:7049–7060.
Rone, M. and Boye, H. A. 1977. Cell wall synthesis in Allomyces macrogynus. Cytobios. 19:159–170.
Rosas, A. L., Nosanchuk, J. D., and Casadevall, A. 2001. Passive immunization with melanin-binding
monoclonal antibodies prolongs survival of mice with lethal Cryptococcus neoformans infection.
Infect. Immun. 69:3410–3412.
Ruiz-Herrera, J. 1967. Chemical components of the cell wall of Aspergillus species. Arch. Biochem.
Biophys. 122:118–125.
Ruiz-Herrera, J. and Ortiz-Castellanos, L. 2010. Analysis of the phylogenetic relationships and evolu-
tion of the cell walls from yeasts and fungi. FEMS Yeast Res. 10:225–243.
Ruiz-Herrera, J., Leon, C. G., Carabez-Trejo, A., and Reyes-Salinas, E. 1996. Structure and chemical
composition of the cell walls from the haploid yeast and mycelial forms of Ustilago maydis.
Fungal Genet. Biol. 20:133–142.
Ruiz-Herrera, J., Martínez, A. I., and Sentandreu, R. 2002. Determination of the stability of protein
pools from the cell wall of fungi. Res. Microbiol. 153:373–378.
Sakaguchi, O., Yokota, K., and Suzuki, M. 1969. Immunochemical and biochemical studies of fungi.
13. On the galactomannans isolated from mycelia and culture filtrates of several filamentous
fungi. Jap. J. Microbiol. 13:l–7.
San-Blas, G. and Carbonell, L. M. 1974. Chemical and ultrastructural studies on the cell walls of the
yeast-like and mycelial forms of Histoplasma farciminosum. J. Bacteriol. 119:602–611.
San-Blas, F., San-Blas, G., and Cova, L. J. 1976. A morphological mutant of Paracoccidioides brasiliensis
strain IVIC Pb9. Isolation and wall characterization. J. Gen. Microbiol. 93:209–218.
San-Blas, G., San-Blas, F., and Serrano, L. E. 1977. Host-parasite relationship in the yeastlike form of
Paracoccidioides brasiliensis strain IVIC Pb9. Infect. Immun. 15:343–346.
Schneider, E. F., Barran L. R., Wood P. J., and Siddiqui, I. R. 1977. Cell wall of Fusarium sulphureum II.
Chemical composition of the conidial and chlamydospore walls. Can. J. Microbiol. 23: 763–769.
Schnitzler, N., Peltroche-Lacsahuanga, H., Bestier, N., Zündorf, J., Lütticken, R., and Haase, G. 1999.
Effect of melanin and carotenoids of Exophiala (Wangiella) dermatitidis on phagocytosis, oxida-
tive burst, and killing by human neutrophils. Infect. Immun. 67:94–101.
Selitrennikoff, C. P., Allin, D., and Sonneborn D. R. 1976. Chitin biosynthesis during Blastocladiella
zoospore germination: Evidence that the hexosamine biosynthetic pathway is post-translation-
ally activated during cell differentiation. Proc. Natl. Acad. Sci. USA 73:534–538.
Sentandreu, R. and Ruiz-Herrera, J. 1978. In situ study of the localization and regulation of chitin
synthetase in Mucor rouxii. Curr. Microbiol. 1:77–80.
Sentandreu, R., Elorza, M. V., Mormeneo, S., Sanjuan, R., and Iranzo, M. 1993. “Possible Roles of
Mannoproteins in the Construction of Candida albicans.” In Candida albicans: Cellular and
Molecular Biology. Edited by R. Prasad, 72–88. Berlin: Springer-Verlag.
Sentandreu, R., Elorza, M. V., Valentín, E. and Ruiz-Herrera, J. 2004. “The Structure and Composition
Blas and R. Calderone, 3–39. Norfolk, UK: Caister Academic Press.
Sentheshanmuganathan, S. and Nickerson, W. J. 1962. Composition of cells and cell walls of triangu-
lar and ellipsoidal forms of Trigonopsis variabilis. J. Gen. Microbiol. 27:451–464.
Shahinian, S. and Bussey, H. 2000. β-1,6-Glucan synthesis in Saccharomyces cerevisiae. Mol. Microbiol.
35:477–489.
Szaniszlo P. J., Ed. 1983. Fungal Dimorphism: With Emphasis on Fungi Pathogenic to Humans. New York:
Plenum Publishing.
Tung, K. K. and Nordin, J. H. 1967. Evidence for a buried location of nigeran in the cell wall of
Aspergillus niger. Biochem. Biophys. Res. Commun. 28:519–524.
Urbanus, J. F. L. M., Van Den Ende, H. V. D., and Koch, B. 1978. Calcium oxalate crystals in the wall
of Mucor mucedo. Mycologia 70:829–842.
Vail, W. J. and Lilly, V. G. 1968. The location of of carotenoid pigments and the thickness of the cell
wall of light- and dark-grown cells of Dacryopinax spathularia. Mycologia 60:902–907.
Van Laere, A. J., Van Assche, J. A., and Furch, B. 1987. “The Sporangiospore: Dormancy and
Germination.” In Phycomyces. Edited by E. Cerdá-Olmedo and E. D. Lipson, 247–279. Cold
Spring Harbor Laboratory.
Vanden Bossche, H., Odds, F. C., and Kerridge, D., Eds. 1993. Dimorphic Fungi in Biology and Medicine.
New York and London: Plenum Press.
Vega, R. and Dominguez, A. 1986. Cell wall composition of the yeast and mycelial forms of Yarrowia
lipolytica. Arch. Microbiol. 144:124–130.
Verrecchia, E. P., Dumont, J. L., and Verrecchia, K. E. 1993. Role of calcium oxalate biomineralization
by fungi in the formation of calcretes: A case study from Nazareth, Israel. J. Sedimentary Res.
63:1000–1006.
Villa, T. G., Notario V., and Villanueva, J. R. 1980. Chemical and enzymic analyses of Pichia polymor-
pha cell walls. Can. J. Microbiol. 26:169–174.
Wang, M. C. and Bartnicki-García, S. 1970. Structure and composition of walls of the yeast form of
Verticillium albo-atrum. J. Gen. Microbiol. 64:41–54.
Wessels, J. G. H. 1997. Hydrophobins, proteins that change the nature of the fungal surface. Adv.
Microbial Physiol. 38:1–45.
Wessels, J. G. H. and Koltin. Y. 1972. R-glucanase activity and susceptibility of hyphal walls to
degradation in mutants of Schizophyllum with disrupted nuclear migration. J. Gen. Microbiol.
71:471–475.
chapter three
Structure of the fungal cell wall

3.1 Introduction
A measure of the importance of the cell wall as a structure that provides protection for the
cell is provided by its weight relative to that of the whole cell. It has been described that the
wall accounts for about 20% of the dry weight of the fungal cells, although values near 40%
were reported for Trichoderma viride (Benitez, Villa, and Garcia-Acha 1976). Besides, it is
important to recall the characteristics of the cell wall important for fulfilling its functions;
mainly, its chemical composition, its structure, and its mechanical properties.
The fungal cell wall is a coherent structure made up of the physical and chemical asso-
ciation of a number of different substances that include polysaccharides, glycoproteins,
lipids, and some other minor components that are linked together by covalent bonds,
hydrogen bonds, hydrophobic interactions, or ionic associations. Each one of these types of
substances plays specific roles, either passive or active. Structural polysaccharides consti-
tute the scaffold of the wall. Amorphous polysaccharides bind the wall structure provid-
ing specificity for the recognition of chemical components from other cells. Glycoproteins
have enzymatic activities necessary for selective and controlled degradation of the wall
components, for degradation of foreign compounds for nutrition and/or cell protection,
and for establishing contact with the medium. Lipids provide hydrophobic characteristics
and surface protection, and they may play a role in growth and signaling. Pigments pro-
tect the cell from damaging radiation and foreign enzymatic activities, and they also have
an important role in protecting the cell from high turgor pressure, which occurs in the case
of appressoria, the invading structures of a number of plant pathogenic species.
Repeatedly, the structure of the cell wall has been considered a two-phase system made
of polysaccharide microfibrils immersed in an amorphous matrix made of the rest of the
components of the wall. This structure can be compared to man-made composites. These
are made mainly of glass, carbon, or boron fibers, or different polymers that are embedded
into matrices of metal, plastic, or ceramic. The properties of the composites are not the sum
of the ones from the isolated components, but they complement the advantages of each one,
reducing their weaknesses. Thus, the fibers provide the structural characteristics of the
composite and the resistance to tension, whereas the matrix provides resistance to stress
other than tension, and protects the fibers from the harmful conditions of the environment
and from fracture. The fungal cell wall has characteristics superior to man-made compos-
ites; it is lighter, more complex, and has a higher tensile strength (Figure 3.1).
3.2 Methods for the study of cell wall structure

Analysis of cell wall structure involves three questions: (i) how are the different compo-
nents organized and disposed across the wall? (ii) what is the organization of the cell wall
microfibrillar polysaccharides? and (iii) how do amorphous and microfibrillar wall com-
ponents organize to produce a coherent structure?
To answer the first question, different methods have been developed. Microscopy
(light, electron, and atomic force) has been extremely useful in these studies. Electron
29
B C
A
GPI PROTEIN-S-S-PROTEIN
PIR PROTEIN-S-S-PROTEIN
O
P
O O
β-1,6 GLUCANS β-1,3 GLUCANS
CHITIN
Figure 3.1 Schematic representation of the fungal cell wall structure. (A) Scheme representing the
association of bound proteins and polysaccharides. Noncovalently bound proteins are not repre-
sented in the scheme. (B) Representation of the organization of the fungal cell wall. Thin waving
lines, chitin; straight red lines, β-glucans; circles, proteins. (C) Electron microscopy section of a ger-
minating spore of Mucor rouxii showing the structure of the cell walls, both original and from the
germ tube. (See color insert.)
microscopy (EM) studies of the structure of the fungal cell walls started more than 45
years ago, and the procedures have been improving continuously (see Osumi 1998; Wright
2000 for reviews). Electron microscopy of longitudinal sections of the cells has been a
common method for observing how the wall components that react with the different
stains used are disposed along the wall width. Using this technique, it has been observed
that the wall does not stain uniformly; instead several concentric layers can be distin-
guished, since the several wall components react differently with the contrasting agents
used. Thus, proteins and the polar heads of phospholipids readily react with osmium
used in the most widely employed method to stain EM sections, appearing more elec-
tron dense, whereas microfibrillar polysaccharides appear electron transparent because
they do not bind osmium. Early studies on Saccharomyces cerevisiae showed that the wall
appeared divided in three layers, of variable electron density (Darling, Theilade, and
Birch-Andersen 1972; Linnemans, Boer, and Elbers 1977). In the case of Candida albicans,
the wall showed from four to eight layers depending on the technique used (Chattaway
et al. 1976; Poulain et al. 1978; Cassone, Mattia, and Boldrini 1978; Tronchin, Poulain, and
Biguet 1979; Tronchin et al. 1981; Tronchin, Poulain, and Vernes 1984). This variation is not
rare and has been described in other fungi, including Schizosaccharomyces pombe (Poole
and Lloyd 1973; Johnson et al. 1982; Walther, Muller, and Schweingruber 1984; Humbel et
al. 2001), Cryptococcus neoformans (Cassone, Simonetti, and Strippoli 1974), Histoplasma far-
ciminosum (San-Blas and Carbonell 1974), Agaricus bisporus (Rast and Hollenstein 1977) and
Trichophyton mentagrophytes (Wu-Yuan and Hashimoto 1977).
More refined EM techniques have made use of the specific reactivity of different wall
components. One of these techniques is the widely used procedure developed by Thiery
(1967), where polysaccharides made of sugars containing vicinal hydroxyl groups are
Chapter three: Structure of the fungal cell wall 31
oxidized with periodate and stained with thiocarbohydrazide and silver proteinate, lead-
ing to in situ silver precipitation. As expected this technique does not label β-1,3-glucans
or chitin. The use of lectins labeled with colloidal gold or ferritin that react with selective
sugar residues permitted the location along the width of the cell wall of specific polysac-
charides or glycoproteins (Horisberger, Rosset, and Bauer 1975; Horisberger and Rosset
1977). A good example of this method is the detection of chitin by treatment with colloidal
gold-labeled wheat germ lectin.
Use of specific antibodies or lectins to locate specific antigens buried in the cell wall
was originally limited because the hard resins used to prepare EM samples avoided the
reaction between lectins or antibodies (normally labeled with colloidal gold or ferritin)
with the wall components but the problem was solved later by the introduction of low
viscosity resins. The other solution to reach compounds in the internal layers of the wall
was the treatment of the cells with some enzymes that partially digested the wall struc-
ture, before incubation with lectins or antibodies. For example, the presence of glycopro-
teins in the inner layers of the cell walls of several fungi including S. cerevisiae and C.
albicans was demonstrated by their labeling with the ferritin-labeled lectin concanavalin
A (ConA), which recognizes mannose residues after treatment with glucanase (Valentin
et al. 1987). Another technique used to analyze the distribution of the different chemi-
cal components along the cell wall width was the use of specific enzymes to degrade the
components, followed by EM to observe the changes occurring in the treated cells. By use
of this procedure, Jeenah, Davidson, and Boothby (1982) suggested that the cell wall from
Trichoderma pseudokoningii was made by an outer layer of β-1,3-glucans that covered an
inner layer of chitin microfibrils immersed in a protein matrix; and Rast and Hollernstein
(1977) hypothesized that the cell wall from Agaricus bisporus was made by three layers con-
taining respectively, melanin and α-1,3-glucan, proteins and microfrillar polysaccharides,
and mucilaginous polysaccharides. The use of labeled antibodies has also been extended
to the analysis of the changes in cell wall structure occurring during differentiation and
morphogenesis. By this method and using monoclonal antibodies directed against cell
wall fragments of Neurospora crassa (S4D1, S3B3, and SIE5), it was possible to demonstrate
that the distribution of the different epitopes recognized by them was different in hyphae,
septa, and conidia, suggesting important changes of the structure of the cell wall during
development (Marshall, Gull, and Jeffries 1997). These authors also observed that a single
epitope could be differentially located in distinct fungi; accordingly, a monoclonal anti-
body raised against the wall of Paxillus involutus, an ectomycorrizal Basidiomycota fungus,
was able to recognize the mycelial and spore wall of distinct fungi, including N. crassa,
Penicillium chrysogenum (only the conidial wall), and P. involutus, and the basidiospores
but not the mycelium of Amanita muscaria (Marshall, Gull, and Jeffries 1997). According to
the results, the authors concluded that the epitopes from N. crassa were located, not in the
surface, but in internal layers of the cell wall of this fungi.
3.3 Organization of the cell wall

In accord with the accumulated data, a general scheme of the organization of the fungal cell
wall has been outlined. It is generally accepted that glycoproteins are enriched in the outer
surface of the wall. On the other hand, the structural polysaccharides, chitin and β-glucans,
accumulate in the inner or median layers of the cell wall, which appear less electron-dense
as indicated above. However, this image is in my opinion an oversimplification, that should
not lead us to conclude that proteins or structural polysaccharides are absent in other lay-
ers of the fungal walls. It is important to stress that the concept of the location of certain
components in different layers of the wall is not a rigid one. Accordingly, chitin appears to
be distributed in different layers of the wall as shown by labeling with Au-wheat germ lec-
tin (Marcilla et al. 1991), and convincing evidence that proteins are present along the whole
wall width was obtained by use of different techniques, such as labeling with the lectin
ConA (see above) or with specific antibodies (Marcilla et al. 1991; Kapteyn et al. 2000). These
data were confirmed in S. cerevisiae, C. albicans, and other fungi, by partial digestion of the
glucans, a treatment that permitted access of ferritin-labeled ConA, resulting in a heavy
labeling of the median layer (Valentin et al. 1987) (see Figure 3.1).
3.4 The cell wall surface

Particular interest has been focused on components present on the surface of the wall,
because they are responsible for the recognition of surfaces with which fungi make con-
tact. These components include possible hosts in the case of pathogenic fungi, predators,
or a surface to which the fungus may attach to grow, for example, during the formation
of biofilms. Information on particular aspects of the wall surface has been obtained by
different methods; scanning electron microscopy (SEM) being one of the most important
ones. Using this technique, it was observed that the surface of some yeasts cells includ-
ing Candida, Leucosporidium, and S. cerevisiae appeared smooth except for the bud scars
that looked rough (Watson and Arthur 1977; Pringle, Forsdyke, and Rose 1979; Koch and
Rademacher 1980). Removal of the superficial mannan layer in S. cerevisiae and C. albicans
cells gave rise to the appearance of a rough and eroded aspect of the surface of the cell
that was more prominent in the bud scar (Koch and Rademacher 1980). These results led
the authors to suggest that the structure of the scars should be different to the rest of the
cell wall. The difference in appearance of the surface of the bud scars in S. cerevisiae (and
probably other yeasts) to the rest of the cell can be explained by the fact that they are richer
in chitin in contrast with the rest of the cell surface, as demonstrated by Bacon et al. (1966).
Atomic force microscopy (AFM) is another extremely useful technique for the analysis
of the cell surface that has provided important information on the surface characteristics
of the wall and its structure (see Dague et al. 2007 for review). Using this technique some
results previously obtained by scanning electron microscopy were confirmed and refined,
for example, the texture of the surface of the cell wall from yeasts, among others (Ahimou,
Touhami, and Dufrene 2003). The previously mentioned structural difference between
the bud scars and the rest of the wall surface in yeast was confirmed by AFM, which
also revealed a distinct stiffness between both cell surface regions (Touhami, Nysten, and
Dufrene 2003). Other results obtained by use of AFM techniques on the mechanical prop-
erties of the cell wall are discussed below.
Specific chemical methods have been employed to gain information on the molecular
bases of some properties of the cell wall surface, for example, by the use of labeled pro-
teins or antibodies, as indicated above. Fluorescent microscopy (either simple or confo-
cal) has been extremely useful in the location of different compounds in the walls (for
example, see Ramon et al. 1996; Poulain et al. 2002). Specific examples of the identification
of surface polysaccharides are the results obtained from ConA binding by S. cerevisiae,
Saccharomyces bayanus, and Candida mycoderma, and binding of peanut lectin by S. pombe
that revealed respectively the presence of mannans or galactans in the wall surface of
these fungi (Barkai-Golan and Sharon 1978). In C. albicans yeast cells, anionic proteins were
detected on the surface of the cell wall by their reaction with colloidal gold-labeled chito-
san that has a positive charge (Horisberger and Clerc 1988). It was hypothesized that these
glycoproteins contained phosphodiester linkages that conferred a negative charge and
hydrophilic properties to the cell surface, as occurs in S. cerevisiae (Jigami and Odani 1999).
The presence of other surface glycoproteins that contain phosphodiester-linked acid-labile
β-1,2-mannan, suggested that, on the contrary, they probably conferred hydrophobic prop-
erties to C. albicans cells, as suggested by the observation that these molecules were larger
and more abundant in hydrophobic than in hydrophilic cells (Masuoka and Hazen 1999).
Of particular importance was the observation that by use of freeze-etching analy-
sis a large number of fungal spores from Ascomycota, Basidiomycota, and Zygomycota
species, showed the presence of a layer of regularly arranged structures named rodlets
(Hess, Sassen, and Remsen 1968; Hess and Stocks 1969; Bronchart and Demoulin 1971;
Hashimoto, Wu-Yan, and Blumenthal 1976; Dempsey and Beever 1979: Hallett and Beever
1981; Gardner, Hess, and Tripathi 1983). These data were later confirmed by use of AFM
techniques; it was observed that the layer of rodlets from the spores of Aspergillus oryzae
changed drastically during germination; they were substituted with some softer material
with a different spatial arrangement (Van der Aa, Michel, and Asther 2001). Similar results
were reported by Dufrene et al. (1999). These authors observed that the rodlet-covered sur-
face of the dormant spores from Phanerochaete chrysosporium changed drastically during
germination; the germinating spores appearing covered with a smooth surface with rough
granular structures. Their results also revealed that during spore germination, a change
in the molecular interactions of the wall surface took place. These results confirmed the
observation that during germination, distinct proteins were incorporated into the cell wall
from Microsporum gypseum (Page and Stock 1974).
Isolation of the rodlet layer from the wall of Trichophyton mentagrophytes conidia was
performed by treatment with urea, mercaptoethanol, and sodium dodecyl sulfate (SDS)
followed by enzymatic treatment with β-1,3-glucanase and chitinase. This layer, which
accounted for about 10% of the weight of the cell wall, demonstrated that it was extremely
insoluble and was made of more than 80% protein and smaller amounts of glucomannan
and melanin (Hashimoto, Wu-Yan, and Blumenthal 1976). A different and milder proce-
dure was applied to isolate the rodlet layer from the cell wall of conidia from N. crassa.
Simply, conidia were vigorously shaken in water giving rise to the liberation of the rodlets
that appeared as a thick layer. After removal of the conidia by filtration, the rodlets were
sedimented by centrifugation, freeze-dried, and subject to analysis. It was found that these
structures were made of ca. 90% protein and small amounts of lipids and carbohydrate
(Beever, Redgewell, and Dempsey 1979).
Further work led to the identification of the proteins making the rodlets as hydropho-
bins. Hydrophobins are small hydrophobic proteins carrying a signal peptide for secretion,
a majority of hydrophobic amino acids, and eight cysteine residues at conserved positions.
The proteins belonging to this group receive their name because they confer hydrophobic-
ity to the cell surface of different fungi. The role of hydrophobins was analyzed by J. H.
Wessels and his collaborators in Schizophyllum commune mainly. They observed that the
gene encoding one type of hydrophobin (Sc3) was expressed during the formation of the
mycelium and other reproductive structures, leading to secretion of large amounts of the
protein in a soluble form that bound to hyphae in the form of insoluble aggregates. Other
hydrophobins were synthesized during the formation of fruit bodies. Interestingly, it was
observed that hydrophobins could be bound to the surface of gas bubbles or hydrophobic
surfaces. EM observation of the layers formed by hydrophobins under these conditions
showed that they had assembled in the form of rodlets. These could be dissociated by
treatment with trifluoroacetic acid and reassembled in a layer of rodlets when the acid was
removed (Wösten, De Vries, and Wessels 1993). The authors suggested that submerged
hyphae secreted a soluble form of the hydrophobin molecules, which became attached to
the surface of the aerial hyphae through noncovalent bonds during phorogenesis when
the spore-bearing structures leave the aqueous medium, and become arranged in the form
of a hydrophobic layer of rodlets. In this way, the nascent aerial hyphae could break the
surface tension of the water-air interface, and grow into the air. This role of hydrophobins
for providing spores with a hydrophobic surface appears to be a general phenomenon. The
human opportunist pathogen Aspergillus fumigatus was demonstrated to contain two genes
encoding hydrophobins that were isolated and disrupted. Mutants in the gene encoding
one of them, RODA, were not affected in virulence in a mouse model nor in their capacity
to bind to pneumocytes, fibrinogen or laminin, but the spores devoid of the rodlet layer,
were hydrophilic and presented problems for their dissemination (Thau et al. 1994). On
the other hand, mutants in the other gene, RODB, conserved the normal rodlet layer in the
spores, indicating that the gene was not responsible for the formation of the rodlet layer,
but the surface of the spores was altered, revealing some role in the structure of the spore
surface (Paris et al. 2003). Interestingly, conidia from rodA and double rodA/rodB mutants,
and to a lesser extent rodB mutants, were more sensitive than the parental strain to killing
in a mouse system, showing a relationship between the hydrophobins with the potential
of host invasion by the fungus.
More direct evidence for hydrophobins as an important virulence factor was reported
for a phytopathogenic fungus. Accordingly, it was demonstrated that virulence in
Magnaporthe grisea, the causal agent of rice blast, was reduced in mutants deficient in gene
MGP1, which encodes a hydrophobin (Talbot, Ebbole, and Hamer 1993). These mutants
showed a noticeable reduction in their capacity to form appressoria, the penetration organ
of many phytopathogenic fungi. Simultaneous alterations in the conidial properties led
the authors to conclude that the hydrophobin encoded by MGP1 was involved in conidial
morphogenesis and appressoria formation.
An interesting hypothesis for a peculiar role of the hydrophobin layer that covers most
fungal spores was raised by Aimanianda et al. (2009), who wondered about the peculiarity
of fungal spores, which do not induce inflammatory responses though they may be abun-
dant in the air that we breath. The hypothesis stated that hydrophobins somehow mask
the recognition of the spores by the immune system. As a test for this hypothesis, they
analyzed the capacity of proteins from RodA and rodA mutants from Aspergillus parasiti-
cus (see above) to induce the activation in vitro of several members of the immune system.
Whereas protein RodA and native spores of the fungus were inert, the rodletless mutant
was effective in inducing the immune response. Nevertheless, it is important to recall that,
as described above, spore germination leads to shedding of the rodlet layer exposing the
underlying wall to the components of the immune system. Masking elements present in
the cell surface of other fungi have also been described, the most prominent example is
the capsule from C. neoformans, which although it is not exactly a wall component, can be
regarded as an extension of some wall components. The mannan moiety of the surface
glycoproteins from C. albicans has also been thought to be a masking agent for other wall
components from the fungus.
In direct contrast to this apparent inertness of the spores for recognition by the immune
system of humans, other fungal cell wall components present in pathogenic fungi are rec-
ognized by the immune system of mammals (see Levitz 2010 for review). The author indi-
cates that fungi are recognized by the innate immune system, and that it is the cell wall
which is, as would be anticipated, the structure involved in the recognition process, or
more specifically, its three most important cell wall polysaccharides: β-1,3-glucans, man-
nan, and chitin. These are recognized by different receptors: β-1,3-glucans by Decti-1, CR3
(CDC11c/CD18, CD5, CD36, and SCARF1); mannans by the mannose receptor (CD206),
DC-SIGN (CD209), Langerin (CD207) and DECTIN-2, and chitin by the mannose receptor
(CD206). The fate of the fungus after recognition is variable and will depend on the type of
receptor involved. Additionally, in some fungi α-1,3-glucans, phospholipomannans, and
peculiarly enough, the heat shock protein 60 (HSP60, a moonlighting protein associated with
the wall from Histoplasma capsulatum) can be recognized by the innate immune system.
Earlier it was mentioned that although the general scheme of the cell wall organization
reveals a different enrichment of the several components along its width, its fine structure
might be substantially different among the several fungal taxa, and even among species. A
noticeable example of this difference was observed by staining the wall from Ascocalix abi-
etina, Ophiostoma ulmi and Vericillium alboatrum with the lectin from Aplisia depilans gonads
(that binds to galacturonate) complexed with colloidal gold. Whereas the lectin labeled a
fibrillar sheet covering the mycelium of the first species, it labeled the outermost layer of O.
ulmi, and the innermost layer of the wall from V. alboatrum (Benhamov 1989).
This same concept of lack of homogeneity of chemical composition along the width of
the cell wall can be applied also to the composition of the cell wall along the length of the
cell surface. What we have described is the average of the components that make up the cell
wall; but some major or slight differences have been noticed in the composition of the cell
wall along its full length. The most prominent one is the physical nature of the structure of
the nascent cell wall as compared to the mature one. Nascent wall is viscoelastic, whereas
the mature wall is elastic (see below). But, besides this physical characteristic, some more
subtle differences have been recorded along the wall surface; for example, a nonuniform
distribution of mannoproteins on the surface of S. cerevisiae cells was demonstrated by
AFM using gold tips functionalized with ConA, that measured the binding force between
the lectin and the mannan present in the surface of the cell wall (Gad, Itoh, and Ikai 1997).
3.5 Microfibrilar structure of the cell wall

Of particular importance for cell wall structure are the microfibrilar polysaccharides,
mainly chitin because of their skeletal role. The presence of chitin in the cell wall has
been revealed by different methods; an interestingly one using optical or electron micros-
copy was by deacetylation into chitosan by alkaline treatment and staining with osmium
tetroxide (Pearlmutter and Lembi 1978). Using this method the authors demonstrated the
existence of three layers distinctly stained in the wall of Ceratocystis ulmi, the outer one
containing a larger amount of the polysaccharide, in contrast to what appears the rule
in fungal cell walls, where chitin appears to accumulate at the inner layers (see above).
The microfibrilar appearance of chitin in the wall was noticeable. The width of the chitin
microfibrils, which is a reflection of the number of polysaccharide chains, was determined
in N. crassa and Schizophylum commune hyphae after treatment with potassium perman-
ganate, revealing different values between the subapical and apical zones of the hyphae.
These were respectively of 18.9 and 10.4 nm for N. crassa, and 11.9 and 9.3 nm for S. com-
mune (Hunsley and Burnett 1968). Values of the same order were reported for P. blakesleea-
nus and Polyporus by Scurfield (1967). All these values are similar to those obtained in a
more systematic study where widths varying between 7 and 27 nm were calculated (Gow
and Gooday 1983). These data suggest that although synthesis of chitin occurs at the apex,
addition of polysaccharide chains to the synthesized microfibrils may occur at the sub-
apical region, or that less likely the structure of the microfibrils change, becoming more
bulky at the subapical region due to relaxation of the hydrogen bonding that maintains
the association of the polysaccharide molecules. With regard to the length of the microfi-
brils, a value more difficult to measure because they are interwoven in the wall, maximal
values of 109–250 μm were obtained for C. albicans, Paracoccidioides brasiliensis, H. capsula-

tum, and Blastomyces dermatitidis, respectively; whereas those from Coprinus cinereus and
Mucor mucedo measured 1310 and 990 μm, respectively (Gow and Gooday 1983).
Interestingly, more recently it was reported that chitin microfibrils in the fungal cell
wall might not be homogeneous. Thus, two forms of chitin microfibrils were detected in
the wall of C. albicans: short microcrystalline rodlets and long microfibrils, whose loca-
tion was different. The long microfibrils were located at the bud scars and primary septa,
whereas the short microfibrils comprised the bulk of the chitin in the wall (Lenardon et
al. 2007). By use of mutants in different CHS genes of the fungus, the authors concluded
that the different forms were synthesized by different enzymes, the short ones by Chs3,
and the long microfibrils by Chs8. The authors suggested two hypotheses to explain their
results; either the two different enzymes synthesized chitin molecules that folded over
themselves at variable rates giving rise to microfibrils of different width and length, or
that in their synthesis there intervened more than one enzyme. In either case, it must be
recalled that C. albicans contains two other classes of chitin synthases, Chs1 and Chs2, that
synthesize chitin whose molecular structure remains unknown.
The arrangement of chitin microfibrils in the wall has been a matter of discussion. Most
reports have described microfibrils as oriented in a random order, with the exception of
chitin in septa which appeared with a parallel circular orientation, as described in Polyporus
millitae (Scurfield 1967), C. albicans (Gooday and Gow 1986), Fusarium sulphureum (Schneider
and Wardrop 1979), and Trichophyton mentagrophytes (Pollack, Lange, and Hashimoto 1983).
Of particular interest in this sense is the study of chitin microfibril orientation around the
pits existing in the resistant sporangia of Allomyces neo-moniliformis measuring 0.25 μm in
diameter and located at a distance of about 1 μm on average. Microfibrils appear concentri-
cally arranged (Dodge and Lawes 1969) around these structures. These two observations of
microfibril orientation in septa and pits suggest the existence of an orientation mechanism
for chitin synthases when growth of a chitin layer occurs in a constrained surface.
Although very old and not confirmed by other methods, the study of the orientation
of chitin microfibrils during the growth of the sporangiophore of P. blakesleanus by bire-
fringence is classical. To better understand these studies, some definitions are necessary.
A positive birefringence of the cell wall means that the larger refraction index is parallel
to the cell long axis, and, in contrast, negative birefringence corresponds to a higher bire-
fringence index forming a right angle to the long axis of the cell. Taking into consideration
that the long axis of the cell wall corresponds to the orientation of the microfibrils, a direct
correlation of these two parameters has been assumed. Oort and Roelofsen (1932) observ-
ing P. blakesleeanus stage 1 sporangiophores under the polarizing microscope noticed that
the most apical zone was positive, appearing bright, that is, birefringent; but at the end of
the growing zone, a short and defined zone appeared dark, that is, nonbirefringent (isotro-
pic). This zone was followed immediately below by another birefringent region. However,
whereas the apical birefringency present at the growing apical region was negative, the
one below the isotropic zone was positive. These data were interpreted by Frey-Wyssling
(1936) to be due to the different orientation of microfibrils, transversally oriented in the
growing zone, and longitudinally arranged in the subapical zone, with equilibrium due
to their reorientation at the isotropic (neutral) region. These data and interpretation were
confirmed by Castle (1938) who used squeezed sporangiophores stained with lugol, and
who also demonstrated that the distance of the isotropic zone to the tip corresponded
to the size of the growing zone of the sporangiophore. Similar results were obtained by
Middlebrook and Preston (1952) who measured the orientation of chitin microfibrils in
Phycomyces nitens. These data suggest that although most of the chitin microfibrils might
be randomly oriented, a main vector for the chitin exoskeleton exists in the fungal wall. In
this sense the data obtained for the chitin microfibril arrangement in the wall of Allomyces
macrogynus (Aronson and Preston 1960) are relevant. Using electron microscopy and elec-
tron diffraction analysis, these authors observed the existence of two layers containing
chitin in the mycelium wall. In the most internal one, microfibrils appeared randomly
oriented, whereas those present in the most superficial layer appeared mostly parallel to
the longitudinal axis of the wall.
3.6 Associations of the components of the cell wall

It would be a mistake to consider that the structure of the fungal cell wall, and the prop-
erties of the wall itself depend on the isolated properties of the several components that
make it. It has been demonstrated by recent studies, that the cell wall components associate
by hydrophobic, hydrophilic, and most noticeable covalent bonds. It is the three-dimen-
sional structure resulting from these associations that is responsible for the properties
and characteristics of the cell wall. In other chapters, I describe these concepts in further
detail (for a review of some of these aspects see Bowman and Free 2006), and analyze how
important classes of the wall proteins (GPI proteins and Pir proteins) associate with the
polysaccharides through covalent bonds, and also the existence of covalent bonds join-
ing β-glucans and chitin that permit the cell wall to reach its complex characteristics and
specific functions. The existence of strong associations among proteins themselves either
directly or indirectly is a possibility for which indirect evidence exists (R. Sentandreu and
E. Valentin, personal communication; also see Chapter 9 on wall assembly) (see Figure 3.2).
Indirect evidence for the existence of an association between chitin and β-1,3-glucans
through a covalent linkage has existed for a long time, mainly in the observation of two
forms of β-glucans, one soluble in alkali and another resistant to alkali, the former being
more abundant in the apical zone of the hyphae. The observation that digestion of chitin
with chitinase led to solubilization of glucan strongly suggested that the alkali-insolubility
of a fraction of the β-glucans was due to their covalent association in the wall (Sietsma and
Wessels 1979). The existence of this bond between both polymers was demonstrated more
recently by solid nuclear magnetic resonance (NMR) studies of the cell wall of Aspergillus
niger (Heux et al. 2000). The demonstration for the existence of a reaction catalyzing the
binding of chitin and β-1,6-glucan in S. cerevisiae was accomplished by observing their
covalent association in vitro, using chito-oligosaccharides labeled with sulforhodamine as
substrate, due to natural chitin insolubility (Cabib et al. 2008). By using different mutants,
the authors suggested that the catalyzing enzyme involved in this reaction was encoded by
the CHRP gene. With further studies, it was suggested that a similar reaction was involved
in the covalent binding between chitin and β-1,3-glucan (Cabib 2009).
It is interesting to notice that the ultrastructure of the cell wall may not be constant
during the different stages of development. Examples of changes in structure occurring
during development have been described by different authors: the case of dimorphic fungi
is noticeable. The yeast form of Mucor rouxii was described as noticeably much thicker than
the mycelial wall, although its difference in density was not as large, an indication that a
change in packing of the wall components of the two forms also took place during wall syn-
thesis. Additionally, important changes in the chemical composition between both forms
were observed (Bartnicki-García and Nickerson 1962). Similarly, changes in structure and
composition among the cell walls of the yeast and mycelial forms of Ustilago maydis (Ruiz-
Herrera et al. 1996) have been described. In the case of another smut fungus, Anthracoidea
elynae, it was observed that during the maturation process of the teliospores, important
Figure 3.2 Cell walls isolated from Mucor rouxii yeast cells. Cell walls were isolated from broken
yeast cells, thoroughly washed, embedded, and sectioned. Notice the different layers of the walls.
Electron microscopy of sections stained with osmium tetroxide. (Photographs obtained by Charles
Bracker. With permission.)
changes in the number and stain characteristics of wall layers occurred, although no
chemical analyses were performed (Parvu et al. 2009). The case of phytopathogenic fungi
is of particularly importance in this regard, since a large number of them produce spe-
cial structures for penetration and invasion of their particular hosts, such as appresoria,
infection pegs, haustoria, and microhyphae. Changes in the structure of the wall of these
invasive elements are notorious as are also the secretion of different enzymes associated
with the wall that may be involved in overwhelming the plant defenses (see Nicole, Ruel,
and Oulette 1994 for review).
3.7 Mechanical characteristics of the fungal cell wall

In the previous edition of this book (Ruiz-Herrera 1992), I complained about the scant stud-
ies on the structure of the fungal cell wall, and indicated that comprehension of this issue
was necessary to understand the ultrastructure of the molecular components, the dynamic
molecular processes, and the growth by elongation which arises from the mechanical
deformation induced by the turgor pressure of the protoplast. The lack of studies on this
subject contrasts with the abundant literature on other aspects of the cell wall, for example,
the knowledge we have reached on the molecular aspect of the biosynthesis of the cell wall
and its regulation. This asymmetry is probably due to the trends normally introduced in
any branch of science. Unfortunately, the problem remains about the same, and the ques-
tions raised by Gordon (1980), who wondered about the sophistication of natural versus
synthetic materials, as in the present case of the cell wall, which is light but resistant to
chemical and physical aggression, and designed to permit the expansion necessary for
the increase in mass of the protoplast without suffering any damage, remain rather unan-
swered. Of importance in this aspect is the fact that chitin, the most important structural
component of the cell wall, shows an extremely high tensile strength, even higher than
man-made fibrous materials (see Table 3.1).
As far as we know, the nascent and mature cell wall have different physical properties.
Whereas the mature wall is elastic (from a practical point of view, rigid), the nascent cell
wall is viscoelastic. The best demonstration of this property was obtained by analysis of
the behavior of the giant sporangiophores from P. blakesleenus glued to an Instron machine
that pulled the sporangium head with a nichrome wire hook. Measurement of the strain
resulting from the stress revealed that deformation of the sporangiophore occurred only
in the growing zone, a property that was stimulated by illumination (Ortega, Garnow, and
Ahlquist 1975). This experiment provided the clearest evidence of the different mechanical
Table 3.1 Tensile Strength of Natural and Artificial
Fibrous Materials
Natural materials Synthetic materials Tensile strength (MPa)
Chitin — 4000
Cellulose — 900
Collagen — 100
— Glass fibers 1500–2000
— Carbon fibers 1900–2600
— Steel 2800
— Boron fibers 3400
Source: Taken from Ruiz-Herrera, J., 1992, Fungal Cell Wall: Structure,
Synthesis, and Assembly. Boca Raton, FL: CRC Press. With permission from
Taylor & Francis.
property of the cell wall in a fungus. In a more recent experiment Zhao, Schaefer, and
Marten (2005) demonstrated that surface modifications may affect the mechanical char-
acteristics of the whole fungal cell wall, an observation that should make us aware of the
necessity to have a deeper knowledge of the mechanical properties of the wall to under-
stand its behavior. These authors measured the mechanical properties of the spores of
A. nidulans by use of AFM. The authors used a rigid probe denominated an indenter that
applies force at the surface, measuring the response to the force applied. With this arrange-
ment, spores from wild-type cells, sonicated spores (a method that eliminates the layer of
rodlets from the spore surface) (see above), and a rodA- mutant were analyzed, obtaining
the following results for stiffness: 110 ± 10, 120 ± 10, and 300 ± 20 N/m; and 6.6 ± 0.4, 7.0 +
0.7, and 22 + 2 GPa of elastic module for wild-type, rodA+ and rodA– strains, respectively.
According to these data, covering of the wall surface by the rodlets changes the mechani-
cal properties of the whole wall of the fungal spores.
References
Ahimou, F., Touhami, A., and Dufrene, Y. F. 2003. Real-time imaging of the surface topography of
living yeast cells by atomic force microscopy. Yeast 20:25–30.
Aimanianda, V., Bayry, J., Bozza, S., Kniemeyer, O. A., Perruccio, K., Elluru, S. R., Clavaud, C., Paris,
S., Brakhage, A. A., Kaveri, S. V., Romani, L., and Latgé, J. P. 2009. Surface hydrophobin pre-
vents immune recognition of airborne fungal spores. Nature 460:1117–1122.
Aronson, J. M. and Preston, R. D. 1960. The microfirillar structure of the cell walls of the filamentous
fungus Allomyces. J. Biophys Biochem. Cytol. 8:247–256.
Bacon, J. S. D., Davidson, E. D., Jones, D., and Taylor, I. F. 1966. The location of chitin in the yeast cell
wall. J. Biochem. 101:36C–38C.
Barkai-Golan, R. and Sharon, N. 1978. Lectins as a tool for the study of yeast cell walls. Exp. Mycol.
2:110–113.
Bartnicki-García, S. and Nickerson, W. J. 1962. Isolation, composition and structure of cell walls of
filamentous and yeast-like forms of Mucor rouxii. Biochim. Biophys. Acta 58:102–119.
Beever, R. E., Redgwell, R. J., and Dempsey, G. P. 1979. Purification and chemical characterization of
the rodlet layer of Neurospora crassa conidia. J. Bacteriol. 140:1063–1070.
Benhamov, N. 1989. Ultrastructural study of galacturonic acid distribution in some pathogenic fungi
using gold-complexed Aplysia depilans gonad lectin. Can. J. Microbiol. 35:349–358.
Benitez, T., Villa, T. G., and Garcia-Acha, I. 1976. Some chemical and structural features of the conid-
ial wall of Trichoderma viride. Can. J. Microbiol. 22:318–321.
Bowman, S. M. and Free, S. J. 2006. The structure and synthesis of the fungal cell wall. BioEssays
28:799–808.
Bronchart, R. and Demoulin, V. 1971. Ultrastructure de la paroi des basidiospores de Lycoperdon et
de Scieroderma (Gasteromycetes) comparee a celle de quelques autres spores de champignons.
Protoplasma 72:179–189.
Cabib, E. 2009. Two novel techniques for determination of polysaccharide cross-links show that
Crh1p and Crh2p attach chitin to both β(1-6)- and β(1-3) glucan in the Saccharomyces cerevisiae
cell wall. Eukaryot. Cell 8:1606–1636.
Cabib, E., Farkas, V., Kosík, O., Blanco, N., Arroyo, J., and McPhie, P. 2008. Assembly of the yeast cell
Wall Crh1p AND Crh2p act as transglycosylases in vivo and in vitro. J. Biol. Chem. 283:29859–29872.
Cassone, A., Mattia, E., and Boldrini, L. 1978. Agglutination of blastospores of Candida albicans by
concanavalin A and its relationship with the distribution of mannan polymers and the ultra-
structure of the cell wall. J. Gen. Microbiol. 105:263–273.
Cassone, A., Simonetti, N., and Strippoli, V. 1974. Wall structure and bud formation in Cryptococcus
neoformans. Arch. Microbiol. 95:205–212.
Castle, E. S. 1938. Orientation of structure in the cell wall of Phycomyces. Protoplasma 31:331–345.
Chattaway, F. W., Shenolikar, S., O’Reilly, J., and Barlow, A. J. 1976. Changes in the cell surface of the
dimorphic forms of Candida albicans by treatment with hydrolytic enzymes. J. Gen. Microbiol.
96:335–337.
Dague, E., Gilbert, Y., Verbelen, C., Andre, G., Alsteens, G., and Dufrene, Y. F. 2007. Towards a
nanoscale view of fungal surfaces. Yeast 24:229–237.
Darling, S., Theilade. J., and Birch-Andersen, A. 1972. Structure and chemical composition of pros-
pheroplast envelopes of Saccharomyces cerevisiae and Hansenula anomala. J. Bacteriol. 110:336–450.
Dempsey, G. P. and Beever. R. E. 1979. Electron microscopy of the rodlet layer of Neurospora crassa
conidia. J. Bacteriol. 140:1050–1062.
Dodge, J. D. and Lawes, J. B. 1969. The arrangement of microfibrils in sporangial walls of Allomyces.
Planta 84:134–140.
Dufrene, Y. F., Boonaert, C. J. P., Gerin, P. A., Asther, M., and Rouxhet, P. G. 1999. Direct probing of
the surface ultrastructure and molecular interactions of dormant and germinating spores of
Phanerochaete chrysosporium. J. Bacteriol. 181:5350–5354.
Frey-Wyssling, A. 1936. Uber den optischen Nachweis der Turgorstreckung. Ber. Deutsch. Bot. Ges. 54:445.
Gad, M., Itoh, A., and Ikai, A. 1997. Mapping cell wall polysaccharides of living microbial cells using
atomic force microscopy. Cell Biol. Int. 21:697–706.
Gardner, J. S., Hess, W. M., and Tripathi, R. K. 1983. Surface rodlets of Tilletia indica teliospores. J.
Bacteriol. 502–504.
Gooday, G. W. and Gow, N. A. R. 1983. A model of the hyphal septum of Candida albicans. Exp. Mycol.
7:370–373.
Gordon, J. E. 1980. “Biomechanics; the Last Stronghold of Vitalism.” In The Mechanical Properties
of Biological Materials. Edited by J. F. V. Vicent and J. D. Currey, 1–11. Cambridge: Cambridge
University Press.
Gow, N. A. R. and Gooday, G. W. 1983. Ultrastructure of chitin in hyphae of Candida albicans and
other dimorphic and mycelial fungi. Protoplasma 115:52–58.
Hallett, I. C. and Beever, R. E. 1981. Rodlets on the surface of Neurospora conidia. Trans. Br. Mycol. Soc.
77:662–665.
Hashimoto, T., Wu-Yuan, C. D., and Blumenthal, H. J. 1976. Isolation and characterization of the rod-
let layer of Trichophyton mentagrophytes microconidial wall. J. Bacteriol. 127:1543–1549.
Hess, W. M. and Stocks, D. L. 1969. Surface characteristics of Aspergillus conidia. Mycologia 61:560–571.
Hess, W. M., Sassen, M. M. A., and Remsen, C. C. 1968. Surface characteristics of Penicillium conidia.
Mycologia 60:290–303.
Heux, L., Brugnerotto, J., Desbriéres, J., Versali, M. F., and Rinaudo, M. 2000. Solid state NMR for
determination of degree of acetylation of chitin and chitosan. Biomacromolecules 1:746–751.
Horisberger, M. and Clerc, M. F. 1988. Ultrastructural localization of anionic sites on the surface of
yeast, hyphal and germ-tube forming cells of Candida albicans. Eur. J. Cell Biol. 46:444–452.
Horisberger, M. and Rosset, J. 1977. Localization of α−galacto mannan on the surface of
Schizosaccharomyces pombe cells by scanning electron microscopy. Arch. Microbiol. 112:123–126.
Horisberger, M., Rosset, J., and Bauer, H. 1975. Colloidal gold granules as markers for cell surface
receptors in the scanning electron microscope. Experientia 31:1147–1149.
Humbel, B. M., Konomi, M., Takagi, T., Kamasawa, N., Ishijima, S. A., and Osumi, M. 2001. In situ
localization of beta-glucans in the cell wall of Schizosaccharomyces pombe. Yeast 18:433–444.
Hunsley, D. and Burnett, J. H. 1968. Dimensions of microfibrillar elements in fungal cells. Nature 218:462.
Jeenah, M., Davidson, B. E., and Boothby, D. 1982. Layered cell wall of Trichoderma cell walls. Arch.
Microbiol. 133:330–331.
Jigami, Y. and Odani, T. 1999. Mannosylphosphate transfer to yeast mannan. Biochim Biophys Acta.
1426:335–345.
Johnson, B. F., Calleja, G. B., Yoo, B. Y., Zuker, M., and McDonald, I. J. 1982. Cell-division-key to cel-
lular morphogenesis in the fission yeast, Schizosaccharomyces. Int. Rev. Cytol. 75:167–208.
Kapteyn, J. C., Hoyer, L. L., Hecht, J. E., Muller, W. H., Andel, A., Verkleij, A. J, Makarow, M., Van Den
Ende, H., and Klis, F. M. 2000. The cell wall architecture of Candida albicans wild-type cells and
cell wall-defective mutants. Mol. Microbiol. 35:601–611.
Koch, Y. and Rademacher, K. H. 1980. Chemical and enzymatic changes in the cell walls of Candida albi-
cans and Saccharomyces cerevisiae by scanning electron microscopy. Can J. Microbiol. 26:965–970.
Lenardon, M. D., Whitton, R. K., Munro, C. A., Marshall D., and Gow, N. A. R. 2007. Individual chi-
tin synthase enzymes synthesize microfibrils of differing structure at specific locations in the
Candida albicans cell wall. Mol. Microbiol. 66:1164–1173.
Levitz, S. M. 2010. Innate recognition of fungal cell walls. PLoS Pathogens 6:e1000758
Linnemans, W. A., Boer, P., and Elbers, P. F. 1977. Localization of acid phosphatase in Saccharomyces
cerevisiae: A clue to cell wall formation. J. Bacteriol. 131:638–644.
Marcilla, A., Elorza, M. V., Mormeneo, S., Rico, H., and Sentandreu R. 1991. Candida albicans
mycelial wall structure: Supramolecular complexes released by zymolyase, chitinase and
β-mercaptoethanol. Arch. Microbiol. 155:312–319.
Marshall, M., Gull, K., and Jeffries, P. 1997. Monoclonal antibodies as probes for fungal wall structure
during morphogenesis. Microbiology 143:2255–2265.
Masuoka, J. and Hazen, K. C. 1999. Differences in the acid-labile component of Candida albicans man-
nan from hydrophobic and hydrophilic yeast cells. Glycobiology 9:1281–186.
Middlebrook, M. J. and Preston, R. D. 1952. Spiral growth and spiral structure. III. Wall structure in
the growth zone of Phycomyces. Biochim. Biophys. Acta 9:32–48.
Nicole, M., Ruel, K., and Ouellette, G. B. 1994. “Fine Morphology of Fungal Structures Involved in
Host Wall Alteration.” In Host Wall Alterations by Parasitic Fungi. Edited by O. Petrini and G. B.
Ouellette, 13–30. St. Paul, MN: APS Press.
Oort, A. J. P. and Roelofsen, P. A. 1932. Spiralwachstum. Wandbau und Plasmatromung bei
Phycomyces. Proc. Roy. Soc. Amsterdam 35:898.
Ortega, J. K. E., Gamow, R. I., and Ahlquist, C. N. 1975. Phycomyces: A change in the mechanical prop-
erties after a light stimulus. Plant Physiol. 55:333–337.
Osumi, M. 1998. The ultrastructure of yeast cell wall structure and formation. Micron 29:207–233.
Page, W. J. and Stock, J. J. 1974. Changes in Microsporum gypseum mycelial wall and spore coat glyco-
proteins during sporulation and spore germination. J. Bacteriol. 119:44–49.
Paris, S., Debeaupuis, J. P., Crameri, R., Carey, M., Charlès, F., Prévost, M. C., Schmitt, C., Philippe, B.,
and Latgé, J. P. 2003. Conidial hydrophobins of Aspergillus fumigatus. Appl. Environ. Microbiol.
69:1581–1588.
Parvu, M., Parvu, A. E., Craciun, C., Barbu-Tudoran, L., and Puscas, M. 2009. Ultrastructure and
development of Anthracoidea Elynae ustilospores. Not. Bot. Hort. Agrobot. 37:41–44.
Pearlmutter, N. L. and Lembi, C. A. 1978. Localization of chitin in algal and fungal cell walls by light
and electron microscopy. J. Histochem. Cytochem. 26:782–791.
Pollack, J. H., Lange, C. F., and Hashimoto, T. 1983. “Nonfibrillar” chitin associated with walls and
septa of Trichophyton mentagrophytes arthrospores. J. Bacteriol. 154:965–975.
Poole, R. K. and Lloyd, D. 1973. Effect of 2-deoxy-D-glucose on growth and cell walls of
Schizosaccharomyces pombe 972h. Arch. Mikrobiol. 88:257–272.
Poulain, D., Slomianny, C., Jouault, T., Gomez, J. M., and Trinel, P. A. 2002. Contribution of phos-
pholipomannan to the surface expression of β-1,2-oligomannosides in Candida albicans and its
presence in cell wall extracts. Infect. Immun. 8:4323–4328.
Poulain, D., Tronchin, G., Dubremetz, J. F., and Biguet, J. 1978. Ultrastructure of the cell wall of
Candida albicans blastospores: Study of its constitutive layers by the use of a cytochemical tech-
nique revealing polysaccharides. Ann. Microbiol. 129:141–145.
Pringle, A. T., Forsdyke, J., and Rose, A. H. 1979. Scanning electron microscope study of Saccharomyces
cerevisiae spheroplast formation. J. Bacteriol. 140:289–293.
Ramon, A. M., Gil, R., Burgal, M., Sentandreu, R., and Valentin, E. 1996. A novel cell wall protein
specific to the mycelial form of Yarrowia lipolytica. Yeast 12:1535–1548.
Rast, D. and Hollenstein, G. O. 1977. Architecture of the Agaricus bisporus spore wall. Can. J. Bot.
55:2251–2262.
Ruiz-Herrera, J., Leon, C. G., Carabez-Trejo, A., and Reyes-Salinas, E. 1996. Structure and chemical
composition of the cell walls from the haploid yeast and mycelial forms of Ustilago maydis.
Fungal Genet. Biol. 20:133–142.
Schneider, E. F. and Wardrop, A. B. 1979. Ultrastructural studies on the cell walls of Fusarium sulphu-
reum. Can. J. Microbiol. 25: 75–85.
Scurfield, G. 1967. Fine structure of the cell walls of Polyporus myllitae Cke. et Mass. J. Linn. Soc. Bot.
60:159–166.
Sietsma, J. H. and Wessels, J. G. H. 1979. Evidence for covalent linkages between chitin and β-glucan
in a fungal wall. J. Gen. Microbiol. 114:99–108.
Talbot, N. J., Ebbole, D. J., and Hamer, J. E. 1993. Identification and characterization of MPG1, a gene
involved in pathogenicity from the rice blast fungus Magnaporthe grisea. Plant Cell 5:1575–1590.
Thau, N., Monod, M., Crestani, B., Rolland, C., Tronchin, G., Latgé, J. P., and Paris, S. 1994. Rodletless
mutants of Aspergillus fumigatus. Infect. Immun. 62:4380–4388.
Thiery, J. P. 1967. Mise en evidence des polysaccharides sur coupes fines en microscopie électronique.
J. Microsc. Paris. 6:987–1018.
Touhami A., Nysten B., and Dufrene Y. F. 2003. Nanoscale zapping of the elasticity of microbial cells
by atomic force microscopy. Langmuir 19:4539–4543.
Tronchin, G., Poulain, D., and Biguet, J. 1979. Cytochemical and ultrastructural studies of the cell
wall of Candida albicans. Arch. Microbiol. 123:245–249.
Tronchin, G., Poulain, D., and Vernes, A. 1984. Cytochemical and ultrastructural studies of Candida
albicans. III. Evidence for modifications of the cell wall coat during adherence to human buccal
epithelial cells. Arch. Microbiol. 39:221–224.
Tronchin, G., Poulain, D., Herbaut, J., and Biguet, J. 1981. Localization of chitin in the cell wall of
Candida albicans by means of wheat germ agglutinin. Fluorescence and ultrastructural studies.
Eur. J. Cell. Biol. 26:121–128.
Valentin, E., Herrero, E., Rico, H., Miragall, F., and Sentandreu, R. 1987. Cell wall mannoproteins dur-
ing the population growth phases in Saccharomyces cerevisiae. Arch. Microbiol. 148:88–94.
Van der Ende, B. C., Michel, R. M., Asther, M., Torres-Zamora, M., Rouxhet, P. G., and Dufrêne,
Y. F. 2001. Stretching cell surface macromolecules by atomic force microscopy. Langmuir
17:3116–3119.
Walther, P., Muller, M., and Schweingruber, M. E. 1984. The ultrastructure of the cell surface and
plasma membrane of exponential and stationary phase cells of Schizosaccharomyces pombe,
grown in different media. Arch. Microbiol. 137:128–134.
Watson, K. and Arthur, H. 1977. Cell surface topography of Candida and Leucosporidium yeasts as
revealed by scanning electron microscopy. J. Bacteriol. 130:312–317.
Wösten, H. A. B., De Vries, M. H., and Wessels, J. G. H. 1993. Interfacial self-assembly of a fungal
hydrophobin into a hydrophobic rodlet layer. Plant Cell 5:1567–1574.
Wright, R. 2000. Transmission electron microscopy of yeast. Microsc. Res. Tech. 51:496–510.
Wu-Yuan, C. D. and Hashimoto, T. 1977. Architecture and chemistry of microconidial walls of
Trichophyton mentagrophytes. J. Bacteriol. 129:1584–1592.
Zhao, L., Schaefer, D., and Marten, M. R. 2005. Assessment of elasticity and topography of Aspergillus
nidulans spores via atomic force microscopy. Appl. Environ. Microbiol. 71:955–960.
chapter four
Chitin
4.1 Introduction
Chitin is a linear polysaccharide made of N-acetylglucosamine (2-acetamido-2-deoxy-D-
glucose, GlcNAc) joined through β-1,4-linkages. Considering that the type of bonding
among the sugar units is β, it is more properly considered made not of monomeric units,
but of units of the disaccharide, diacetyl chitobiose. This polysaccharide has been consid-
ered the most abundant nitrogenous substance in nature, and the second most abundant
organic compound on earth, yielding only to cellulose. It has been estimated that in a year,
at least 10 gigatons of chitin are recycled (Muzzarelli 1999). This immense amount of chitin
is synthesized by a large number of organisms, all of them eukaryotes, since no prokary-
iote is known to possess the capacity to synthesize this polysaccharide.
Chitin was originally described by Braconnot (1811), who isolated it from several
mushrooms, including Agaricus volvaceous. This author demonstrated that the polysaccha-
ride contained nitrogen and acetyl groups, and that it was distinct from cellulose; but the
name Braconnot coined, fungine, was forgotten, and its present name is derived from the
French chitine (χιτων, “tunic” in Greek) given by Odier (1823), who isolated the polysaccha-
ride from the elytra of May beetles in 1823. It is important to notice that the polysaccharide
from fungi and animals has no structural difference.
Chitin and some of its derivatives, normally obtained from brine shrimp and crab
waste (although the use of fungi as source of chitin may be an attractive possibility), have
interesting technological applications including the elaboration of biodegradable packag-
ing, surgical dressings, the preservation of agricultural commodities, to recover proteins
from waste effluents, and so forth. For further information on this aspect of chitin, several
reviews can be consulted (Muzzarelli 1977; 1993a; 1993b; Domard et al. 1996; Muzzarelli
and Peter 1997). On the other hand, the presence of chitin in fungi and insects, and its
absence in plants or higher animals, makes chitin synthesis the ideal target for the control
of these organisms (see Ruiz-Herrera and San-Blas 2003).
4.2 Structure of chitin

The molecular size of chitin chains was determined essentially by two methods, which
gave approximately the same values. In one of them, we measured the ratio between [3H]
GlcNAc of chitin synthesized in vitro and [14C]galactose added at the nonreducing end of
the chitin chains by galactosyl transferase (Calvo-Mendez and Ruiz-Herrera 1987). The
value obtained revealed that chitin chains were made of 2220–2250 GlcNAc units. The
second method involved deacetylation of chitin, and its solubilization in acid. Light scat-
tering or viscosimetry of these solutions rendered a degree of polymerization of 2250 ± 50
(cited by Muzzarelli 1984).
It has been described that about 20–400 chitin chains associate by hydrogen bond-
ing to form microfibrils with a crystalline structure, as revealed by X-ray diffraction (see
below). Due to this crystallinity, chitin is one of the most insoluble natural substances.
45
Chitin is only slightly soluble (5%) in CaCl2 containing ethanol, in LiCl containing
dimethylacetamide, or LiCl containing N-methyl-2-pyrrolidone (Muzzarelli 1999). The
organization of the chitin chains in microfibrils gives rise to three different arrange-
ments: β-chitin, where all chains are parallel (i.e., all chains keeping the same reduc-
ing end to nonreducing end orientation); α-chitin, where chains are antiparallel (i.e., in
which a chain in one direction alternates with another chain in the opposite direction);
and, γ−chitin, where two parallel chains alternate with one antiparallel chain (Blackwell,
Minke, and Gardner 1978). Interestingly, these three forms of chitin are present in nature,
while in contrast, only one crystalline form (I) of cellulose, where the chains are parallel
exist in plants and algae. Of the three chitin forms, α-chitin is the most abundant one,
and γ-chitin the rarest. Because of their different arrangement, the three chitin forms dis-
play distinct physicochemical characteristics, α-chitin, being the stiffest one (Hepburn
and Chandler 1978), probably because the packing of the chains excludes water from its
structure. This form of chitin is extremely insoluble, and with a high-tensile strength,
higher than for man-made fibers (steel, carbon, or boron fibers) (see Table 3.1) (reviewed
by Ruiz-Herrera 1992; Ruiz-Herrera and Ruiz-Medrano 2004). For a thorough review on
the structure, crystallinity, and microfibril characteristics of the isoforms of chitin, see
Salmon and Hudson (1997).
4.3 Chitin distribution in nature

As mentioned above, chitin is a molecule specific to eukaryotes. Chitin is amply distrib-
uted in nature. In Protista, it constitutes the structural component of the cysts of amoeba
(Arroyo-Begovich, Carabez-Trejo, and Ruiz-Herrera 1980), the loricae of some ciliates
(Herth, Mulisch, and Zugenmaier 1986; Jeauniaux 1978), and the stalks or stems of hydro-
zoan colonies. Chitin’s presence in some green algae has also been reported, and, interest-
ingly, diatoms and chrysoflagellates contain β-chitin (Herth, Mulisch, and Zugenmaier
1986). In the fungal-related group of Stramenopiles, small amounts of chitin have been
detected in the cell wall of Oomycetes (Dietrich 1975), with the exception of members of
the order Leptomitales, which accumulate chitin in intracellular granules, denominated
cellulin granules of obscure genesis and function (Lee and Aronson 1975).
In the animal kingdom, chitin is present in members of all phyla with the exception
of echinoderms and chordates, and it is most abundant in the exoskeleton of arthropods.
Interestingly, it is in invertebrates where the three forms of chitin are present. The most
abundant form is α-chitin, but β-chitin is present in a very pure form in the pen of squids,
in chaeta from Aphrodite, and in pogonophore tubes; while the γ-form occurs in the stom-
ach lining of Loligo and the cocoon fibers of the beetle Ptinus.
Chitin, and, interestingly, only the α-form, is the most important structural compo-
nent of the cell walls of fungi. Apparently, and as indicated above, the β- and γ-forms are
only present in members of other phyla. Chitin is present in amounts varying from 2% in
the Saccharomyces cerevisiae cell wall to about 60% in the walls of Allomyces macrogynus and
Sclerotium rolfsii, with an average content of about 20% in the different fungal walls (see
Ruiz-Herrera 1978.) As previously indicated, the content of chitin in the fungal cell wall
can vary depending on the stage of the organism: spore, vegetative, aerial mycelium, or
morphology, for example, yeast or mycelium.
Different observations have revealed that chitin in the fungal cell wall is present in
the form of microfibrils. As was discussed in Chapter 3, whether microfibrils follow a
defined orientation in the fungal cell wall is an unsettled matter. In the giant sporangio-
phore of Phycomyces blakesleeanus, it was described that nascent microfibrils were deposited
Chapter four: Chitin 47
transversally, and during growth they became perpendicular (Roelofsen 1965), while in
septa they are circularly arranged (see Scurfield 1967 and other citations in Chapter 3).
4.4 Chitin biosynthesis

4.4.1 General properties of fungal chitin synthases
Chitin is synthesized by a group of enzymes that have been denominated chitin synthases
(Chs). Unfortunately, chitin synthases have not been purified thus far, and their biochemi-
cal properties have been analyzed in partially purified preparations, such as particulate
fractions, preparations extracted with detergents, and chitosomes (see below). Nor, unfor-
tunately has a major advance taken place on this aspect of the study of chitin during recent
years, and the status of the field remains at the same stage as reported in previous reviews
on the subject (Munro et al. 2001; Ruiz-Herrera, Gonzalez-Prieto, and Ruiz-Medrano 2002;
Ruiz-Herrera and San-Blas 2003; Ruiz-Herrera and Ruiz-Medrano 2004; Ruiz-Herrera et
al. 2006).
Two important points regarding the determination of the kinetic properties of chitin
synthases are important to recall at this time. First, fungal species have been shown to
have more than one chitin synthase (see below). Accordingly, since only crude or par-
tially purified samples have been used up to now to determine the catalytic properties of
chitin synthases, the results obtained thus far represent an average of the properties of
the chitin synthases present in the preparations used, or the enzymes corresponding to
the most active or abundant one(s). In this sense, it is fortunate that the general proper-
ties of most chitin synthases are similar, thus the results obtained are useful. The second
point is to recall that the basic aspects of this biosynthetic reaction were reported in the
pioneering work of Glaser and Brown (1957) on chitin synthase(s) from Neurospora crassa.
These authors described the substrate for chitin synthase(s) as uridine diphosphate-N-
acetylglucosamine (UDPGlcNAc), an observation that was confirmed in all subsequent
work done with Chs from distinct fungal and animal origin (see Figure 4.1). UDPGlcNAc
is synthesized from uridine triphosphate (UTP) and GlcNAc-1-phosphate by an enzyme
named pyrophosphorylase. Using cell free extracts from Mucor rouxii, it was observed
that the only sugar nucleotide synthesized by incubation of GlcNAc-1-phosphate and
different nucleotide triphosphates was UDPGlcNAc (McMurrough, Flores-Carreon, and
Bartnicki-García 1971). Following the initial report from Glaser and Brown, it was also
confirmed that all of the enzymes analyzed thus far require a divalent metal ion for activ-
ity. Some early negative reports (Moore and Peberdy 1975; Peberdy and Moore 1975) were
probably due to the existence of firmly bound residual amounts of metal ions, which
could only be removed by dialysis against ethylene diamine tetraacetate (EDTA) solu-
tions (Ruiz-Herrera 1982). In the majority of analyzed systems, Mg2+ was found to be the
most efficient ion metal, although Mn2+ was more active in some systems (Ruiz-Herrera,
Lopez-Romero, and Bartnicki-García 1977), whereas chitin synthase 3 from S. cerevisiae
requires Co2+ for activity (Sburlati and Cabib 1986). All analyzed chitin synthases display
a sigmoidal response to substrate concentration, indicating a cooperative effect, and they
all present Km´s for UDPGlcNAc in the millimolar range. Optimal pH of chitin synthases
ranges between 5.8 and 8.0, and optimal temperatures oscillate between 15 and 40ºC
(reviewed by Ruiz-Herrera and Ruiz-Medrano 2004).
Glaser and Brown (1957) also described the activation of chitin synthases by GlcNAc,
which was not incorporated into the product. GlcNAc reduces sigmoidicity of enzyme
kinetics (McMurrough and Bartnicki-García 1971), but contrary to an initial suggestion
Figure 4.1 Comparative chemical structures of the chitin synthase substrate UDPGlcNAc (1), the
competitive inhibitors Nikkomycin Z (2), and X (3), and Polyoxins A, B, and D (4). (Modified from
Ruiz-Herrera, J., 1992, Fungal Cell Wall: Structure, Synthesis, and Assembly, Boca Raton, FL: CRC Press.
With permission from Taylor & Francis.)
that the sugar just mimicked the role of the substrate at an allosteric site, the Doris Rast
group demonstrated that GlcNAc indeed bound to a specific allosteric site in the enzyme
(Horsch and Rast 1993; Horsch, Mayer, and Rast 1996).
Chitin synthases are hydrophobic proteins with several transmembrane domains, a
fact that has probably hampered their isolation and purification. This property also sug-
gests that a hydrophobic environment may be important for enzyme activity. In this sense,
it must be indicated that the analysis of their dependence on incubation temperature by
means of Arrhenius plots revealed break points (Montgomey and Gooday 1985; Vermeulen
and Wessels 1983; reviewed by Ruiz-Herrera, Sentandreu, and Martinez 1992), suggesting
the transition of the lipids associated with the enzyme from a solid crystalline stage to a
liquid one. Additionally, it has been reported that several lipids stimulate the activity of
different enzyme preparations (Montgomey and Gooday 1985; Vermeulen and Wessels
1983), and that the efficiency of the lipids on activity depends on the nature of the fatty
acids that they contain.
4.4.2 CHS gene multiplicity in fungi

Since the early, original work done by the Cabib and Bulawa groups, it has been observed
that Saccharomyces cerevisiae contained not one, but three genes encoding chitin synthases
(CHS) (Bulawa et al. 1986; Sburlati and Cabib 1986; Bulawa 1992). By means of comple-
mentation of mutants affected in in vitro synthesis of the polysaccharide, it was possible
to clone the first CHS gene (CHS1) in S. cerevisiae (Bulawa et al. 1986). This gene encodes a
polypeptide with a theoretical Mr of 130 kDa. Surprisingly, mutation of this gene did not
affect viability of the yeast, nor its capacity to synthesize a normal cell wall. These results
made the authors conclude that another gene was responsible for the synthesis of most of
the wall chitin in the yeast. Further studies led to the isolation of a second gene (CHS2)
(Silverman, et al. 1988) that nevertheless was found to be not essential since chs1/chs2
double mutants were viable. A third CHS gene (CHS3) was identified and cloned later by
two independent groups using different experimental methods (Bulawa 1992; Valdivieso
et al. 1991). Mutation of CHS3 has demonstrated that the enzyme encoded by this gene
is responsible for the synthesis of most of the chitin present in the wall of S. cerevisiae,
although chs3 mutants are viable.
The characteristic observation of CHS gene multiplicity was extended later to all
fungal species analyzed thus far (see Ruiz-Herrera, Gonzalez-Prieto, and Ruiz-Medrano
2001, for review). On the contrary, analysis of the presence of chitin synthases from dif-
ferent animal species, has demonstrated that they contain at most two CHS genes (see
Merzendorfer and Zimoch 2003 for review). It is interesting to note that in fungi, members
of Zygomycota contain the highest numbers of CHS genes among fungi, around 20 (Ma,
Ashraf, Ibrahim, et al. 2009). Most Ascomycota and Basidiomycota contain an interme-
diate number of genes. There is no satisfactory hypothesis to explain the multiplicity of
CHS genes, although several possibilities have appeared in the literature. In the case of S.
cerevisiae, it was suggested that most of the chitin in the wall was synthesized by Chs3p,
whereas Chs2p was involved in the synthesis of the chitin ring present in the septum
that separates the bud from the mother cell. Chs1p was hypothesized to play a simple
reparatory function during the final steps of the separation of the bud from the mother
cell (Shaw et al. 1991). Nevertheless, it is important to emphasize that only the triple chs
mutant is lethal. Specific functions for some chitin synthases have been also described in
several other species. Noticeable in this sense is Aspergillus nidulans. In this species, it was
described that chsB was required for normal hyphal growth and development (Borgia et
al. 1996), whereas chsA was required for optimal conidiation (Culp et al. 2000) and conidio-
phore formation (Fujiwara et al. 2000). ChsDp seemed to be involved in the wall structure
of swelling spores (Specht et al. 1996), as occurs with ChsA from Colletotrichum graminicola
(Amnuaykanjanasin and Epstein 2003). Of special interest is the case of CHS3 from N.
crassa, whose mutant spores fail to germinate, this probably being the sole example of an
essential CHS gene reported in fungi (Beth-Din and Yarden 2000). A further example is C.
albicans Chs1p reported to be involved in septa formation (Munro et al. 2001). Mutation of
other CHS genes led to alterations in osmotic sensibility; an example of this is ChsAp from
A B C D
Figure 4.2 Role of Ustilago maydis chitin synthase Chs6 in pathogenesis. Maize plantlets were
inoculated with different mixtures of sexually compatible strains of U. maydis, incubated in the
greenhouse for 2 more weeks, and photographed. (A) Plants inoculated with a mixture of wild
type strains FB1 and FB2. (B) Plants inoculated with a mixture of a wild type strain (FB1) and an
a2b2 Δchs6 mutant. (C) Plants inoculated with a mixture of a1b1 and a2b2 Δchs6 mutants. (D) Control
plants that were injected with sterile distilled water only. (Modified from Garcerá-Teruel, A. et al.,
2004, Loss of Virulence in Ustilago maydis by Umchs6 Gene Disruption, Res. Microbiol., 155: 87–97.)
Glomerella graminicola (Amnuaykanjanasin and Epstein 2003) whose mutant spores lyse
under hypotonic conditions. Other chs mutants from phytopathogenic fungi have been
found to be avirulent or with reduced virulence, and, interestingly, most of them are defec-
tive in CHS genes belonging to division 2 (see below for Chs classification). Among these
mutants, we may cite several examples. In Fusarium oxysporum, two class V chitin syn-
thases were described as critical for pathogenicity (Madrid, Di Pietro, and Roncero 2003;
Martín-Urdíroz et al. 2008), and in the related species Fusarium asiaticum, chs1 mutants also
displayed reduced virulence and alterations in the cell wall structure (Xu et al. 2010). U.
maydis chs6 mutants displayed abnormal morphology and chitin distribution in the cell
wall, and were avirulent to maize (Garcerá-Teruel et al. 2004) (see Figure 4.2). The same
occurred with chs7 mutants (Weber et al. 2006). Further examples are chsV mutants of the
maize pathogen Colletotrichum graminearum, which have reduced pathogenicity (Werner et
al. 2007).
Considering that, in opposition to the examples cited above, most single and even
double mutants show almost no phenotypic alterations, and the large number of CHS
genes in Zygomycota species makes almost impossible the assignment of specific roles for
the encoded enzymes, we have alternatively suggested that fungal CHS gene multiplicity
in most fungi probably constitutes a safety or compensatory mechanism that protects the
cell integrity, permitting the organism to dwell in different environments and under dif-
ferent conditions (Chavez-Ontiveros, Martinez-Espinoza, and Ruiz-Herrera 2000; Ruiz-
Herrera et al. 2001). This idea has its roots in the concept that selection tends to conserve
beneficial traits and eliminate redundancy during evolution, unless it proves to be useful
for competence. If we accept this idea, it may be concluded that chitin synthases mul-
tiplicity is beneficial for fungal survival. The fact that loss of an apparently redundant
chitin synthase may affect survival of a fungus in a competing natural environment is
illustrated by the following example: U. maydis chs3 mutants display only minor pheno-
typic alterations (Xoconostle-Cázares, León-Ramirez, and Ruiz-Herrera 1996) and grow
at about the same rate when inoculated together with a wild-type strain in synthetic
medium. On the other hand, when the mutant was inoculated into a maize competing
with the wild-type strain for a sexually compatible strain, it was almost completely elimi-
nated from the plants (J. Ruiz-Herrera and C. Leon, unpublished). In this regard, the
observation that mutation in each one of the CHS genes from C. albicans led to an increase
in the transcriptional levels of the rest of the genes and an increase in chitin content of the
cells (Munro et al. 2007), is relevant.
4.4.3 Classification and possible evolution of fungal chitin synthases

Bowen et al. (1992) reported a preliminary classification of three classes of fungal chitin
synthases from data obtained by sequence analysis of CHS gene fragments synthesized by
polymerase chain reaction (PCR) from several species. A classification into five classes was
provided when a larger number of genes were sequenced (Specht et al. 1996; Xoconostle-
Cázares et al. 1997). By in silico analysis of complete gene sequences including a thorough
analysis of the structure of Chs which identified conserved motifs in chitin synthases
(see below), we classified fungal chitin synthases into two divisions (1 and 2), each one
enclosing three (I, II, and III), and two (IV and V) classes, respectively (Ruiz-Herrera et al.
2002). This classification agrees with the observation that division 1 enzymes possess an
activity-related motif named chitin synthase 1, different to the equivalent motif of enzymes
belonging to division 2, named chitin synthase 2 (see http://pfam.sanger.ac.uk/). Other
authors have added two further classes to this classification, although in my opinion the
differences of these two new classes are not supported by evolutive considerations (Ruiz-
Herrera and Ortiz-Castellanos 2010).
It is probable that separation into divisions initiated the diversification of fungal chitin
synthases. When the location of the conserved motifs in the different classes of fungal Chs
is analyzed, one can observe that the motifs are restricted to a short peptide stretch located
towards the carboxy terminus in division 2 Chs. Based on these results, we have suggested
that separation of Chs into divisions involved a rearrangement of the N- and C-termini
from division 2 Chs (Ruiz-Herrera et al. 2002).
Our studies on the phylogenetic relationships and the evolution of the fungal cell wall
have revealed that evolution of CHS genes followed the accepted taxonomy of the king-
dom Fungi. According to the results of a study on this matter, it was concluded that genes
of class IV were the most primitive ones, present in all phyla, whereas genes belonging to
class V were absent in Microsporidia. Regarding CHS belonging to division I, those from
class II were common to Zygomycota, Basidiomycota, and Ascomycota, whereas class III
genes were present only in Basidiomycota and Ascomycota, and class I genes were specific
to Ascomycota (Ruiz-Herrera and Ortiz-Castellanos 2010). The observation that chitin syn-
thases present in members from other Opisthokont groups and Protista are related to Chs
belonging to class IV agrees with these ideas on the evolution of chitin synthases (Ruiz-
It is also important to recall that chitin is only present in eukaryotes, but not in plants;
and that cellulose is absent in Opisthokonts, Fungi included, and that the correspond-
ing synthesizing enzymes (chitin and cellulose synthases) have structures in common
to processive transglycosidases (see above). Accordingly, we have suggested that chi-
tin synthase is probably an ancient enzyme that appeared once Opisthokonts diverged
from other eukaryotic groups, a process occurring approximately 1 billion years ago (see
Ruiz-Herrera et al. 2001, 2002). The observation that chitin synthases from Oomycetes
(Chromista or Stramenopiles) are related to fungal division 1 (Ruiz-Herrera et al. 2002;
Ruiz-Herrera and Ortiz-Castellanos 2010), suggests that the corresponding encoding
genes derive from a gene possibly captured by an ancestor of these groups through hori-
zontal transfer.
4.4.4 Structure of chitin synthases

Unfortunately, our knowledge of the structure of chitin synthases is meager since none has
been purified yet as described above. For this reason, all the corresponding information has
been obtained by in silico procedures. By use of hydrophobic cluster analysis (HCA) of pro-
tein sequences, it was concluded that three aspartic acid residues plus a QXXRW motif were
involved in the catalytic activity of processive β-glycosyl-transferases, including S. cerevi-
siae Chs1 (Saxena et al. 1995). Further crystallographic analysis of a β-glycosyl-transferase
from Bacillus subtilis indicated that the three aspartic residues identified by Saxena et al.
(1995), were involved in binding of the substrate and a divalent cation at the active site of
the enzyme (Charnock and Davies 1999). Use of the QSLAVE (Johnson, Overington and
Blundell 1993) program confirmed the importance of the QXXRW motif in catalysis (Horsch
and Sowdhamini 1996). These authors also suggested that chitin synthases displayed an
(α/β)8-barrel type of fold. Additionally, it may be cited that Nagahashi et al. (1995), by use of
site-directed mutagenesis, confirmed the role of residues Asp441, Asp562, and the QRRRW
motif amino acids Gln601, Arg604, and Trp605 in the catalytic activity of S. cerevisiae Chs2.
In agreement with these data, by alignment of chitin synthases belonging to different fun-
gal species and classes, we found that all of them possessed three strategically located
Asp (or Asn) residues, and the QXRRW motif (Ruiz-Herrera et al. 2002). In this study, we
also identified three conserved motifs besides QXRRW in all the analyzed Chs: QXXEY,
LP(G/A), and LXEDRXL, plus the invariant location of amino acids K662, E665, G669, L714,
L720, T744, and P747 in the core region of the enzymes (Ruiz-Herrera et al. 2002). Moreover,
we demonstrated that the above-mentioned motifs were strategically located at the same
position in the enzymes: the QXXEY pentapeptide was located at the second helix of the
conserved region, LP(G/A) appeared at the fifth coil, LXEDRXL was located at the start of
the sixth helix, and QXRRW appeared between two coils at the end of the seventh helix.
Analysis of the hydrophobic profile of the enzymes showed that this critical pentapeptide
was positioned in a flexible region located at the surface of a transmembrane helix sur-
rounded by hydrophylic stretches, and by three transmembrane helices, two towards the
amino terminus, and one towards the carboxy terminus (Ruiz-Herrera et al. 2002).
An in silico study devoted to the analysis of the structure of the active site of chitin
synthases made in comparison with a known enzyme, offered the possibility of gaining
further information on this subject. The authors (Merz et al. 1999) used a template of the
crystalline structure of a T4 DNA-modifying β-glucosyl-transferase analyzed by Vrielink
et al. (1994) in their comparison. They came to the interesting conclusion that residues
Asn215 (or Asp), Tyr 261, Arg269 (or Lys), and Glu272 (or Gln) made direct contact with
the substrate, whereas Trp341 from the QXRRW motif was an important part of the hinge
region that formed the substrate binding cleft.
4.4.5 Regulation of chitin synthases

According to data obtained in the analysis of different systems, it may be concluded that
chitin synthase activity in vivo depends on complex interactions between transcriptional
and posttranscriptional regulatory mechanisms, whose final result probably depends on
the environmental conditions and the specific organism (see Ruiz-Herrera et al. 2001). With
an in silico approach, we analyzed the sequence of the upstream regulatory regions belong-
ing to the different classes of Chs genes (Ruiz-Herrera et al. 2002). We observed the pres-
ence of canonical sites for the binding of several transcription factors that are known to be
involved in cellular response to heat shock, stress, pH, nitrogen deprivation, and phosphate
levels, among others, suggesting their regulation by these agents. These theoretical results
agree with some experimental data obtained for CHS3 from Exophiala dermatitidis. This
gene that presents Stre01, PacC, and Nit2 (AreA) motifs at its regulatory region is up-regu-
lated by high temperature, acidic pH, or nitrogen deprivation (Wang and Szaniszlo 2000).
It has also been demonstrated that the eight CHS genes from U. maydis were differentially
regulated in response to stress by salts, during the pathogenic process in maize, and dur-
ing dimorphic growth (Robledo-Briones 2009). Other CHS genes reported to be regulated
by heat stress during the dimorphic transition are CHS4 and CHS5 from Paracoccidioides
brasiliensis (Nino-Vega et al. 2000), whereas reports of other genes regulated during sev-
eral differentiation processes are: CHS2 from C. albicans (Chen-Wu et al. 1992); CHS1 from
Mucor circinelloides (Lopez-Matas, Eslava, and Diaz-Minguez 2000) during dimorphism;
and, CHS genes from Tuber borchii during fruit body formation (Balestrini et al. 2000). LacZ
was used as a reporter gene to measure transcriptional regulation of the four CHS genes
encoding the catalytic peptides (CHS1, CHS2, CHS3, and CHS8) from C. albicans (Munro
et al. 2007). The authors reported that all the genes were activated by damaging agents,
including mutation in each one of the CHS genes. This regulation was coordinated by the
Pkc, Map Hog, and Calcium-calmodulin pathways, and led to an increase in the chitin
content of the wall.
An interesting result was the observation that treatment of C. albicans with subin-
hibitory concentrations of echinocandins, known inhibitors of β-1,3-glucan biosynthesis,
stimulated chitin biosynthesis through their effect at three levels: stimulation of transcrip-
tion of CHS1, CHS2, and CHS8 genes, activity of the enzymes encoded by these genes,
and chitin content of the cell (Walker et al. 2008). Interestingly, CHS3 gene expression was
increased only when cells were treated with anidulafungin. Data collected by the authors
suggested that the observed effects were mediated, the same as indicated above, through
the operation of several signaling pathways; Hog, Pkc, and calcium-calcineurin.
The five CHS genes from A. nidulans were also found to be regulated at the transcrip-
tional level during the cell cycle (Park, Maeng, and Park 2001). According to the authors,
transcription of chsA and chsC genes decreased only at the S-phase, whereas chsb and chsD
genes decreased at the start of G2 phase and increased at the M phase. In addition, regula-
tion of transcription of CHS genes of this fungus by the transcriptional regulator AbaA
occurred during sporulation (Park, Park, and Park 2003).
An interesting characteristic of most chitin synthases is their activation by protease
treatment as initially described for Chs from Saccharomyces carlsbergensis by Keller and Cabib
(1971), who interpreted the process as the activation of a zymogen. The behavior in vitro of
a large number of chitin synthases is similar, as they are activated by limited proteolysis.
Examples of the few exceptions to this rule are Chs3 from S. cerevisiae (Valdivieso et al.
1991), and chitin synthase from U. maydis (Xoconostle-Cázares, León-Ramirez, and Ruiz-
Herrera 1996). The mechanism of this phenomenon is not understood yet, contrary to its
earlier interpretation, mainly because of the lack of a purified chitin synthase to compare
its Mr before and after proteolysis. As an example of data that do not fit into a simple zymo-
genic activation, I would cite the result obtained with P. blakesleeanus Chs. The enzyme(s)
present in cell-free extracts from the sporangiophores of the fungus was (were) activated
in vitro by light or by trypsin, and this activity was inhibited by iodoacetamide, which is
not an inhibitor of this protease (Herrera-Estrella and Ruiz-Herrera 1983; Reyna and Ruiz-
Herrera 1987), and by benzamidine, an inhibitor of trypsin, but that bound irreversibly to
chitin synthase. Additionally, it was observed that Chs from the mycelium of the fungus
was activated by calcium-calmodulin, and that this process was inhibited by trifluopera-
zine, an inhibitor of calmodulin (Martinez-Cadena and Ruiz-Herrera 1987). These results
were reproduced in Chs from N. crassa (Suresh and Subramanyam 1997), suggesting the
possibility that activation may occur by a reaction in cascade started by unspecific prote-
olysis of the real Chs activator.
Posttranscriptional regulation of chitin synthases has also been described to occur
by several effectors. Thus, as described above, Chsp activity from wild-type strains of
P. blakesleeanus was activated by light in vitro and in vivo, but such activation did not
occur in madb “blind” mutants, revealing the specificity of the process (Herrera-Estrella
and Ruiz-Herrera 1983). It was also described that Chs activity in Benjaminiella poitrasii
responded positively to salt stress (Deshpande, O’Donnell, and Gooday 1997). It has also
been reported that alterations in cell wall synthesis may affect Chs regulation, probably
by a compensatory mechanism. It was thus observed that ggp1/gas1 mutants of S. cerevisiae,
which contain reduced contents of β-glucans, synthesize higher amounts of chitin than the
parental strain (Popolo et al. 1997). It could be hypothesized that the mechanism of this
effect would be similar to the effect of echinocandins as described above (Walker et al.
2008), but this may only be a wild speculation. Mutants in FKS1 (one of the genes encod-
ing β−1,3-glucan synthase in S. cerevisiae) contain higher amounts of chitin in the cell wall
(Garcia-Rodriguez et al. 2000), which agrees with the results of Popolo et al. (1997). It was
suggested these changes were due to misslocalization of Chs3 and by Chs4, a regulator
of chitin synthase activity. Mutation in KNR4, another CHS regulator of S. cerevisiae, con-
tained severely reduced CHS transcripts levels, but was only moderately affected in its
chitin content. This phenotype was explained as due to accumulation of inactive forms of
Chs within chitosomes (Martin et al. 1999).
More recently, a possible additional level of regulation was described for Chs3 from C.
albicans, neither of gene transcription nor enzymatic activity. Accordingly, it was observed
that this protein is phosphorylated at a single serine residue (Ser 139) independently that
the enzyme contains 77 possible phosphorylation sites: 43 Ser, 16 Thr, and 18 Tyr resi-
dues (Lenardon et al. 2010). Although the kinase responsible for the phosphorylation of
Chs3 remains unknown, the authors indicated that the phosphorylation site is a consen-
sus of the sites phosphorylated by cyclin-dependent kinases and theorized that possibly
the kinase involved in the process was Cdc28, the single cyclin-dependent kinase of C.
albicans. In studying the localization of Chs3 in the cell, the enzyme was found located at
the tip of growing buds and hyphae, but before cytokynesis occurs, it becomes relocated
at the site of septum development. More interesting was the observation that when the
Ser139 residue was changed by directed mutagenesis by an Ala residue, and therefore the
molecule was not subject to phosphorylation, Chs3 became delocalized, and the amount
of chitin decreased in the cell wall. Use of another version of the enzyme where S139 was
substituted by an aspartic residue to mimick a phosphorylated enzyme led to inhibition of
its relocation to the septum. The authors interpreted all these data as indicative that loca-
tion and relocation of Chs3 in the C. albicans cell depends on the correct phosphorylation
and dephosphorylation of the enzyme. Whether this is a general phenomenon existing in
other fungal chitin synthases remains unknown.
4.4.6 The mechanism of chitin biosynthesis

From a chemical point of view, the process of chitin biosynthesis can be described as
a simple transglycosylation reaction in which N-acetylglucosaminyl residues are trans-
ferred from a sugar donor; UDP-N-acetylglucosamine (UDP-GlcNAc) to the growing
chain of the polysaccharide:
UDP-GlcNAc + (GlcNAc)n → (GlcNAc)n+1 + UDP
Since the GlcNAc units in chitin are β−bound, it was suggested that each step involved
transfer of two GlcNAc residues, and that the enzyme possessed two active sites (see Ruiz-
Herrera 1992; Saxena et al. 1995). However Imai et al. (2003) obtained evidence that at each
step of growth of β-chitin chains from diatoms, a single unit of GlcNAc was added to
the chitin chain. Whether these contrasting data are due to the different forms of chitin
considered (α or β), or if a misinterpretation of either mechanism was responsible for the
different points of view, remains unknown. In this regard, it may be indicated that the
reported number of Hill’s coefficients, which may be indicative of the number of active
sites, oscillates between 4 at low substrate concentrations and 1 at high concentrations
(Rousset-Hall and Gooday 1975; Hanseler, Nyhlen, and Rast 1983). Taking into consider-
ation the allosteric nature of the enzyme, some of these sites might be allosteric, but pos-
sibly more than one would be catalytic.
In contrast to this simple mechanism, Merz et al. (1999) published an interesting
hypothesis suggesting that chitin biosynthesis occurred in two steps. According to these
authors, the first step was a priming reaction, not stimulated by GlcNAc (see above), but
absolutely dependent on controlled proteolysis and UDPGlcNAC. This reaction was inter-
preted as an initial glycosylation of a protein acceptor. According to the authors, the sec-
ond step always involved further addition of glycosyl units to this acceptor to produce
the normal long chitin chains. Using 16 S subunits (see below) of M. rouxii, we observed
that the effect of specific inhibitors and metal ions produced results that partially agreed
with this hypothesis (reviewed by Ruiz-Herrera and Ruiz-Medrano 2004). With regard to
the complexity of this hypothetical mechanism, it should be recalled that a similar one
occurs in the synthesis of glycogen (see Roach and Skurat 1997; Skurat et al. 2002) and
β-1,3-glucans (Andaluz et al. 1988; Ruiz-Herrera and Larriba 1995), where the presence of
primer proteins has been demonstrated. Interestingly, Bartnicki-García, Lippman et al.
(1979) demonstrated that chitin synthesized in vitro by M. rouxii preparations contained a
polypeptide joined to its reducing end. Similarly, Imai et al. (2003) presented evidence that
the reducing end of the α-chitin chains in microcrystals from Lamellibrachia satsuma and
microfibrils from the diatom Thalassiosira weissflogii were bound to a nonchitinous mate-
rial. These data strongly suggest the role of a primer, probably of proteinaceous nature,
during chitin biosynthesis.
The role of a lipid serving as an intermediate in chitin synthesis, as occurs during the
synthesis of glycoproteins (see the corresponding chapter), was also proposed in the older
literature. Data from Mills and Cantino (1980) on the isolation of labelled lipids during
chitin biosynthesis by Blastocladiella emersonii zoospores led the authors to suggest that
glycosyldiacylglycerol was a lipid carrier involved in the formation of the polysaccharide.
Similarly, it was suggested that a dolichol derivative was a sugar carrier during chitin bio-
synthesis by the brine shrimp (Horst 1983). These results have not been confirmed since
then, nor has this line of investigation been pursued further. Using chitin synthase prepa-
rations from M. rouxii, we clearly ruled out the role of a high-energy intermediate in chitin
biosynthesis (reviewed by Ruiz-Herrera 1982).
It has been demonstrated, and is generally accepted, that polysaccharides are synthe-
sized in the direction: reducing end → nonreducing end (for example, Koyama et al. 1997;
DeAngelis 1999; Lai-Kee-Him et al. 2002). Using electron microscopy crystallography tech-
niques, the same directionality was demonstrated to occur during the synthesis of β-chitin
from Lamellibrachia satsuma and Thalassiosira weissflogii (Imai et al. 2003). Taking into con-
sideration that polysaccharide chains have the same polarity in β-chitin, the process of
synthesis and microfibril formation occurred simultaneously, as occurs with cellulose,
whose chains are also parallel. This process is opposite to what occurs during chitin bio-
synthesis in fungi. As already described, chitin chains in fungi adopt the α-configuration,
that is, they are antiparallel (see above); accordingly, synthesis of the polysaccharide and
the microfibril formation can not be a simultaneous process and must be separated in
time, as has been demonstrated by different pieces of evidence. Accordingly, it is known
that nascent and mature chitin are differentially sensitive to chitinase and chitin deacety-
lase, the former being more sensitive to either enzyme (Lopez-Romero, Ruiz-Herrera, and
Bartnicki-García 1982; Calvo Mendez and Ruiz-Herrera 1987); and that Calcofluor white
and Congo red, substances that interact with the chitin chains, interfere with microfibril
formation and crystallization (Elorza, Rico, and Sentandreu 1983; Herth 1980; Vermeulen
and Wessels 1986).
4.5. Inhibitors of chitin synthases

Taking into consideration that chitin is absent in higher animals and plants, it has been
considered an ideal target for the control of pathogenic fungi and insects. Great efforts
have been spent in the search of substances with the required characteristics to be used
in the clinic, although with poor success until now (see Ruiz-Herrera and San-Blas 2003
for a thorough review of the subject). Nevertheless, these studies have provided important
clues to understanding the mechanism of chitin synthesis and some properties of the bio-
synthetic enzymes.
Rast et al. (2000) published a classification of the groups of substances known to inhibit
chitin synthases, including their Ki and IC50 values. Their list included the following chem-
ical compounds: (i) nucleoside peptides with Ki values from 0.2 to 7 μM, (ii) polyene mac-
rolides, (iii) chlorinated benzenes (Ki = 1–75 μM), (iv) phenylphosphorodithioates (Ki =
50 mM), (v) morpholines, (vi) polyaromatics, (vii) triterpenoid saponins, (viii) sterol gyco-
sides, (ix) pseurotins (Ki = 93 mM) for pseurotin A), (x) flavonoids, (xi) dibenzazepines, (xii)
aryliminodithiazoles (Ki = 16–25 mM), (xiii) pyrimidines (Ki = 14 mM for 4-imidazoyl-5-
phenyl-pyridine), and (xiv) tetrahydropyranes (Ki = 4–39 mM). Most of these compounds
were originally described as antifungals, and later found to inhibit chitin synthase. A list
of inhibitors more recently elaborated appears in Ruiz-Herrera and San Blas (2003).
Undoubtedly, the best known and most effective inhibitors of chitin synthase are the
peptidyl nucleoside antibiotics named polyoxins and nikkomycins, produced by differ-
ent species of Streptomyces: Streptomyces cacaoi var. asoensis (Endo and Misato 1969; Isono,
Asahi, and Suzuki 1969) , and Streptomyces tendae, respectively (Dahn et al. 1976) (see
Figure 4.1). These compounds have Ki values in the micromolar range when analyzed
against the enzymatic activity of cell-free extracts or partially purified preparations. When
compared with the Km of the substrate that is in the millimolar range, this means a dif-
ference of 1000. However, their minimal inhibitory concentrations (MIC) are significantly
higher when tested against whole cells, probably due to their impaired permeability into
the cells, a process that was suggested to utilize a dipeptide (Hori, Kakiki, and Misato
1974a) or a tripeptide carrier system (Furter and Rast 1985). Kinetic studies of the binding
of polyoxins and several derivatives to the active site of chitin synthases demonstrated that
they behave as competitive inhibitors (Hori, Kakiki, and Misato 1974a; 1974b). This behav-
ior was later observed for chitin synthases from all fungi analyzed thus far (Ruiz-Herrera
and San-Blas 2003). Nevertheless some data suggest that some specific structural char-
acteristics of Chs are responsible for the inhibitory mechanism of peptidyl nucleosides,
taking into consideration that they are specific inhibitors of chitin synthases, and do not
inhibit other reactions that utilize UDPGlcNAc as substrate, such as murein biosynthesis
in bacteria and glycoprotein biosynthesis in eukaryotes. Also, the differential sensitivity
of several Chs to polyoxins and nikkomycins, the different sensitivity of chitin synthases
dependent on Mg2+ or Co2+, and the observation that the Ki for the DG42 protein (involved
in the synthesis of a chitin-like oligosaccharide during Xenopus development) is very high,
ca. 50 mM (Semino and Robbins 1995), are very difficult to explain only on the basis of a
competitive inhibitory mechanism of action of both types of compounds (see Ruiz-Herrera
and San-Blas 2003, for discussion).
It is known that the inhibitory effect of polyene macrolide antibiotics depends on the
alterations they bring about on the selective permeability of the membranes by their asso-
ciation to sterols (see Récamier et al. 2010); therefore, no easy explanation can be given for
their inhibitory effect on chitin synthase reported by Rast and Bartnicki-García (1981) and
Merz et al. (1999). The observed complex kinetics and high Ki exhibited by polyenes on Chs
can only be explained as due to their interactions with the sterols located in the chitosomal
membrane (Lopez-Romero, Monzon, and Ruiz-Herrera 1985).
A study of the mode of action of pentachloronitrobenzene (PCNB) on chitin syn-
thase activity had an interesting connotation with regard to the possible structure of the
enzyme (Merz et al. 1999). The observation that this inhibitor has no structural similarity
with the substrate (UDPGlcNAc) but forms a stable complex with chitin synthase, led the
authors to suggest that it binds tightly to a tyrosine residue located at the active site of the
chitin synthases.
During synthetic reaction, besides chitin, uridine diphosphate (UDP) is produced
from UDPGlcNAc as a byproduct. It was demonstrated that this nucleotide inhibits chitin
synthases with complex kinetics, which may be suggestive of allosteric interactions and
Ki values in the mmolar range (Lopez-Romero and Ruiz-Herrera 1976). Besides UDP, other
nucleotides inhibit chitin synthases with lower or higher Ki than UDP; among these we
can mention the following in order of efficiency: uridine monophosphate (UMP), uridine,
UDPMan, UDPGlc, deoxythimidine, guanosine monophosphate (GMP), dTMP, GDPGlc,
GDPMan, and dTPGLc with Ki values from about 1.5 to 30 mM for Piricularia oryzae (Hori
et al. 1974a).
4.6 Cytology of chitin biosynthesis

The biochemical mechanism of chitin biosynthesis was originally considered a simple
transglycosylation reaction, but later it was revealed to be difficult to understand how
the biosynthetic process occurs in vivo, taking into consideration that chitin is one of the
most insoluble compounds in nature, and accumulates in the cell walls of fungi, whereas
the substrate is present in the cytoplasm. Two solutions have been suggested for the pro-
cess: that the polysaccharide is synthesized at the site of its final deployment; or else that
a noncrystalline form of chitin is transferred from the cytoplasm to the exterior. Most
authors who have written on the process favor the first hypothesis. The fact that chitin
synthases contain several hydrophobic transmembrane stretches has led to the suggestion
that the enzyme is present in the plasma-lema and transports GlNAc residues donated by
UDPGlcNAc from the cytoplasmic face to the external one, where the biosynthetic process
takes place. As already described, fungi contain α-chitin, whose chains are antiparallel
(see above); accordingly, synthesis of the polysaccharide and microfibril formation can
not be simultaneous processes, as some authors have suggested, and must be separated
in time. Also, taking into consideration that no protein synthesis occurs at the plasma
membrane, it has to be accepted that chitin synthase must be synthesized in a different
compartment of the cell.
Early studies, originally on M. rouxii, and later with other fungal species belonging
to different taxa, revealed that most of the chitin synthase was present in a cytoplasmic
population of microvesicles of regular size and density. These microvesicles were desig-
nated as chitosomes (Bracker, Ruiz-Herrera, and Bartnicki-García 1976; Bartnicki-García et
al. 1978; Bartnicki-García, Ruiz-Herrera et al. 1979; Ruiz-Herrera 1984). Chitosomes have a
diameter of about 40–70 nm, and possess a very thin double membrane measuring 6.5–7.0
nm. Chitosomes were found to be made of two-parts protein of a unique composition, and
one-part lipids with a composition different to the rest of the cell membranes (Hernandez
et al. 1981). When chitosomes are incubated in vitro with GlcNAc and activators (a protease
and GlcNAc), they suffer an interesting series of morphological modifications, accumu-
lating extremely thin microfibrils inside, and they finally give rise to chitin microfibrils
essentially identical to those existing in the fungal cell wall (Bracker, Ruiz-Herrera, and
Bartnicki-García 1976) (see Figure 4.3). Interestingly, chitosomes can be dissociated by
treatment with digitonin into 500 kDa subunits that retain enzymatic activity, and reas-
semble in the form of chitosome-like aggregates when digitonin is removed.
Although the concept that fungal chitin synthases are accumulated into chitosomes
was slowly accepted, the great amount of evidence that was published supporting the
concept that chitosomes are a specialized type of microvesicles carrying chitin synthase
to the cell surface, where synthesis of chitin takes place finally led to its acceptance, even
by the most severe critics. Among the accumulated pieces of evidences, the following
may be cited: (i) data revealing that chitin synthases were accumulated in the cytoplasm;
for example, the observation that chitin synthesis by toluene-permeabilized cells of M.
rouxii occurred in the cytoplasm, but not in the plasma membrane (Sentandreu, Martinez-
Ramon, and Ruiz-Herrera 1984) and (ii) the observation that cytoplasm from the giant
sporangiophores of P. blakesleeanus extracted with a microsyringe or extruded by pressure,
contained microvesicles, which when incubated with UDPGlcNAc and activators, synthe-
sized typical chitin microfibrils (Herrera-Estrella and Ruiz-Herrera 1982). To these I would
add: (iii) electron microscopic immunochemical studies showing that chitin synthase was
localized in apical microvesicles of N. crassa (Sietsma et al. 1996) and (iv) the demonstration
that specific antibodies raised against three of the eight Chs from U. maydis labeled with
colloidal gold recognized microvesicles emerging from tubular structures and fusing with
the plasma membrane (Ruiz-Herrera et al. 2006).
The origin of chitosomes was originally difficult to trace. It was hypothesized that they
were formed either in the endoplasmic reticulum (ER), or in the Golgi. Furthermore, their
B C
D E
Figure 4.3 Chitosomes from Mucor rouxii and microfibrils synthesized in vitro. Negatively stained
preparations with uranyl acetate. (A) Sample of purified chitosomes. (B) Chitosomes incubated for a
short time with substrate. Chitin microfibrils crystallized inside chitosomes acquiring the figure we
call fibroids. (C) Fibroids and microfibrils synthesized after a longer time of incubation. (D) A fibroid
with an extension of a rather straight microfibril. (E) Robust microfibrils accumulated after a long
incubation period. Magnification bars: A, 300 nm; B through E, 100 nm. (Materials and photographs
courtesy of C. Bracker, S. Bartnicki-García, and J. Ruiz-Herrera.)
presence in multivesicular bodies was entertained, until it was demonstrated that these
organelles are involved in endocytic processes. By use of selective labeling of Chs3 from
S. cerevisiae, data were obtained supporting the concept that they were synthesized in the
rough endoplasmic reticulum (RER), and followed the normal exocytic route (see Santos
and Snyder 1997). These studies led to the description of several genes (CHS4 to CHS7)
possibly involved in the mobilization of chitosomes to the cell surface in S. cerevisiae. It has
been shown that chs5 mutants are affected in Chs3 polarization and in the localization of
the enzyme at the growing sites of the cell. However, the role of Chs5 does not appear to
be specific since chs5 mutants also present misplacement of Fus1p and Fus2p, two proteins
involved in cell fusion during mating (Santos and Snyder 1997). CHS4 encodes another pro-
tein involved in the correct localization and activation of Chs3 (Trilla, Duran, and Roncero
1997; Ono et al. 2000), whereas the role of Chs6 was suggested in the observation that chs6
mutants contained reduced amounts of chitin accumulated in the chitosomes (Ziman et
al. 1998). Regarding Chs7, it has been proposed that it behaves as a specific chaperone for
Chs3 (Trilla, Duran, and Roncero 1999).
However, a problem exists that makes difficult to accept the concept that chitin syn-
thases synthesized by membrane-bound ribosomes in the rough ER follow the normal
exocytic route. The main obstacle is the observation by in silico analysis of gene sequences
that no chitin synthase contains a signal peptide (J. Ruiz-Herrera and L. Ortiz-Castellanos,
unpublished), which constitutes the passport utilized by the proteins that enter the exo-
cytic route (discussed in Chapter 8). In a very important paper, Riquelme et al. (2007)
described the route followed by Chs3 and Chs6 labeled with the green fluorescent protein
(GFP), from the subapical region to the apex of N. crassa hypha. These authors observed
that in contrast to the previous reports on S. cerevisiae Chs3 (Santos and Snyder 1997), the
enzyme did not colocalize with the ER. Instead, labeling identified the presence of the
enzymes in large membranous and tubular structures that gave rise to vesicles (chito-
somes), which accumulated at the Spitzenkörper (see Figure 4.4). With these results, they
suggested a novel mechanism for Chs synthesis and delivery, distinct from the classical
ER-Golgi route. Mobilization of chitosomes to the cell surface has been shown to depend
on actin filaments. It is known that some chitin synthases belonging to division 2 possess
a myosin-like motif at their N-termini, but this is not functional and does not play a role in
chitosome displacement in the cytoplasm (for example, see Treitschke et al. 2010).
It is known that chitin delivered to the extracellular space may be subjected to dif-
ferent modifications; for example, deacetylation to form chitosan, covalently binding to
other wall components, and crystallization into microfibrils (reviewed by Ruiz-Herrera
and Martinez-Espinoza 1999). Briefly, we can summarize the in vivo synthetic process of
chitin as follows: (i) synthesis of sugar chains at the plasma-lemma-cell wall interface, (ii)
chemical modifications of certain amounts of the polymer, (iii) establishment of covalent
and noncovalent associations with other wall components, (iv) microfibril formation, and
finally (v) organization into the wall composite.
Figure 4.4 Laser scanning confocal microscopy (LSCM) of a hyphal apex of Neurospora crassa
expressing chitin synthase 1 tagged with green fluorescent protein (Chs 1-GFP). (A) Phase-contrast
showing mitochondria, the Spitzenkörper (Spk) at the apical dome and other high-density intracel-
lular organelles. (B) Fluorescent channel showing strong localization of Chs1-GFP at the Spk. (C)
Merged images of A and B. Scale bar: 5 μm. (Courtesy of Meritxel Riquelme.) (See color insert.)
References
Amnuaykanjanasin, A. and Epstein, L. 2003. A class V chitin synthase gene, chsA is essential for
conidial and hyphal wall strength in the fungus Colletotrichum graminicola (Glomerella gramini-
cola). Fungal Genet. Biol. 38:272–285.
Andaluz, E., Ridruejo, J. C., Ramirez, M., Ruiz-Herrera, J., and Larriba, G. 1988. Initiation of glucan
synthesis in yeast. FEMS Microbiol. Lett. 49:251–255.
Arroyo-Begovich, A., Carabez-Trejo, A., and Ruiz-Herrera, J. 1980. Identification of the structural
component in the cyst wall of Entamoeba invadens. J. Parasitol. 66:735–741.
Balestrini, R., Mainieri, D., Soragni, E., Garnero, L., Rollino, S., Viotti, A., Ottonello, S., and Bonfante,
P. 2000. Differential expression of chitin synthase III and IV mRNAs in ascomata of Tuber borchii
Vittad. Fungal Genet. Biol. 31:219–232.
Bartnicki-García, S., Bracker, C. E., Reyes, E., and Ruiz-Herrera, J. 1978. Isolation of chitosomes from
taxonomically diverse fungi and synthesis of chitin microfibrils in vitro. Exp. Mycol. 2:173–192.
Bartnicki-García, S., Lippman, E., and Heick, J. 1979. Evidence for a poly-peptide acceptor in fungal
chitin biosynthesis. Abstr. Annu. Meet. Am. Soc. Microbiol. 106.
Bartnicki-García, S., Ruiz-Herrera, J., and Bracker, C. E. 1979. “Chitosomes and Chitin Synthesis.” In
Fungal Walls and Hyphal Growth. Edited by J. H. Burnett and A. P. J. Trinci, 149–168. Cambridge:
Cambridge University Press.
Beth-Din, A. and Yarden, O. 2000. The Neurospora crassa chs3 gene encodes an essential Class I chitin
synthase. Mycologia 92:65–73.
Blackwell, J., Minke, R., and Gardner, K. H. 1978. “Determination of the Structure of α- and β-chitins
by X-Ray Diffraction.” In Proceedings of the First International Conference on Chitin/Chitosan.
Edited by R. A. A. Muzzarelli and E. R. Pariser, 108–123. Cambridge: Massachusetts Institute
of Technology Press.
Borgia, P. T., Iartchouk, N., Riggle, P. J., Winter, K. R., Koltin, Y., and Bulawa, C. E. 1996. The chsB gene
of Aspergillus nidulans is necessary for normal hyphal growth and development. Fungal. Genet.
Biol. 20:193–203.
Bowen, A. R., Chen-Wu, J. L., Momany, M., Young, R., Szaniszlo, P. J., and Robbins, P. W. 1992.
Classification of fungal chitin synthases. Proc. Natl. Acad. Sci. USA 89:519–523.
Bracker, C. E., Ruiz-Herrera, J., and Bartnicki-García, S. 1976. Structure and transformation of chitin
synthetase particles (chitosomes) during microfibril synthesis in vitro. Proc. Natl. Acad. Sci. USA
73:4570–4574.
Braconnot, H. 1811. Ann. Chim. (Paris) 79:265. Cited in Foster, A. B. and Webber, J. M. Chitin. Adv.
Carb. Chem. 15:371–396.
Bulawa, C. E. 1992. CSD2, CSD3, and CSD4, genes for chitin synthesis in Saccharomyces cerevisiae: The
CSD gene product is related to chitin synthases and to developmentally regulated proteins of
Rhizobium species and Xenopus levis. Mol. Cell. Biol. 12:1764–1776.
Bulawa, C. E., Slater, M., Cabib, E., Au-Young, J., Sburlati, A., Adair, W. L., and Robbins, P. W. 1986.
The Saccharomyces cerevisiae structural gene for chitin synthase is not required for chitin synthe-
sis in vivo. Cell 46:213–225.
Mycol. 11:128–140.
Charnock, S. J. and Davies, G. J. 1999. Structure of the nucleotide-diphospho-sugar transferase, SpsA
from Bacillus subtilis, in native and nucleotide-complexed forms. Biochemistry 38:6380–6385.
Chavez-Ontiveros, J., Martinez-Espinoza, A. D., and Ruiz-Herrera, J. 2000. Double chitin synthetase
mutantes from the corn smut fungus Ustilago maydis. New Phytol. 146:335–341.
Chen-Wu, J. L., Zwicker, J., Bowen, A. R., and Robbins, P. W. 1992. Expression of chitin synthase
genes during yeast and hyphal growth phases of Candida albicans. Mol. Microbiol. 6:497–502.
Culp, D. W., Dodge, C. L., Miao, Y., Li, L., Sag-Ozkal, D., and Borgia, P. T. 2000. The chsA gene from
Aspergillus nidulans is necessary for maximal conidiation. FEMS Microbiol. Lett. 182:349–353.
Dahn, U., Hagenmaier, H., Höhne, H., Konig, W. A., Wolf, G., and Zähner, H. 1976. Stoffwechsel
produkte von Mikroorganismen. 154 Mitteilung. Nikkomycin, ein neuer Hemmstof der
Chitinsynthese bei Pilzen. Arch. Microbiol. 107:143–160.
DeAngelis, P. L. 1999. Molecular directionality of polysaccharide polymerization by the Pasteurella
multocida hyaluronan synthase. J. Biol. Chem. 274:26557–26562.
Deshpande, M. V., O’Donnell, R., and Gooday, G. W. 1997. Regulation of chitin synthase activity in
the dimorphic fungus Benjaminiella poitrasii by external osmotic pressure. FEMS Microbiol. Lett.
152:327–332.
Dietrich, S. M. C. 1975. Comparative study of hyphal wall components of Oomycetes: Saprolegniae
and Pythiaceae. An. Acad. Brasil. Cien. 47:155–162.
Domard, A., Jeuniaux, C., Muzzarelli, R. A. A., and Roberts, G. 1996. Advances in Chitin Science, Vol
1. France: Jacques André.
Elorza, M. V., Rico, H., and Sentandreu, R. 1983. Calcofluor white alters the assembly of chitin fibrils
in Saccharomyces cerevisiae and Candida albicans cells. J. Gen. Microbiol. 129:1577–1582.
Endo, A. and Misato, T. 1969. Polyoxin, D., a competitive inhibitor of UDP-N-actylglucosamine: Chitin
N acetylglucosaminyl transferase in Neurospora crassa. Biochem. Biophys. Res. Commun. 37:718–722.
Fujiwara, M., Ichinomiya, M., Motoyama, T., Horiuchi, H., Ohta, A., and Takagi, M. 2000. Evidence
that the Aspergillus nidulans class I and class II chitin synthase genes, chsC and chsA, share criti-
cal roles in hyphal wall integrity and conidiophore development. J. Biochem. 127:359–366.
Furter, R. and Rast, D. M. 1985. A comparison of the chitin synthase-inhibitory and antifungal effi-
cacy of nucleoside-peptide antibiotics: Structure-activity relationship. FEMS Microbiol. Lett.
28:205–211.
Garcerá-Teruel, A., Xoconostle-Cázares, B., Rosas-Quijano, R., Ortiz, L., León-Ramírez, C., Specht,
C. A., Sentandreu, R., and Ruiz-Herrera, J. 2004. Loss of virulence in Ustilago maydis by Umchs6
gene disruption. Res. Microbiol. 155:87–97.
Garcia-Rodriguez, L. J., Trilla, J. A., Castro, C., Valdivieso, M. H., Duran, A., and Roncero, C. 2000.
Characterization of the chitin biosynthesis process as a compensatory mechanism in the fks1
mutant of Saccharomyces cerevisiae. FEBS Lett. 478:84–88.
Glaser, L. and Brown, D. H. 1957. The synthesis of chitin in cell-free extracts of Neurospora crassa. J.
Biol. Chem. 228:729–742.
Hanseler, E., Nyhlen, L. E., and Rast, D. M. 1983. Isolation and properties of chitin synthetase from
Agaricus bisporus mycelium. Exp. Mycol. 7:17–30.
Hepburn, H. R. and Chandler, H. D. 1978. “Tensile Mechanical Properties and Transconformational
Changes of Chitins.” In Proceedings of the First International Conference on Chitin/Chitosan. Edited
by R. A. A. Muzzarelli and E. R. Pariser, 124–143. Cambridge: Massachusetts Institute of
Technology Press.
Hernandez, J., Lopez-Romero, E., Cerbon, J., and Ruiz-Herrera, J. 1981. Lipid analysis of chitosomes,
chitin-synthesizing microvesicles from Mucor rouxii. Exp. Mycol. 5:349–356.
for roles of chitin biosynthesis and breakdown. Exp. Mycol. 7:362–369.
Herth, W. 1980. Calcofluor white and Congo red inhibit chitin microfibril assembly of
Poteriochromonas: Evidence for a gap between polymerization and microfibril formation. J.
Cell. Biol. 87:442–450.
Herth, W., Mulisch, M., and Zugenmaier, P. 1986. “Comparison of Chitin Fibril Structure and
Assembly in Three Unicellular Organisms.” In Chitin in Nature and Technology. Edited by R.
Muzarelli, C. Jeauniaux, and G. W. Gooday, 107–120. New York and London: Plenum Press.
Hori, M., Kakiri, K., and Misato, T. 1974a. Further study on the relation of polyoxin structure to chitin
synthetase inhibition. Agr. Biol. Chem. 38:691–698.
Hori, M., Kakiki, K., and Misato, T. 1974b. Interaction between polyoxin and active center of chitin
synthetase. Agr. Biol. Chem. 38:699–705.
Horsch, M. and Rast, D. M. 1993. “Allosteric Activation of Chitin Synthase by N-acetylglucosamine.
A Mechanistic Study.” In Chitin Enzymology. Edited by R. A. A. Muzzarelli, 47–56. Lyon and
Ancona: European Chitin Society.
Horsch, M. and Sowdhamini, R. 1996. “A Fold Prediction for the Catalytic Domain of Chitin
Synthases.” In Chitin Enzymology, Vol. 2. Edited by R. A. A. Muzzarelli, 447–458. Grottammare:
Atec Edizioni.
Horsch, M., Mayer, C., and Rast, D. M. 1996. Stereochemical requirements of chitin synthase for
ligand binding at the allosteric site for N-acetylglucosamine. Eur. J. Biochem. 237:476–482.
Horst, M. N. 1983. The biosynthesis of crustacean chitin. Isolation and characterization of polypre-
nol-linked intermediates from brine shrimp microsomes. Arch. Biochem. Biophys. 223:254–263.
Imai, T., Watanabe, T., Yui, T., and Sugiyama, J. 2003. The directionality of chitin biosynthesis: A
revisit. Biochem. L. 374:755–760.
Isono, K., Asahi, K., and Suzuki, S. 1969. Studies on polyoxins, antifungal antibiotics. XIII. The struc-
ture of polyoxins, J. Am. Chem. Soc. 91:7490–7505.
Jeauniaux, C. 1978. “Distribution and Quantitative Importance of Chitin in Animals. In Proceedings
of the First International Conference on Chitin-Chitosan. Edited by R. A. A. Muzzarelli and E. R.
Pariser, 5–10. Cambridge, MA: MIT Sea Grant Program.
Johnson, M. S., Overington, J. P., and Blundell, T. L. 1993. Alignment and searching for common pro-
tein folds using a data bank of structural templates. J. Mol. Biol. 231:735–752.
Keller, F. A. and Cabib, E. 1971. Chitin and yeast budding. Properties of chitin synthetase from
Saccharomyces carlsbergensis. J. Biol. Chem. 246:160–166.
Koyama, M., Helbert, W., Imai, T., Sugiyama, J., and Henrissat, B. 1997. Parallel-up structure evi-
dences the molecular directionality during biosynthesis of bacterial cellulose. Proc. Natl. Acad.
Sci. USA 94:9091–9095.
Lai-Kee-Him, J., Chanzy, H. Müller, M., Putaux, J. L., Imai, T., and Bulone, V. 2002. In vitro versus
in vivo cellulose microfibrils from plant primary wall synthases: Structural differences. J. Biol.
Chem. 277:36931–36939.
Lee, H. Y. and Aronson, J. M. 1975. Composition of cellulin, the unique chitin-glucan granules of the
fungus Apodachlya sp. Arch. Microbiol. 102:203–208.
Lenardon, M. D., Milne, S. A., Mora-Montes, H. M., Kaffarnik, F. A. R., Peck, S. C., Brown, A. J. P.,
Munro, C. A., and Gow, N. A. R. 2010. Phosphorylation regulates polarization of chitin synthe-
sis in Candida albicans. J. Cell Sci. 123:2199–2206.
Lopez-Matas, M. A., Eslava, A. P., and Diaz-Minguez, J. M. 2000. Mcchs1, a member of a chitin syn-
thase gene family in Mucor circinelloides, is differentially expressed during dimorphism. Curr.
Lopez-Romero, E., Monzon, E., and Ruiz-Herrera, J. 1985. Sterol composition of chitosomes from
yeast cells of Mucor rouxii: Comparison with whole cells. FEMS Microbiol. Lett. 30:369–372.
Lopez-Romero, E. and Ruiz-Herrera, J. 1976. Synthesis of chitin by particulate preparations from
Aspergillus flavus. Ant. v. Leeuwenhoek 42:261–276.
Lopez-Romero, E., Ruiz-Herrera, J., and Bartnicki-García, S. 1982. The inhibitory protein of chitin
synthetase from Mucor rouxii is a chitinase. Biochim. Biophys. Acta 702:233–236.
Ma, L. J., Ashraf, S., Ibrahim, A. S. et al. 2009. Genomic analysis of the basal lineage fungus Rhizopus
oryzae reveals a whole-genome duplication. PLoS Genetics 5:e1000549.
Madrid, M. P., Di Pietro, A., and Roncero, M. I. 2003. Class V chitin synthase determines pathogenesis
in the vascular wilt fungus Fusarium oxysporum and mediates resistance to plant defence com-
pounds. Mol. Microbiol. 47:257–266.
Martin, H., Dagkessamanskaia, A., Satchanska, G., Dallies, N., and Francois, J. 1999. KNR4, a sup-
pressor of Saccharomyces cerevisiae cwh mutants, is involved in the transcriptional control of
chitin synthase genes. Microbiology 145:249–258.
Martín-Urdíroz, M., Roncero, M. I. G., González-Reyes, J. A., and Ruiz-Roldan, C. 2008. ChsVb, a
Class VII chitin synthase involved in septation, is critical for pathogenicity in Fusarium oxyspo-
rum. Eukaryot. Cell 7:112–121.
Martinez-Cadena, G. and Ruiz-Herrera, J. 1987. Activation of chitin synthetase from Phycomyces
blakesleeanus by calcium and calmodulin. Arch. Microbiol. 148:280–285.
McMurrough, I. and Bartnicki-García, S. 1971. Properties of a particulate chitin synthetase from
Mucor rouxii. J. Biol. Chem. 246:4008–4016.
McMurrough, I., Flores-Carreon, A., and Bartnicki-García, S. 1971. Pathway of chitin synthesis and
cellular localization of chitin synthetase in Mucor rouxii. J. Biol. Chem. 246:3999–4007.
Merz, R. A., Horsch, M., Nyhlén, L. E., and Rast, D. M. 1999. “Biochemistry of Chitin Synthase.” In
Chitin and Chitinases. Edited by P. Jolles and R. A. A. Muzzarelli, 9–37. Basel: Birkhäuser.
Merzendorfer, H. and Zimoch, L. 2003. Chitin metabolism in insects: Structure, function, and regula-
tion of chitin synthases and chitinases. J. Exp. Biol. 206:4393–4412.
Mills, G. L. and Cantino, E. C. 1980. The glycolipid involved in chitin synthesis by zoospores of
Blastocladiella emersonii is a monoglucosyldiacylglycerol. Exp. Mycol. 4:175–180.
Montgomery, G. W. G. and Gooday, G. W. 1985. Phospholipid-enzyme interactions of chitin synthase
of Coprinus cinereus. FEMS Microbiol. Lett. 27:29–33.
Moore, P. M. and Peberdy, J. F. 1975. Biosynthesis of chitin by particulate preparations from
Cunninghamella elegans. Microbios. 12:29–39.
Munro, C. A., Selvaggini, S., De Bruijn, I., Walker, L., Lenardon, M. D., Gerssen, B., Milne, S., Brown,
A. J. P., and Gow, N. A. R. 2007. The PKC, HOG and Ca2++ signalling pathways co-ordinately
regulate chitin synthesis in Candida albicans. Mol. Microbiol. 63:1399–1413.
Munro, C. A., Winter, K., Buchan, A., Henry, K., Becker, J. M., Brown, A. J., Bulawa, C. E., and Gow,
N. A. 2001. Chs1 of Candida albicans is an essential chitin synthase required for synthesis of the
septum and for cell integrity. Mol. Microbiol. 39:1414–1426.
Muzzarelli, R. A. A. 1977. Chitin. London, England: Pergamon Press.
Muzarelli, R. A. A. 1984. “Chitin.” In The Polysaccharides, Vol. 3. Edited by G. O. Aspinall, 417–450.
New York: Academic Press.
Muzzarelli, R. A. A. 1993a. Chitin Enzymology. Italy: Atec Grottammare.
Muzzarelli, R. A. A. 1993b. Chitin Enzymology, Vol 2. Italy: European Chitin Society, Ancona.
Muzzarelli, R. A. A. 1999. “Native, Industrial and Fossil Chitins.” In Chitin and Chitinases. Edited by
P. Jolles and R. A. A. Muzzarelli, 1–6. Basel, Switzerland: Birkhäuser.
Muzzarelli, R. A. A. and Peter, M. G. 1997. Chitin Handbook. Italy: European Chitin Society, Ancona.
Nagahashi, S., Sudoh, M., Ono, N., Sawada, R., Yamaguchi, E., Uchida, Y., Mio, T., Takagi, M., Arisawa,
M., and Yamada-Okabe, H. 1995. Characterization of chitin synthase 2 of Saccharomyces cere-
visiae: Implication of two highly conserved domains as possible catalytic sites. J. Biol. Chem.
270:13961–13967.
Nino-Vega, G. A., Munro, C. A., San-Blas, G., Gooday, G. W., and Gow, N. A. R. 2000. Differential
expression of chitin synthase genes during temperature-induced dimorphic transitions in
Paracoccidioides brasiliensis. Med. Mycol. 38:31–39.
Odier, A. 1823. Memoire sur la composition chemique des parties cornees des insects. Mem. Soc. Hist.
Nat. Paris. 1:29–42.
Ono, N., Yabe, T., Sudoh, M., Nakajima, T., Yamada-Okabe, T., Arisawa, M., and Yamada-Okabe,
H. 2000. The yeast Chs4 protein stimulates the trypsin-sensitive activity of chitin synthase 3
through an apparent protein-protein interaction. Microbiology 146:385–391.
Park, B. C., Maeng, P. J., and Park, H. M. 2001. Cell cycle-dependent expression of chitin synthase
genes in Aspergillus nidulans. J. Microbiol. 39:74–78.
Park, B. C., Park, Y. H., and Park, H. M. 2003. Activation of chsC transcription by AbaA during
asexual development of Aspergillus nidulans. FEMS Microbiol. Lett. 220:241–246.
Peberdy, J. F. and Moore, P. M. 1975. Chitin synthase in Mortierella vinacea, properties, cellular loca-
tion and synthesis in growing cultures. J. Gen. Microbiol. 90:228–236.
Popolo, L., Gilardelli, D., Bonfante, P., and Vai, M. 1997. Increase in chitin as an essential response
to defects in assembly of cell wall polymers in the ggp1D mutant of Saccharomyces cerevisiae. J.
Bacteriol. 179:463–469.
Rast, D. M. and Bartnicki-García, S. 1981. Effects of amphotericin B, nystatin, and other polyene anti-
biotics on chitin synthase. Proc. Natl. Acad. Sci. USA 78:1233–1236.
Rast, D., Merz, R. A., Jeanguenat, A., and Mösinger, E. 2000. “Enzymes of Chitin Metabolism for the
Design of Antifungals.” In Advances in Chitin Sciences, Vol 4. Edited by M. G. Peter and R. A. A.
Muzzarelli, 479–505. Postdam: University of Postdam.
Récamier, K. S., Hernández-Gómez, A., González-Damián, J., and Ortega-Blake, I. 2010. Effect of
membrane structure on the action of polyenes: I. Nystatin action in cholesterol and ergosterol-
containing membranes. J. Membrane. Biol. 237:31–40.
Reyna, G. and Ruiz-Herrera, J. 1987. Fotoactivación in vitro de la quitina sintetasa de los esporangió-
foros de Phycomyces blakesleeanus. Rev. Mex. Micol. 3:249–2623.
Riquelme, M., Bartnicki-García, S., González-Prieto, J. M., Sánchez-León, E., Verdín-Ramos, J. A.,
Beltrán-Aguilar, A., and Freitag, M. 2007. Spitzenkörper localization and intracellular traffic
of green fluorescent protein-labeled CHS-3 and CHS-6 chitin synthases in living hyphae of
Neurospora crassa. Eukaryot. Cell 6:1853–1864.
Roach, P. J. and Skurat, A. V. 1997. Self-glucosylating inhibitor proteins and their role in glycogen
biosynthesis. Prog. Nucl. Acids. Res. Mol. Biol. 57:289–316.
Robledo-Briones, A. M. 2009. “Análisis de la Expresión de las Quitina Sintasas Y β 1,3 Glucana
Sintasa de Ustilago Maydis a Nivel Transcripcional.” (M.Sc. Thesis.) Irapuato, Mexico: Unidad
Irapuato, Centro de Investigación y de Estudios Avanzados, IPN.
Roelofsen, P. A. 1965. “Ultrastructure of the Wall in Growing Cells and Its Relation to the Direction
of Growth.” In Advances in Botanical Research, Vol. 2. Edited by R. D. Preston, 67–149. New York
and London: Academic Press.
Rousset-Hall, A. and Gooday, G. W. 1975. A kinetic study of solubilized chitin synthetase prepara-
tions from Coprinus cinereus. J. Gen. Microbiol. 88:146–154.
Ruiz-Herrera, J. 1978. “The Distribution and Quantitative Importance of Chitin in Fungi.” In
Proceedings of the First International Conference on Chitin/Chitosan. Edited by R. A. A. Muzarelli
and E. R. Pariser, 11–21. Cambridge, MA: MIT Sea Grant Program.
Ruiz-Herrera, J. 1982. “Synthesis of Chitin Microfibrils In Vitro.” In Cellulose and Other Natural Polymer
Systems. Edited by R. M Brown, 207–223. New York: Plenum Press.
Ruiz-Herrera, J. 1984. “The Role of Chitosomes in the Apical Growth of Fungi.” In Microbial Cell Wall
Synthesis and Assembly. Edited by C. Nombela, 113–120. Amsterdam: Elsevier.
Ruiz-Herrera, J., Gonzalez-Prieto, J. M., and Ruiz-Medrano, R. 2001. “Significance of the Multigenic
Control of Chitin Synthase in Fungi.” In Chitin Enzymology. Edited by R. A. A. Muzzarelli,
451–462. Grottammare: Atec Edizioni.
Ruiz-Herrera, J., Gonzalez-Prieto, J. M., and Ruiz-Medrano, R. 2002. Evolution and phylogenetic
reationships of chitin synthases from yeasts and fungi. FEMS Yeast Res. 1(4):247–526.
Ruiz-Herrera, J. and Larriba, G. 1995. High molecular weight precursors of glucans in Saccharomyces
cerevisiae. Ant. v. Leeuwenhoek 68:231–235.
Ruiz-Herrera, J., Lopez-Romero, E., and Bartnicki-García, S. 1977. Properties of chitin synthetase in
isolated chitosomes from yeast cells of Mucor rouxiii. J. Biol. Chem. 252:3338–3343.
Ruiz-Herrera, J. and Martinez-Espinoza, D. 1999. “Chitin Biosynthesis and Structural Organization In
Vivo.” In Chitin and Chitinase. Edited by P. Jolles and R. A. A. Muzzarelli, 39–56. Basel: Birkhäuser.
Biotechnology, 2nd ed. Edited by P. K. Arora. New York: Marcel Dekker.
Ruiz-Herrera, J. and San-Blas, G. 2003. Chitin synthesis as a target for antifungal drugs. Curr. Drug.
Targ. Infect. Disorders 3:77–91.
Ruiz-Herrera, J., Sentandreu, R., and Martinez, J. P. 1992. “Chitin Biosynthesis in Fungi.” In Applied
Mycology, Vol. 4. Edited by D. K. Arora, K. G. Mukerji, and R. P. Elander, 281–312. New York:
Marcel Dekker.
Ruiz-Herrera, J., Xoconostle-Cázares, B., Reynaga-Peña, C. G., León-Ramírez, C., and Cárabez-Trejo,
A. 2006. Immunolocalization of chitin synthases in the phytopathogenic dimorphic fungus
Ustilago maydis. FEMS Yeast Res. 6:999–1009.
Salmon, S. and Hudson, S. M. 1997. Crystal morphology, biosynthesis, and physical assembly of cel-
lulose, chitin, and chitosan. Polymer Rev. 37:199–276.
Santos, B. and Snyder, M. 1997. Targeting of chitin synthase 3 to polarized growth sites in yeast
requires Chs5p and Myo2p. J. Cell Biol. 136:95–110.
Saxena, I. M., Brown, M. R. Jr., Fevre, M., Geremia, R. A., and Henrissat, B. 1995. Multidomain
architecture of β-glycosyl transferases: Implications for mechanisms of action. J. Bacteriol.
177:1419–1424.
Sburlati, A. and Cabib, E. 1986. Chitin synthase 2, a presumptive participant in septum formation in
Saccharomyces cerevisiae. J. Biol. Chem. 261:15147–15152.
Scurfield, G. 1967. Fine structure of the cell walls of Polyporus millitae Cke. et Mass. J. Linn. Soc. Bot.
60:159–166.
Semino, C. E. E. and Robbins, P. W. 1995. Synthesis of “Nod”-like chitin oligosaccharides by the
Xenopus environmental protein DG42. Proc. Natl. Acad. Sci. USA 92:3498–3501.
Sentandreu, R., Martinez-Ramon, A., and Ruiz-Herrera, J. 1984. Localization of chitin synthase in
Mucor rouxii by an autoradiographic method. J. Gen. Microbiol. 130:1193–1199.
Shaw, J. A., Mol, P. C., Bowers, B., Silverman, S. J., Valdivieso, M. H., Duran, A., and Cabib, E. 1991.
The function of chitin synthases 2 and 3 in the Saccharomyces cerevisiae cell cycle. J. Cell Biol.
114:111–123.
Sietsma, J. H., Beth Din, A., Ziv, V., Sjollema, K. A., and Yarden, O. 1996. The localization of chitin
synthase in membranous vesicles (chitosomes) in Neurospora crassa. Microbiology 142:1591–1596.
Silverman, S. J., Sburlati, A., Slater, M. L., and Cabib, E. 1988. Chitin synthase 2 is essential for septum
formation and cell division in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 85:4735–4739.
Skurat, A. V., Dietrich, A. D., Zhai, L., and Peter J. Roach, P. J. 2002. GNIP, a novel protein that binds
and activates glycogenin, the self-glucosylating initiator of glycogen biosíntesis. J. Biol. Chem.
277:19331–19338.
Specht, C. A., Liu, Y., Robbins, P., Bulawa, C. E., Iartchouk, N., Winter, K. R., Riggle, P. J., Rhodes,
J. C., Dodge, C. L., Culp, D. W., and Borgia, P. T. 1996. The chsD and chsE genes of Aspergillus
nidulans and their roles in chitin synthesis. Fungal Genet. Biol. 20:153–167.
Suresh, K. and Subramanyam, C. 1997. A putative role for calmodulin in the activation of Neurospora
crassa chitin synthase. FEMS Microbiol. Lett. 150:95–100.
Treitschke, S., Doehlemann, G., Schuster, M., and Steinberg, G. 2010. The myosin motor domain of
fungal chitin synthase V is dispensable for vesicle motility but required for virulence of the
maize pathogen Ustilago maydis. Plant Cell 22:2476–2494.
Trilla, J. A., Duran, A., and Roncero, C. 1997. Characterization of CHS4 (CAL2), a gene of Saccharomyces
cerevisiae involved in chitin biosynthesis and allelic to SKT5 and CSD4. Yeast 13:795–807.
Trilla, J. A., Duran, A., and Roncero, C. 1999. Chs7p, a new protein involved in the control of protein
export from the endoplasmic reticulum that is specifically engaged in the regulation of chitin
synthesis in Saccharomyces cerevisiae. J. Cell Biol. 145:1153–1163.
Valdivieso, M. H., Mol, P. C., Shaw, J. H., Cabib, E., and Duran, A. 1991. CAL1, a gene required for
activity of chitin synthase 3 in Saccharomyces cerevisiae. J. Cell Biol. 114:101–109.
Vermeulen, C. A. and Wessels, J. G. H. 1983. Evidence for a phospholipid requirement for chitin syn-
thase in Schizophyllum commune. Curr. Mirobiol. 8:67–71.
Vermeulen, C. A. and Wessels, J. G. H. 1986. Chitin synthesis by a fungal membrane preparation.
Evidence for a transient non-crystalline state of chitin. Eur. J. Biochem. 158:411–415.
Vrielink, A., Rüger, W., Driessen, H. P. C., and Freemont, P. S. 1994. Crystal structure of the DNA
modifying enzyme β-glucosyltransferase in the presence and absence of the substrate uridine
diphosphoglucose. EMBO J. 13:3413–3422.
Walker, L. A. Munro, C. A., De Bruijn, I., Lenardon, M. D., McKinnon, A., and Gow, N. A. R. 2008.
Stimulation of Chitin Synthesis Rescues Candida albicans from Echinocandins. PLos Pathog.
E1000040, 1–12.
Wang, Z. and Szaniszlo, P. J. 2000. WdCHS3: A gene that encodes a class III chitin synthase in
Wangiella (Exophiala) dermatitidis, is expressed differentially under stress conditions. J. Bacteriol.
182:874–881.
Weber, I., Assmann, D., Thines, E., and Steinberg, G. 2006. Polar localizing class V myosin chitin syn-
thases are essential during early plant infection in the plant pathogenic fungus Ustilago maydis.
Plant Cell 18:225–242.
Werner, S., Sugui, J. A., Steinberg, G., and Deising, H. B. A. 2007. Chitin synthase with a myosin-like
motor domain is essential for hyphal growth, appressorium differentiation, and pathogenicity
of the maize anthracnose fungus Colletotrichum graminicola. MPMI 20:1555–1567.
Xoconostle-Cázares, B., León-Ramirez, C., and Ruiz-Herrera, J. 1996. Two chitin synthase genes from
Ustilago maydis. Microbiology 142:377–387.
Xoconostle-Cazares, B., Specht., C. A., Robbins, P. W., Liu, Y., Leon, C., and Ruiz-Herrera, J. 1997.
Umchs5, a gene coding for a class IV chitin synthase in Ustilago maydis. Fungal Genet. Biol.
22:199–208.
Xu, Y. B., Li, H. P., Zhang, J. B., Song, B., Chen, F. F., Duan, X. J., Xu, H. Q., and Liao, Y. C. 2010.
Disruption of the chitin synthase gene CHS1 from Fusarium asiaticum results in an altered struc-
ture of cell walls and reduced virulence. Fungal Genet. Biol. 47:205–215.
Ziman, M., Chuang, J. S., Tsung, M., Hamamoto, S., and Scheckman, R. 1998. Chs6p-dependent
anterograde transport of Chs3p from the chitosome to the plasma membrane in Saccharomyces
cerevisiae. Mol. Biol. Cell. 9:1565–1576.
chapter five
Chitosan
5.1 General characteristics and distribution of chitosan
Chitosan is a polysaccharide of basic nature made mostly of glucosamine and a variable
number of GlcNAc residues bound through β-1,4-linkages. This has been demonstrated by
degradation with nitrous acid to anhydromannose of the differentially labeled polysac-
charide in the sugar moiety and the acetyl group with [14C] or [3H], respectively (Calvo-
Mendez and Ruiz-Herrera 1987) (see below). The amounts of GlcNAc in chitosan may vary
depending on the different batches and the origin of the sample of chitosan analyzed.
On this basis, chitosan can be considered a deacetylated product of chitin. Chitosan is
poorly crystalline as demonstrated by X-ray diffraction analysis; it is easily hydrated and
the hydrogen bonding between its neighbor chains is weak. In contrast to the extreme
insolubility of chitin, chitosan is soluble in acid, and, as indicated above, it reacts with
nitrous acid to produce anhydromannose. In addition, in contrast to chitin that cannot be
stained, it gives a purple color when treated in acid conditions with iodine-iodide solution
(lugol). Chitosan isolated from the cell wall of Mucorales has an Mr of 4 x 105–1.2 x 106, cor-
responding to a polymerization value of ca. 2200–6600 glucosamine units.
Chitosan was originally identified by Kreger (1954) in Phycomyces blakesleeanus, and
it was considered a characteristic component of the cell wall from Mucoromycotina
(Zygomycota), being in higher concentrations than chitin in species of this group of fungi.
However, further studies have proved that chitosan is also present in Ascomycota and
Basidiomycota species. In fact, the evidence of the presence of genes encoding chitin
deacetylases (which are involved in chitosan biosynthesis as described below) in almost
all fungi, suggests that chitosan is a universal component of the cell wall of fungi. In
Ascomycota, chitosan was originally detected in the ascospores of Saccharomyces cerevisiae
(Briza et al. 1988), but not in its vegetative walls, nor in the vegetative walls of Candida
albicans (Banks et al. 2005). Chitosan was also found to be a component of the wall in
Schizosaccharomyces pombe ascospores (Matsuo 2005), and of its vegetative cells, although
in lesser amounts (Sietsma and Wessels 1990). Its presence was also reported in the myce-
lium of Aspergillus niger (Pochanavanich and Suntornsuk 2002). In Basidiomycota, it has
been described to be present in Lentinus edodes and Pleurotus sajo-caju (Crestini, Kovac, and
Giovannozzi-Sermanni 1996; Pochanavanich and Suntornsuk 2002). Noticeable was the
description that the cell walls of the vegetative form of Cryptococcus neoformans contained
higher amounts of chitosan than chitin (Banks et al 2005).
5.2 The functions of chitosan

Because of its physical characteristics, it appears that chitosan does not play a structural
role in the cell wall of fungi. It is probable that due to its basic nature it binds to nega-
tively charged molecules, facilitating their accumulation in the cell wall. Another possible
role may be due to its plastic properties, protecting chitin from the enzymatic attack of
69
chitinases. We observed that fluorescent wheat germ lectin did not stain native stage I spo-
rangiophores of P. blakesleeanus, unless they were treated with HNO2, suggesting that chi-
tosan was covering the wall chitin microfibrils (Herrera-Estrella and Ruiz-Herrera 1983).
In S. cerevisiae it was demonstrated that chitosan is accumulated in a median layer
of the ascospores located between the two internal layers made of glucans and man-
nans, and a superficial protein layer (Briza et al. 1988). As described in detail below, it
was demonstrated that two chitin deacetylases were involved in the synthesis of chito-
san. Interestingly, double mutants in the genes (CDA1 and CDA2) encoding the deacety-
lases formed apparently normal ascospores, but these were more sensitive to ether, heat
shock, or lytic enzymes (Christodoulidou, Bouriotis, and Thireos 1996). The observations
described by Coluccio et al. (2008) were more interesting. These authors confirmed that
ascospores from S. cerevisiae were resistant to digestion in the gut of Drosophila spp., which
has been described to feed on yeasts, whereas ascospores from osw1 or mum3 mutants,
impaired in the formation of dityrosine and chitosan layers, were more sensitive. It was
suggested that feeding by flies is a mechanism of dispersal of diploid cells that may help
increased genetic diversity of the yeast population; but regarding the topic here discussed,
it is an evidence of the role of chitosan in the ascospore resistance and dispersion.
5.3 Chitosan biosynthesis and genetic control and

phylogenetic relationships of chitin deacetylases
A large amount of evidence and results obtained with different systems revealed that
chitosan is synthesized by deacetylation of chitin. The first report of the existence of an
enzyme that deacetylated chitin was published by Araki and Ito (1974). This chitin deacet-
ylase (Cda) could use as substrates soluble chito-oligodextrins or glycolchitin, but not
native chitin. Nevertheless the authors suggested their role in the synthesis of chitin. Only
later was it demonstrated that chitosan was indeed not synthesized by a transglycosyl-
ation reaction from a glucosamine-activated donor, when the existence of a glucosamine-
nucleotide in Mucorales was ruled out, and it was demonstrated that the deacetylases were
not active against the preformed polysaccharide, but only on nascent chitin, before the
polymer reached its microfibrillar crystalline structure (Davis and Bartnicki-García 1984a;
1984b). One important observation was that chitosomes did not synthesize chitosan, but
chitin only, demonstrating that they did not contain the deacetylase (Calvo-Mendez and
Ruiz-Herrera 1987). On the other hand, when crude extracts were incubated with a mix-
ture of UDPGlcNAc labeled with [3H] in the sugar moiety or with [14C] in the acetyl group,
two polysaccharides were synthesized, one (chitin) completely insoluble in acetic acid and
resistant to degradation by nitrous acid, and the other one (chitosan) soluble in acetic acid
and degraded by nitrous acid. The products obtained by HNO2 treatment with nitrous
acid were a mixture of monomers (anhydromannose) exclusively labeled with [3H], and
oligomers of different size (anhydromannose bound to GlcNAc) labeled with both [3H]
and [14C] (see Table 5.1).
These data were evidence that the deacetylase did not act on all the GlcNAc residues
during the synthesis of the polysaccharide (Calvo-Mendez and Ruiz-Herrera 1987). The
model proposed by all these studies suggested that during chitin synthesis, deacetylases
transformed a number of GlcNAc units to glucosamine, inhibiting the correct assembly of
the corresponding chains into microfibrils, allowing their further deacetylation to make
chitosan. The coexistence of chitin and chitosan in more or less conserved proportions
in each fungal species suggested that there might be a rather close association of chitin
Chapter five: Chitosan 71
Table 5.1 In Vitro Synthesized Chitosan Is Made by a Mixture of Glucosamine and

N-Acetyl Glucosamine
Spot Distance from Possible identity C Labeled in
14 C Labeled in
14
number origin (cm) of precursor glucosaminea acetateb

1 0 [GlcN/(GlcNAc]>5 ++++ +
2 8 [GlcN/(GlcNAc]5 ++ ++
3 18 [GlcN/(GlcNAc]4 ++ +
4 23 [GlcN/(GlcNAc]3 +++ ++
5 28 [GlcN/(GlcNAc]2 ++ ++
6 34 [GlcN] ++++ –
Source: Modified from Calvo-Mendez and Ruiz-Herrera, 1987, Biosynthesis of Chitosan in
Membrane Fractions from Mucor rouxii by the Concerted Action of Chitin Synthetase and a
Particulate Deacetylase, Exp. Mycol., 11:128–140.
Note: The products were synthesized by extracts from Mucor rouxii using acetyl- or glucos-
amine-labeled UDPGlcNAc. The products were treated with nitrous acid that transforms
GlcN or GlcN polymers into anhydromannose, but does not affect GlcNAc or its poly-
mers. The resulting products were analyzed by paper chromatography.
a Label in glucosamine corresponds to both GlcNAc and anhydromannose.
b Label in acetate corresponds to GlcNAc.
synthase and deacetylase molecules to make chitosan, and that when such association was
not given, the polysaccharide chains were directed to make chitin. These ideas are sup-
ported by the observation that the ratio of GlcN/GlcNAc residues present in the product
depended on incubation conditions, possibly due to the different turnover number and
kinetic behavior of the enzymes, and their relative amounts in the samples. Decreasing
the concentrations of UDPGlcNAc or GlcNAc in the incubation mixtures, or by addition
of limited concentrations of inhibitors, the proportion of GlcN/GlNAc increased in the
products (Calvo-Mendez and Ruiz-Herrera 1987).
Our in silico studies have demonstrated the presence of genes encoding chitin deacety-
lases in all fungal species analyzed (Ruiz-Herrera and Ortiz-Castellanos 2010). As occurs
with chitin synthases, most of the fungi analyzed contain more than one gene (CDS)
encoding chitin deacetylases. The largest numbers correspond to Zygomycota, as occurs
with CHS genes. Accordingly, in the P. blakesleeanus genome sequence, we identified 9
genes encoding chitin deacetylases, 25 in Mucor circinelloides (J. Ruiz-Herrera and L. Ortiz-
Castellanos, unpublished), and 24 in Rhizopus oryzae (Ma, Ashraf, Ibrahim, et al. 2009). In
the case of Basidiomycota, we identified 8 genes encoding chitin deacetylases in Ustilago
maydis, five of them with signal peptides demonstrating that they are present in the plas-
malema or in the cell wall itself (Ruiz-Herrera et al. 2008). The existence of CDS genes in
Chitridiomycota and Microsporidia, as well as in Protists and lower animals reveals that
they are ancient genes that probably appeared right after chitin synthases in Opisthokonts
(Ruiz-Herrera and Ortiz-Castellanos 2010).
The specific role of each one of the encoded deacetylases is difficult to understand.
In the case of S. cerevisiae where CDS1 and CDS2 have been found to be the deacetylases
involved in chitosan biosynthesis during ascospore formation, it is known that only the
cds1/cds2 double mutants lack chitosan (Christodoulidou et al. 1996), and that CHS3 is the
enzyme that provides the chitin for chitosan biosynthesis. Similarly, it was reported that
of the 8 CHS genes present in C. neoformans, only strains with a mutation in gene CHS3
were deficient in chitosan biosynthesis (Banks et al. 2005). Additionally mutants in gene
CSR2 (a growth regulator) also displayed a reduction in chitosan biosynthesis. The authors
suggested that a complex between Chs3 and Csr2 was necessary to recruit a chitin deacet-
ylase for chitosan formation.
References
Araki, Y. and Ito, E. 1974. A pathway of chitosan formation in Mucor rouxii: Enzymatic acetylation of
chitin. Biochim. Biophys. Res. Commun. 56:669–675.
Banks, I. R., Specht, C. A., Donlin, M. J., Gerik, K. J., Levitz, S. M., and Lodge, J. K. 2005. A chitin
synthase and its regulator protein are critical for chitosan production and growth of the fungal
pathogen Cryptococcus neoformans. Eukaryot. Cell 4:1902–1912.
Briza, P., Ellinger, A., Winkler, G., and Breitenbach, M. 1988. Chemical composition of the yeast asco-
spore wall. The second outer layer consists of chitosan. J. Biol. Chem. 263:11569–11574.
Mycol. 11:128–140.
Christodoulidou, A., Bouriotis, V., and Thireos, G. 1996. Two sporulation-specific chitin deacetylase-
encoding genes are required for the ascospore wall rigidity of Saccharomyces cerevisiae. J. Biol.
Chem. 271:31420–31425.
Coluccio, A. E., Rodriguez, R. K., Kernan, J. M., and Neiman, A. M. 2008. The yeast spore wall enables
spores to survive passage through the digestive tract of Drosophila. PLoS ONE 3:e2873.
Crestini, C., Kovac, B., and Giovannozzi-Sermanni, G. 1996. Production and isolation of chitosan
by submerged and solid state fermentation from Lentinus edodes. Biotechnol Bioeng. 50:207–210.
rouxii. J. Gen. Microbiol. 130:2095–2102.
for roles of chitin biosynthesis and breakdown. Experimental Mycol. 7:362–369.
Kreger, D. R. 1954. Observations on cell wall of yeast and some other fungi by X-ray diffraction and
solubility test. Biochim. Biophys. Acta 13:1–9.
Matsuo, Y., Tanaka, K., Matsuda, H., and Kawamukai, M. 2005. CDA1+, encoding chitin deacetylase
is required for proper spore formation in Schizosaccharomyces pombe. FEBS Lett. 579:2737–2743.
Pochanavanich, P. and Suntornsuk, W. 2002. Fungal chitosan production and its characterization.
Lett. Appl. Microbiol. 35:17–21.
Ruiz-Herrera, J., Ortiz-Castellanos, L., Martínez, A. I., León-Ramírez, C., and Sentandreu, R. 2008.
Analysis of the proteins involved in the structure and synthesis of the cell wall of Ustilago may-
dis. Fungal Genet. Biol. 45:571–576.
Sietsma, J. H. and Wessels, J. G. H. 1990. The occurrence of glucosaminoglycan in the wall of
Schizosaccharomyces pombe. J. Gen. Microbiol. 136:2261–2265.
chapter six
Fungal glucans
6.1 Introduction
The name glucans, in its most widely used term, is given to a number of polysaccharides
made solely of glucose. Some glucans have specific names such as cellulose, glycogen,
starch, and so forth. Nevertheless, the number of types of glucans, their chemical and
physical characteristics, and their function and cellular location are extremely varied. In
the previous edition of this book (Ruiz-Herrera 1992), I made a tentative classification of
glucans present in fungi, and their general characteristics. In general, it may be indicated
that glucose units in fungal glucans are joined through α- or β-bonds, mainly 1,3; 1,6,
and more rarely 1,4. The physical characteristics of fungal glucans are also variable; some
appear in the form of microfibrils, whereas others appear mucilaginous. Their solubility
is also different depending on their size or their binding to other polysaccharides; some
are water soluble, soluble only in alkali, or completely insoluble. Their cellular location
may be different; some are intracellular and may serve as reserve material, while others
are secreted to the medium, and the most important ones for the purposes of this volume
are those located in the cell wall and those having a structural role (see Ruiz-Herrera 1992;
Sentandreu et al. 2004). Interestingly, analysis of their distribution in fungi revealed that
they are absent in the vegetative forms of the Zygomycota, being present only in their spo-
rangiospores (Bartnicki-García 1968).
6.2 Structure of glucans

According to their structure, glucans can be divided into two large groups: α-glucans
and β-glucans. Both are present in a large number of fungi as decribed below. With a
few exceptions from some Basidiomycota species, for example, Armillaria mellea (Sanchez-
Hernandez et al. 1993), almost all studies on these polysaccharides have been realized with
Ascomycota fungi. Most α-glucans are made of glucosyl units joined by α-1,3-linkages.
Cell wall-bound α-1,3-glucans are water insoluble, linear homopolymers; but soluble in
cold alkali, a property that has been used to extract them from the cell wall (Sanchez-
Hernandez et al. 1993). Some display a structure in the form of 10–14 nm thick microfibrils
when observed by electron microscopy, for example, those from Paracoccidioide brasiliensis,
Aspergillus niger, Polyporus betulinus, and Histoplasma farciminosum. The α-1,3-glucan from
Schizosaccharomyces pombe was described as made from two interconnected large linear
chains of about 120–135 α-1,3 bound glucose units or building blocks, linked by a low
number of α-1,4-bound glucose units (Bacon et al. 1968; Grün et al. 2005). A different type
of α-glucans is the one generically known as nigerans that was originally described in
Aspergillus and Penicillum species. These polysaccharides are made of D-glucopyranosyl
moieties joined through alternating α-1,3- and α-1,4-linkages (Barker et al. 1957). The pres-
ence of nigeran in isolated cell walls and autoradiography observations in cell sections
(Gold et al. 1973) was contrary to the idea that nigeran was accumulated inside the cells
and ruled out this possibility. To these two types of polysaccharides, we may add glyco-
gen, which was described in the cell wall of S. cerevisiae covalently bound to β-1,3-glucans
73
(Arvindekar and Patil 2002); and elsinan, a mucillaginous polysaccharide, with a similar
structure to nigeran, synthesized by Elsinoe leucospila. Elsinan is made of repeating α-1,4-
bound trisaccharides joined by α-1,3-bonds (Tsumuraya et al. 1978).
The second group is made of β-glucans. In fungi, these are mostly made of gluco-
syl units bound by β-1,3- and/or β-1,6-linkages. β-1,3-glucans are the most abundant
polysaccharides present in the fungal cell walls, with the above-indicated exception of
Zygomycota, which contain the polysaccharide only in the cell wall of the sporangio-
spores. The structure of β-1,3-glucans was determined by the electron microscopic obser-
vation of S. cerevisiae protoplasts synthesizing the unbranched polysaccharide in the form
of microfibrils (Kreger and Kopecka 1975). Similar microfibrils measuring about 0.5 mm in
length were synthesized by incubation of cell-free extracts from S. cerevisiae with uridine
diphosphate glucose (UDPGlc) (Larriba, Morales, and Ruiz-Herrera 1981). This product
was completely solubilized by an exo β-1,3-glucanase giving rise to glucose as the only
product. The absence of gentobiose (a disaccharide made of glucose units bound by β-1,6-
linkages) in the product of hydrolysis, revealed the absence of β-1,6-branches. All these
β-1,3-glucans are poorly crystalline and similar to the so-called hydroglucan obtained by
boiling the branched β-glucan from yeast with mineral acids, a treatment that eliminates
the β-1,6-bound branches. It has been suggested that the microfibrils of the β-1,3-glucans
synthesized by yeast protoplasts, or cell-free extracts, or the β-1,3-glucan curdlan from
Alcaligenes faecalis var. myxogenes (Harada et al. 1979) are similar to lentinan, a β-1,3-glucan
from L. edodes (Bluhm and Sarko 1977). It was suggested that the structure of lentian was
composed of three intertwined helical chains whose association is stabilized by extensive
hydrogen bonding at the C-2 hydroxyl residue and which contains six glucose moieties per
turn (Kopecka and Kreger 1986). Nevertheless, small changes in the number of glucose
units per turn or the hydrogen bonding that keeps the chains associated may occur (see
Ruiz-Herrera 1992 for discussion).
A large amount of the β-1,3-glucans from the fungal cell walls are covalently bound
to β-1,6 glucans in the form of branching polysaccharides, although, interestingly, it has
been described that the human opportunist pathogen A. fumigatus does not contain β-1,6-
glucans in the cell wall (Fontaine et al. 2000). These multibranched β-1,3/β-1,6-glucans are
common in fungi, and have different general properties; some are loosely bound to the cell
wall and accumulate in the culture medium as slime or gelatinous material, while others
are firmly bound to the wall and can be solubilized only by treatment with acid or alkali.
This different behavior may be related to the molecular size and the degree of branch-
ing of the different polymers; thus, the glucans from a mutant of S. cerevisiae containing
the polysaccharide with a higher degree of β-1,6-linkages are more alkali-soluble than
the ones from the wild-type strain (Ha et al. 2002). The size of the branches is also vari-
able; for example, a glucan synthesized by Monilinia fructigena is made of a linear chain of
β-1,3 bound glucose units with β-1,6-branches made of single glucose units every second
glucose unit (Santamaría, Reyes, and Lahoz 1978). This polysaccharide is similar to a wall-
bound alkali-soluble glucan from Coprinus lagopus that contains β-1,6 branches made of a
single glucose unit (Schaefer 1977). In contrast, we may cite the glucans from the cell wall
of S. cerevisiae where three different fractions have been sequentially extracted that con-
tain variable but high amounts of β-1,6-branches (Manners, Masson, and Patterson 1973).
S. pombe has been shown to contain different types of β-glucans similar to S. cerevisiae in
the cell wall: alkali-soluble and alkali-insoluble except that the alkali-soluble component
had a smaller degree of polymerization (DP) of about 544, contained a higher degree of
branching, and had a minor proportion of β-1,6-linked glucose units in comparison to the
S. cerevisiae glucans (Manners and Meyer 1977). Furthermore, in the cell wall of Candida
Chapter six: Fungal glucans 75
albicans the existence of both β-1,3 and β-1,6-glucans has been demonstrated. Among them,
an acid-soluble fraction was found to consist mainly of highly branched β-1,6-glucan,
whereas the alkali-insoluble glucan from the walls of the yeast-like form contained dif-
ferent proportions of β-1,3 and β-1,6-linkages (Gopal et al. 1984). Analysis by nuclear mag-
netic resonance (NMR) of the glucans isolated from the yeast or hyphal forms of C. albicans
demonstrated that they were different from S. cerevisiae glucans in side-chain branching
and reducing termini, and that the yeast glucan had a higher average Mr than that from
the mycelial forms (Lowman, Ferguson, and Williams 2003).
From the examples cited above, it may be concluded that the existence of populations
of β-glucans with different solubility properties forming part of the cell wall structure
is a common feature, but this feature is not specific to Ascomycota species, which are the
most widely studied examples, but also of Basidiomycota species. One example is the wall
from the maize pathogen Ustilago maydis that was shown to have an alkali-soluble glucan
made of glucose only joined by β-1,3 linkages, and an alkali-insoluble glucan containing
both β-1,3 and β-1,6-linkages (Fonseca-García 2010). Mutants of this fungus defective in the
members of the Pal/Rim pathway of pH control, secreted to the medium large amounts of
a polysaccharide of mucilaginous consistency (Aréchiga-Carvajal and Ruiz-Herrera 2005;
Cervantes-Chávez et al. 2010) identified as an unbranched β-1,3-glucan (Fonseca-García et
al. 2011), a peculiar characteristic, since as indicated above, all β-1,3-glucans are branched
polymers. The differential solubility of apparently similar polysaccharides might be
related to the different size of the polymers. However, although glucans with a low DP are
readily soluble in water, some mucillagenous glucans which are secreted to the medium
may have rather high Mr values, and in contrast part of the glucans from the yeast cell
walls have low DP and are alkali insoluble. One possibility is that strong hydrogen bond-
ing limits the access to water of the organized chains reducing their solubility. A further
possibility is that insoluble β-glucans are covalently bound to chitin, which is known to
be extremely insoluble, even in alkali. This possibility was discussed above by Stagg and
Feather (1973) who described how the alkali-insoluble β-glucan from the cell walls of A.
niger and Penicillium chrysogenum became alkali soluble when treated with nitrous acid.
As described in Chapter 5 on “Chitosan,” it is known that alkali deacetylates GlcNAc into
glucosamine GlcN, which is converted into anhydromannose when treated with HNO2,
depolymerizing the polysaccharide (Mol and Wessels 1987).
It is also important to notice that other β-1,3-glucans present in some fungal species
contain glucose bound by different kinds of linkages. Examples of these are the glucans
synthesized by M. fructigena (Feather and Malek 1972) and Armillaria mellea (Sanchez-
Hernandez et al. 1993) that contain not only β-1,3- and β-1,6-linkages, but also β-1,4-bonds,
and a linear cell wall polysaccharide from Aspergillus nidulans that contains glucose bound
by β-1,3- and β-1,4 -linkages (Fontaine et al. 2000).
In the cell wall, β-1,3-glucans appear under the electron microscope in the form of
microfibrils with a diameter of 7.5–10 nm that associate into meshes 20–60 nm thick (Kreger
and Kopecka 1975). Interestingly, as demonstrated in a few examples, it appears that the
distribution of β-glucans in the fungal cell walls may be not uniform. Selective localiza-
tion of the different β-glucans in the cell wall was described for Schizosaccharomyces pombe
by use of immuno-electron microscopy. Thus, β-1,6-branched/β-1,3-glucan was located in
the electron transparent layer of the cell wall, whereas unbranched β-1,3-glucan appeared
located exclusively in the primary septum of the dividing cells, and β-1,6-glucan was
located in the secondary septum (Humbel et al. 2001).
The best known β-1,6-glucan is the one present in the cell wall of S. cerevisiae. This is a
highly branched glucose polysaccharide with a size of about 350 glucose units covalently
linked to β-1,3-glucan and corresponding to about 10% of the total glucans in the cell
wall (Shahinian and Bussey 2000). Besides fungi, β-1,6-glucans have been found only in
members of the phylum Chromista. These polysaccharides have been analyzed mostly
in Ascomycota, and only a few reports on Basidiomycota species have been published;
Sclerotium rolfsii, Schizophyllum commune, some higher fungi (Daba and Ezeronye 2003;
Rau 2004), and U. maydis (see above). According to our preliminary experimental and in
silico analyses, β-1,6-glucans are absent in Microsporidia, Chitridiomycota, and possibly
Zygomycota (Ruiz-Herrera and Ortiz-Castellanos 2010; see below).
6.3 Role of fungal glucans

Considering that glucans are the most abundant polysaccharides in fungi, and that differ-
ent kinds of these polymers with different physical properties exist, it may be anticipated
that they have important and varied functions in the cells. Some glucans have been shown
to accumulate in the cells, possibly serving as storage food material. An example of this is
the role of α-1,3-glucans in development of A. nidulans. It was observed that after glucose
was exhausted in the medium, α-1,3-glucan started to be digested by a glucanase, serv-
ing apparently as energy source for cleistothecia development (Zonneveld 1972). In the
absence of α-1,3-glucans, no cleitothecia were formed.
Another type of function appears to be the case of glucans that are secreted to the
medium in the form of highly hydrated material that may protect the cell from dehydra-
tion or chemical aggression of the medium. In contrast, the role of glucans in virulence of
pathogenic fungi is a well-known phenomenon. For example, the role of the capsule from C.
neoformans as a virulence factor that inhibits phagocytosis may be cited. In this sense, the role
of glucans from pathogenic fungi in the interaction with their hosts has been widely studied
in C. albicans, probably the most important human pathogenic species. It is known that glu-
cans, either alone, or in the form of complexes with mannans are released by C. albicans into
the growth media and into the blood of infected patients. These compounds are toxic and
may induce anaphylactic shock and coronary arteritis in murine models. Also, glucans play
a role in the development of candidiasis suppressing monocyte functions directly, and T cell
function indirectly, a hypothesis supported by the observation that anti-β-glucan antibod-
ies participate in the immune response, recognizing C. albicans and other pathogenic fungi
(Ishibashi et al. 2005). The existence of glucan-specific receptors outside the immune system
is important, since hormones and cytokines may act as regulatory messengers between the
neuroendocrine and immune systems. These receptors recognize pathogen-specific macro-
molecules called pathogen-associated molecular patterns, and fungal cell wall glucans stimulate
various aspects of innate immunity via interaction with membrane receptors on immune-
competent cells. It was hypothesized that glucans might interact directly with pituitary cells
as an early signaling event in fungal infections, and evidence was obtained that pituitary
cells directly recognized and responded to fungal cell wall glucans (Breuel et al. 2004). It is
possible also that wall glucans play a role in the observed phenomenon that the hyphal form
of C. albicans is the invasive stage of the fungus. According to this hypothesis, it has been
suggested that wall glucans remain protected from Dectin-1 (a receptor that binds β-glucans
and is important for phagocytosis of fungi by macrophages) by other components, but dur-
ing budding when scar glucans are exposed, they become accessible to binding of the recep-
tor, and are eliminated. On the other hand, hyphae, lacking a budding mechanism may
escape from the receptor and colonize the host tissues.
Not only β-glucans have been suggested to play a role in fungal pathogenicity; α-1,3-
glucans also appear to be important in the virulence of several fungal species pathogenic
to humans. P. brasiliensis is a dimorphic human pathogen that grows in a mycelial form in

its saprophytic stage, and in a yeast-like form during its pathogenic stage. One of the most
important differences in the cell wall composition of both forms is that the yeast form con-
tains high amounts of an alkali-soluble α-1,3-glucan, whereas the mycelial form contains
instead alkali-soluble β-1,3-glucan (Kanetsuna and Carbonell 1970; Kanetsuna et al. 1974).
A similar situation has been shown to occur in two other dimorphic fungi: Blastomyces
dermatitidis, a human pathogenic fungus (Kanetsuna and Carbonell 1971; Hogan and
Klein 1994), and Histoplasma farciminosum a fungus pathogenic for horses, whose invasive
forms are yeast-like, and contain α-1,3-glucan only in their invasive forms (San-Blas and
Carbonell 1974). In all these fungi, the dimorphic transition from mycelium to yeast can
be induced by cultivation at high temperature, around 37oC, as occurred in P. brasiliensis
(San-Blas, San-Blas, and Serrano 1977). In P. brasiliensis, it was observed that successive
subculturing of the fungus led to a progressive virulence reduction that was accompa-
nied by a simultaneous decrease in the content of α-1,3-glucan. When the avirulent strain
was inoculated into a susceptible host, both characters were recovered again (San-Blas,
San-Blas, and Serrano 1977). Additionally, an isolated avirulent mutant was found to lack
α-1,3-glucan, which was substituted by a novel α-1,3-mannan, whereas other mutants with
increased virulence contained higher amounts of α-1,3-glucan. The observation that the
avirulent mutant was more rapidly destroyed by the action of polymorphonuclears than
the cells of the more virulent strains, led the authors to suggest that the role of α-1,3-glucan
in virulence was related to protection from phagocytosis (San-Blas and San-Blas 1977).
The presence of β-glucans in the cell wall in the form of microfibrils, and its covalent
association to chitin is an indication that they have a structural role. A common observa-
tion is that protoplasts from S. cerevisiae, which contain very low amounts of chitin in the
cell wall, are obtained by digestion with a mixture of β-glucanases. Incubation of different
fungi with glucan synthase inhibitors leads to their lysis or to the transformation of cells
into aberrant forms. One example is the result from Miyata, Kanbe, and Tanaka (1985),
who incubated S. pombe with aculeacin, an inhibitor of glucan synthesis, which led to the
formation of abnormal cells. In this species it was also demonstrated that α-1,3-glucan
was essential for cell wall integrity, as shown by results obtained with a thermosensitive
mutant deficient in a gene putatively encoding the α-1,3-glucan biosynthetic enzyme. It
was observed that at the nonpermissive temperature, the mutant cells were lysed, and that
at an intermediate temperature they appeared with an abnormal morphology and with
low contents of α-1,3-glucan (Hochstenbach et al. 1998).
6.4 Synthesis of glucans

6.4.1 Introduction
Considering the large number of classes of glucans, it is easy to conclude that their synthe-
sis must be complex. In recent years a large amount of information on the synthesis of these
polysaccharides has accumulated, the enzymes involved have been purified, although not
isolated, and their genetic control has been clarified by means of the isolation and disrup-
tion of the corresponding genes. Nevertheless, advances in the study of these processes
have not been uniform. For example the least known process, α-1,3-glucan synthesis,
which lagged behind compared to the study of the process of β-1,3-glucan biosynthesis,
has advanced rapidly, whereas, the mechanism of β-1,6-glucan synthesis remains obscure.
In the following pages, I will try to describe the present stage of our knowledge on the
mechanism of biosynthesis of each one of these different and important polysaccharides.
6.4.2 Introduction to the synthesis of β-1,3-glucans

Synthesis in vitro of β-1,3-glucans was originally reported by Sentandreu, Elorza, and
Villanueva (1975), who used protoplast of S. cerevisiae permeabilized with toluene in order
to permit the entrance of the substrate. The low values of radioactivity incorporated from
UDP[14C]Glc were probably due to the extreme instability of the synthase and the absence
of some activators as was demonstrated subsequently. Using different conditions, we were
able to synthesize significant amounts of the polysaccharide using cell-free extracts from
S. cerevisiae also (López-Romero and Ruiz-Herrera 1977). These seminal studies were fol-
lowed by experiments performed with a large number of fungal species, in most cases using
crude membrane preparations, although in some cases samples were partially purified by
centrifugation in different types of gradients or extracted with detergents. (see reviews
by Ruiz-Herrera 1991; 1992; Douglas 2001). These studies demonstrated that UDPglucose
(UDPGlc) was the universal glycosyl donor for β-1,3-glucan biosynthesis. Some early
studies where GDPGlc or ADPGlc were used as alternative substrates produced negative
results, with the exception of a communication by Balint et al. (1976) who described that
GDPGlc was the most efficient substrate, followed by ADPGlc and UDPGlc. Until now, we
lack a logical explanation for these results, which have never been reproduced.
Affinity of β-1,3-glucan synthase for the substrates is low, with Km values in the
same range as chitin synthases, ca. 0.12–4.5 mM. The observation that the Km values of
some systems varied according to the conditions of growth or the stage of development
(for example, San-Blas and San-Blas 1986) was possibly due to the presence of different
amounts of stimulator or inhibitors in the crude extracts (see below). The only report of the
Hill number for β-1,3-glucan synthase (an indication of the number of substrate binding
sites in the enzyme) was provided for the enzyme from Neurospora crassa, where a value
of 1.2 was obtained, suggesting that the enzyme contains a single binding site for UDPGlc
(Quigley and Selitrennikoff 1984). Optimal pH values for the different enzymes studied
are close to the neutrality, in the range of 5.8 to 7.8, and this value depends somewhat on
the buffer used; normally phosphate buffer gives the best results. The optimal tempera-
ture varied between 20oC and 30oC. Contradictory results have been reported regarding
the effect of divalent metals, but in general, the addition of different divalent metal ions,
Mg++ or Mn++ ions being the most efficient ones, stimulates activity.
With regard to the nature of the synthesized products, the only criterion used in
the initial studies to characterize them as β-1,3-glucans was their insolubility in water,
their resistance to amylase (an α-1,4-hydrolizing enzyme), and their digestion by β-1,3-
glucanases (for example, López-Romero and Ruiz-Herrera 1977). Exhaustive digestion of
the polysaccharide synthesized in vitro by membranous fractions from S. cerevisiae with exo
β-1,3-glucanase gave as products mainly glucose, small amounts of gentiobiose (a β-1,6-
bound disaccharide), and β-1,6-bound oligosaccarides of different size (López-Romero
and Ruiz-Herrera 1977). These results suggested that the crude preparation had at least
three enzymatic activities: β-1,3-glucosyl transferase (β-1,3-glucan synthase), a branching
enzyme and a β-1,6-glucosyl transferase. In further experiments, β-1,3-glucan was syn-
thesized using high-speed supernatants obtained from S. cerevisiae extracts incubated
with UDPGlc. In this case the product, insoluble in water, ethanol, and acid, but soluble
in alkali, appeared under the electron microscope as microfibrils with an average length
of about 0.5 mm (see Figure 6.1). These microfibrils were susceptible to hydrolysis by both
endo and exo β-1,3-glucanases giving glucose as the only product, which indicated that
they consisted of β-1,3-glucan molecules with no detectable branches (Larriba, Morales,
and Ruiz-Herrera 1981), a result that indicated that the enzyme in these presoparations
Figure 6.1 Microfibrils of β-1,3-glucan synthesized by Saccharomyces cerevisiae cell free extracts. Top:
Shadow cast preparation. Bottom: Negative staining with uranyl acetate. Magnification: 95,000X.
(Modified from Ruiz-Herrera, J., 1992, Fungal Cell Wall: Structure, Synthesis, and Assembly, Boca Raton,
FL. With permission from Taylor & Francis.)
contained only β-1,3-glucosyl transferase. An interesting observation was that the β-1,3-
glucan chains did not possess a free reducing end. This observation has relevance for
understanding the mechanism of polysaccharide synthesis as discussed below.
6.4.3 Stimulators and inhibitors of β-1,3-glucan synthases

The observation that nucleoside triphosphates stimulated the activity of β-1,3-glucan syn-
thase (Shematek et al. 1980b) opened the way for the understanding of the mechanism
involved in the catalysis of the enzyme. The best studied system for understanding the
mechanism of activation by ATP and GTP was S. cerevisiae (Shematek, Broatz, and Cabib
1980; Shematek and Cabib 1980). These authors observed that the mode of action of both
nucleotides was different. While ATP was involved in a phosphorylation step, GTP bound
to the enzyme with a dissociation constant of 1–8 × 10 –7 M, and could be substituted by the
nonphosphorylating analog GTP[γS]. Further work refined the possible mechanism of acti-
vation, and the suggestion was made that phosphorylation of the enzyme was not neces-
sary (Notario, Kawai, and Cabib 1982). Using as models Hansenula anomala and N. crassa it
was concluded that the catalytic and activation sites were present in different polypeptides
(Kang and Cabib 1986), which could be separated by treatment with a mixture of Tergitol,
NP40 and NaCl. The protein with the catalytic activity remained in the particulate fraction,
and the activating one (putatively binding GTP) appeared in the supernatant in the form of
three fractions, the main one with a Mr of 300 kDa. The identification of both subunits had
to await the isolation of the catalytic subunit of β-1,3-glucan synthase (see below).
Another substance that in crude extracts behaved as an activator of β-1,3-glucan syn-
thase was NaF (Larriba, Morales, and Ruiz-Herrera 1981). Originally, added as an inhibitor
of phosphatases present in the crude extracts, later work demonstrated that NaF increased
the stimulatory effect of nucleoside triphosphates, being hypothesized that it facilitated
the activation of the catalytic enzyme (Leal et al. 1984). In contrast to chitin synthases that
are activated by limited proteolysis, β-1,3-glucan synthase was found to be insentive to low
concentrations, and was inhibited (probably digested) by higher concentrations of differ-
ent proteases. One common observation was that β-1,3-glucan synthase activity in crude
extracts was extremely sensitive, being lost after short periods of time, even if kept at low
temperature, and partially stabilized by addition of some osmolytes such as sucrose in
high concentrations (reviewed in Ruiz-Herrera 1991).
Undoubtedly the most important inhibitors of β-1,3-glucan synthases are echinocan-
dins and papulacandins, a family of antibiotics, interestingly produced by fungi, some
of which have been synthetically modified further to obtain higher inhibitory activity,
and the absence of secondary effects. This group of substances that act as noncompetitive
inhibitors of β-1,3-glucan synthases are chemically cyclic lipopeptides including: aculeacin
A (from Aspergillus aculeatus), echinocandin B (from Aspergillus rugulovalvus), pneumocan-
din B (from Zalerion arboricola), enfumafungin (from a Hormonema-like fungus), and papu-
lacandins (from Papularia sphaerosperma) (see Denning 2002 for review). These inhibitors
have become the most promising antimycotics and they are now actively being studied
to increase their activity in vivo, and the clinical pharmacology of different echinocandin
derivatives for treatment of patients suffering fungal invasive infections is an important
topic of research (see Gupta and Tomas 2003).
Papulacandins were initially isolated as inhibitors of the growth of yeasts, mainly
C. albicans and S. cerevisiae, and shown to inhibit glucan synthesis in yeast protoplasts
(Baguley et al. 1979; Rommele et al. 1983), demonstrating that glucan synthesis was the tar-
get of the drug. Similar results were obtained with echinocandins, although their chemical
structure is somewhat different to papulacandins. Interestingly, it was a common obser-

vation that a relationship was lacking between the effect of the drugs on cell growth or
on glucan synthesis in vivo, and its in vitro effect on glucan synthase; peculiarly, the latter
was less sensitive than the former (for example, Varona, Perez, and Duran 1983; Quigley
and Selitrennikoff 1984; Davila, San-Blas, and San-Blas 1986; Kang et al. 1986). No definite
explanation has been given for this discrepancy. Nevertheless, the observations that (i)
derivatives of papulacandin and aculeacin devoid of the their fatty acids are not inhibitory
(Rommele et al. 1983; Varona, Perez, and Duran 1983), and (ii) the kinetics of inhibition are
noncompetitive and sigmoidal, may suggest that the inhibitors bind to the enzyme in its
natural hydrophobic environment, different from the one existing in the in vitro conditions.
Besides this chemotherapeutic application, these compounds have helped to identify
the correct polypeptides responsible for β-1,3-glucan synthase activity. Thus, the mutations
responsible for spontaneous echinocandin resistance in C. albicans and to papulacandin B
in S. cerevisiae were pinpointed to the genes putatively encoding their most important glu-
can synthases (Douglas et al. 1997; Castro et al. 1995, respectively) (see below).
As happens with chitin synthases (see Chapter 4), the byproduct of the synthetic reac-
tion (UDP) behaves as a competitive inhibitor of β-1,3-glucan synthases with Ki inhibi-
tion constant values in the mM range (López-Romero and Ruiz-Herrera 1978), the same as
occurred with uridine triphosphate (UTP) and uridine monophosphate (UMP) (Quigley
and Selitrennikoff 1984). Another inhibitor of β-1,3-glucan synthases was found to be glu-
conolactone, which had been described as an inhibitor of glucanases. This substance is a
noncompetitive inhibitor that acts on the enzyme with complex kinetics (López-Romero
and Ruiz-Herrera 1978; Quigley and Selitrennikoff 1984). Finally, I may cite sorbose, known
for a long time to cause an inhibition of colony growth in N. crassa, which behaved as an
uncompetitive inhibitor of glucan synthase with an 18 mM Ki (Mishra and Tatum 1972).
6.4.4 Purification of β-1,3-glucan synthases

All the evidence indicates that the catalytic polypeptide of β-1,3-glucan synthase is mem-
brane-bound, and a number of research groups attempted to extract it by use of different
methods, mainly by use of different detergents, with different degrees of success. Using 13
neutral detergents: Brij 35, Brij 58, Lubrol WX, Nonidet P-40, Tergitol 15-S9, Tergitol NP-10,
Triton 770, Triton WR-1339, Triton X-100, Tween 20, Tween 40, Tween 60, and Tween 80, only
a minimal amount of solubilization was obtained using membrane preparations from P.
brasiliensis (San-Blas and San-Blas 1982). Better results were obtained with the glucan syn-
thase from C. albicans (Frost et al. 1997). These authors used Chaps and demonstrated that
effectiveness in solubilization depended upon the strain and the method used to detect
enzyme activity. The solubilized enzyme was purified over 765-fold using a modified
product entrapment technique. But probably the most successful attempts to isolate and
purify β-1,3-glucan synthase were made by the Selitrennikoff group who used membrane
fractions of N. crassa as starting material. In an initial study, extraction was tried with
Chaps, octylglucoside (OG), deoxycholate, and digitonin, and it was observed that only a
mixture of OG and Chaps was effective solubilizing nearly 50% of the enzyme (Quigley et
al. 1988). The extracted enzyme remained in the supernatant when centrifuged at 100000
g, and its kinetic parameters remained the same as in the membrane-bound enzyme. In a
later study, this research group obtained better results and isolated from a N. crassa extract
the purest soluble form obtained thus far with a specific activity of 14000 nmoles glucose
incorporated per min per mg protein (Awald et al. 1994). In C. albicans, it was possible to
partially identify a polypeptide of Mr 210 kDa as the fungus β-1,3-glucan synthase, since
antibodies raised against the polypeptide precipitated the enzymatic activity. Moreover,
the polypeptides identified by proteolytic digestion of the protein coincided with those
deduced from the sequence of a gene putatively encoding the enzyme (GSC1, see below)
(Mio et al. 1997).
A different approach for the characterization of β-1,3-glucan synthase involved the
attempts to label it, with a modified photosensitive analog of the substrate: 5-azido-(β-
(32P)-UDPG. Nevertheless, problems were encountered, since several polypeptides in
the active preparations became labeled (see Douglas 2001 for review). It was again the
Selitrennikoff group that succeeded in labeling the catalytic polypeptide with the photo-
sensitive analog, using a highly purified sample of β-1,3-glucan synthase from N. crassa
(Schimoler-O’Rourke et al. 2003) This result permitted its unequivocal identification, and
the demonstration that the catalytic polypeptide was the product of the FKS gene, as had
been proposed by genetic and molecular biology approaches (see below).
6.4.5 Genes encoding β-1,3-glucan synthases

In a peculiar way, the gene encoding β-1,3-glucan synthase was identified when it was
realized that genes from S. cerevisiae distinctly named, isolated for variable reasons, and
having different phenotypes corresponded all to the same one(s), now called FKS. These
genes previously designated as CND1, CWH53, ETG1, FKS1, GSC1 and PBR1 corresponded
to genes identified in mutants resistant to inhibitors of β-1,3-glucan synthesis such as pap-
ulacandin B (papulacandin B resistance, pbr mutants) (Castro et al. 1995) and echinocandin
(echinocandin target gene, etg mutants) (Douglas et al. 1994; El-Shebeini and Clemas 1995);
to Calcofluor white hypersensitivity mutants (cwh53 mutants) (Ram et al. 1995) gsc1 mutants
(Abe et al. 2001), to mutants with calcineurin dependent growth (cnd1 mutants) (Garrett-
Engele, Moilanen, and Cyert 1995) or hypersensitive to tracolimus (FK506-supersensitive,
fks1 mutants) (Parent et al. 1993). Why the designation FKS (for FK506-supersensitive) for
the gene encoding β-1,3-glucan synthase remained over the others, when it hardly rep-
resents its function is unknown; other authors used the designation BGS for these genes
(β-glucan-synthase) (see below), that undoubtedly is more explanatory of their function.
With the advent of technique for massive sequencing that led to the publication of the
genomes of a large number of fungal genomes, it was possible to demonstrate that all
of them contain homologs of FKS genes. The exceptions to this rule are Microsporidia
and Chitridiomycota species that apparently lack homologs of FKS genes. We presented
the hypothesis that this observation suggests that the capacity to synthesize β-1,3-glucans
appeared in the kingdom Fungi once these two groups departed from the evolutionary
line of the other phyla (Ruiz-Herrera and Ortiz-Castellanos 2010).
The number of genes encoding β-1,3-glucan-synthases in fungal species is variable,
but contrary to what occurs with chitin synthase encoding genes, there are species that
contain only one encoding gene such as occurs with U.maydis, S. roseus, and Malazessia glo-
bosa. Other species contain more than one gene, but never approaching the large number
of CHS genes that may be present in some fungi, since their numbers oscillate from two
to four. An observation that seems contradictory is that members of Zygomycota, such as
Rhizopus oryzae, whose vegetative phases do not contain β-1,3-glucans in their cell walls,
have up to four genes encoding β-1,3-glucan synthases (Ma et al. 2009). It was originally
described that S. cerevisiae possessed two FKS genes whose encoded products shared 88%
similarity: FKS1 and FKS2. Nevertheless, in further studies, a putative homolog (FKS3),
whose encoded protein shares only 56% identity with Fks1p and Fks2p, was identifed
(Mazur et al. 1995). Mutation of FKS2 or FKS3 had no significant phenotypic alteration in
the yeast (Lesage et al. 2004), whereas deletion of FKS1 led to a decrease in glucans and
mannoproteins. On the other hand, the double mutation of FKS1 and FKS2 was found
to be lethal (Mazur et al. 1995). Regulation of FKS1 and FKS2 is different, thus FKS1 is
expressed under conditions of growth in optimal conditions in the presence of glucose,
and, interestingly its transcription fluctuates periodically throughout the cell cycle. On the
other hand, FKS2 is poorly expressed under optimal growth conditions, and its expres-
sion is calmodulin-dependent. Its expression is stimulated when the cells are incubated
in poorly utilizable carbon sources (Mazur et al. 1995). This effect is probably due to the
absence of glucose that would act as a repressor (Zhao et al. 1998). Expression of the gene
is also stimulated in fks1 mutants, or during mating as a response to mating pheromones,
and is essential for sporulation (Mazur et al. 1995). The function of Fks3 was unknown for
a long time, until it was demonstrated that it is involved in the synthesis of the spore wall,
together with Fks2p, and that it is also involved in the regulation of β-1,3-glucan synthesis
(Ishihara et al. 2007).
In contrast to S. cerevisiae, four genes homologues of FKS1 have been described in S.
pombe: BGS1, BGS2, BGS3, and BGS4, and found to probably have different roles in the
growth and development of this yeast. The authors (Ishiguro et al. 1997) suggested that
BGS1 (originally denominated CPS1) was involved in the formation of β-1,3-glucan located
at the septum. The protein encoded by BGS2 appeared to be involved in the maturation
of the ascospore wall (Liu et al. 2000); whereas BGS3 was reported to be an essential gene
constitutively expressed, and whose product was located at the growing tip and at the
septum during different stages of cell growth (Martín et al. 2003). Finally, BGS4, also an
essential gene, was described to encode a β-1,3-glucan synthase with similar location to
Bgs3p (Cortés et al. 2005). Another well-studied model is C. albicans, which contains three
homologues of yeast FKS1 denominated CaGSC1, CaGSL1 and CaGSL2 (Mio et al. 1997).
The authors described that during growth, only CaGSC1 and CaGSL1 were expressed, but
the in vivo activity was mainly due to Gsc1p, identified as the target of glucan synthase
inhibitors (Douglas et al. 1997). Mio et al. (1997) made the interesting observation that the
CAI4 strain of C. albicans contained, not two, but three alleles of CaGSC1. Attempts to dis-
rupt the three CaGSC1 alleles were unsuccessful but mutation of two alleles reduced the
levels of wall glucan in 50%. These results were in opposition to Douglas et al. (1997) who
described in the same C. albicans strain the existence of two alleles of the gene, as should
be expected, and reported their failure in their attempts to disrupt them. These results are
suggestive that the CaGSC1 gene is essential.
Regulation of β-1,3-glucan synthases is also variable and depends on the fungal spe-
cies concerned. The results cited above for Ascomycota yeasts have been treated with
some detail, since they have been considered the most representative models, although
this supposition may not be strictly correct (for a discussion on this matter see Ruiz-
Herrera and Ortiz-Castellanos 2010). Information on other systems is scant, but I may
cite some examples described in the literature. Thus, the presence of homologues of the
FKS gene in several mushroom species, Auricularia auricula-judae, L. edodes, Pleurotus
eryngii, Stropharia aeruginosa, Agrocybe aegerita, P. pulmonarius, Armillaria mellea, Pleurotus
ferulae, Pleurotus ostreatus, and Pleurotus nebrodensis, demonstrates the wide distribution
of the gene (Reverberi, Mario, and Tomati 2004). With regard to their regulation, it was
described that during infection of susceptible hosts, both FKS and CHS genes were down
regulated in the entomopathogenic fungus Beauveria bassiana (Tartar et al. 2005). A differ-
ent result was observed for the only gene encoding β-1,3-glucan synthase present in the
maize pathogen U. maydis, where its expression was neither affected by different condi-
tions of growth in vitro, nor during maize infection (Robledo-Briones 2009). These results
contrast with data from Lentinula edodes where it was described that olive mill wastewa-
ters and phenols strongly stimulated gene transcription and enzyme activity (Reverberi,
Mario, and Tomati 2004).
6.4.6 Structure and phylogenetic characteristics of β-1,3-glucan synthases

β-1,3-glucan synthases are glycosyltransferase belonging to the 48 family (GT48). This fam-
ily is made by various enzymes including fungal enzymes homologues to Fks from S.
cerevisiae and the enzymes also known as callose synthases that catalyse the formation
of β-1,3-glucan in plants, algae, and Chromista. In silico analysis of the structure of β-1,3-
glucan synthases has revealed that they have a high level of homology among them, and
that independently of their origin, display important structural differences with the rest
of β-glucosyl transferases. Among these differences, we may cite the absence of the charac-
teristic conserved amino acids (three Asp residues) and the QXXRW motif, associated with
the catalytic activity of chitin and cellulose synthases: they also lack the proposed UDP
glucose-binding motif (R/K) XGG that has been identified in glycogen synthases (Farkas
et al. 1990). These data suggest their probable different phylogenetic origin (Campbell et al.
1997, reviewed by Douglas 2001; Ruiz-Herrera et al. 2004). By means of hydropatic analy-
ses, it was established that Fks are membrane bound proteins with 16 transmembrane
helices and a central hydrophilic domain (Douglas et al. 1994,) that probably corresponds
to the catalytic site. By analysis of a group of S. cerevisiae mutants resistant to different
inhibitors of β-1,3-glucan synthase, a critical inhibitor resistance cluster with the consensus
sequence FL(V/I/T/)LS(L/I/F)(R/K)D was defined (Douglas 2001). It is possible that this
motif is associated with activity, but whether it is the catalytic site is unknown.
Starting from the observation that some fks mutants, or mutants in the analogous
genes in other fungal species, show pleitropic defects, which include alteration of other
components of the cell wall such β-1,6-glucans, chitin and mannoproteins, morphologi-
cal alterations, and even endocytosis, Okada et al. (2010) followed a different approach
to understand the relationship between the sequence and function of the FKS1 gene.
The authors proceeded to isolate a number of thermosensitive random mutants from
S. cerevisiae defective in the FKS1 gene in a Δfks2 background, and subjected them to an
exhaustive phenotypic analysis. Their results indicated that all mutations occurred at
the cytoplasmic stretch of the gene, and that the mutants fell into three groups accord-
ing to their main phenotypic differences. Group 1 mutations were accumulated towards
the N-terminus of the gene, whereas group 3 mutations appeared in a zone of the gene
towards the C-terminus putatively corresponding to the catalytic site; and mutations of
members of group 2 appeared in between groups 1 and 3. Members of the first group of
mutants showed normal β-1,3-glucan synthase activity in vitro, but in vivo the polysac-
charide was not deposited at the proper location in the cell. Mutants belonging to group
2 were defective in growth polarization and endocytosis, but displayed normal contents
of β-1,3-glucan, whereas members of group 3, as expected, had lost the catalytic activity
of the enzyme. Based on these results, the authors suggested that Fks1p contains multiple
domains that are required or are involved, not only on catalysis, but also necessary for cell
wall construction and cellular morphogenesis. I could add that the only way by which the
Fks1 (or its analogs) could fulfill these two last proposed properties would be as the result
of the association of the enzyme with other proteins, either catalytic or not, involved in cell
wall formation, and that this would occur only in Basidiomycota, Ascomycota, and the
spores of Zygomycota, considering the probable abscence of β-1,3-glucans in the vegetative
forms of Zygomycota, and in Chitridiomycota and Microsporidia (see above).
To analyze the phylogenetic relationships among β-1,3-glucan synthases from fungi

and plants (where they are denominated callose synthases), we made a comparison of the
sequences of some selected enzymes from both groups. It was observed that plant β-1,3-
glucan synthases separated into a compact group from the fungal enzymes. These results
suggest their early evolutionary departure (Ruiz-Herrera et al. 2004). In this same study,
comparison of the sequence of β-1,3-glucan synthases of fungal origin, confirmed the
high similarity of these enzymes, and distinguished four groups. One group included
sequences from Ascomycota species only, which revealed a noticeable difference in Mr
of β-1,3-glucan synthases; those from Saccharomyces castelli B and Saccharomyces kluyveri
which are made of about 600 amino acids in length, while the rest of the enzymes are made
of about 1900 amino acid residues (size average 1904 ± 32). The second group included
enzymes from Ascomycota and Basidiomycota species whose size was similar to the large
proteins from group 1, with an average of 1698 + 352 amino acid residues. The third group
(the most homogeneous one) was made by enzymes with similarities from 77% to 80%
and a size average of 1911 ± 13 amino acid residues. Fksp (csp1) from S. pombe, due to its
large size (2353 amino acid residues), departed from the rest (Ruiz-Herrera et al. 2004). In a
further study where the phylogenetic analysis of β-1,3-glucans was made, it was observed
that the enzymes from Ascomycota could be subdivided in a larger number of groups
than β-1,3 glucan synthases from Basidiomycota and Zygomycota; whereas as mentioned
above, Chitridiomycota and Microsporidia lacked homologues of those enzymes (Ruiz-
6.4.7 Activation of β-1,3-glucan synthases by a small GTPase

The stimulatory effect of GTP on the activity of β-1,3-glucan synthases (see above) was
pinpointed to a protein different from Fks in S. cerevisiae. This protein was later identi-
fied as a member of the so-called small GTPases: Rho1 (Mazur and Baginski 1996; Qadota
et al. 1996). It was demonstrated that Rho1 could be immunoprecipitated together with
Fks1 by a cross-reactive anti-Fks2 antibody, and that the activating property of Rho1 was
eliminated by its ADP-ribosylation by exoenzyme C3 (toxin) from Clostridium botulinum
(it is known that ADP ribosylation of Rho GTPases causes their inactivation) (Mazur
and Baginski 1996). These initial studies led to a rapid identification of Rho1 homologs
in other fungi. Rho1 from S. pombe was found to have more than 70% identity with the
yeast enzyme (Nakano and Mabuchi 1995), and similar data were obtained for Aspergillus
flavus (Beauvais 2001). Higher identity with the S. cerevisiae enzyme was reported for Rho1
from C. albicans (82.9%) (Kondoh et al. 1997), that was able to complement S. cerevisiae rho1
mutants, the same as occurred with C. neoformans Rho1 (Chang and Penoyer 2000). Rho1p
was found to be essential in C. albicans. With the use of mutants that expressed Rho1p con-
ditionally, it was observed that depletion of the protein in either yeast or hyphal cells led
to cell aggregation, death, and lysis. These mutants were also avirulent in a mouse model
(Smith et al. 2002). Mutation of Rho1p is also lethal in S. cerevisiae and S. pombe (Nakano,
Arai, and Mabuchi 1997), but on the other hand the RHO1 homolog gene from Y. lipolytica,
that was able to complement yeast rho1 mutants, was found to be nonessential, suggesting
the existence of other homologous genes (Leon, Jaafar, and Zueco 2003)
The function of Rho1 is far from being exclusively the activation of β-1,3-glucan syn-
thase. Important observations led to the demonstration that this GTPase is involved in
at least four pathways; (i) it binds and activates Pkc1, which leads to the activation of the
Pkc1-Mapk pathway involved in actin cytoskeleton organization and cell integrity, (ii) it
interacts with Bn1p which regulates the actin cytoskeleton through its binding to profilin,
(iii) it also binds to Skn7, which is involved in at least three functions, oxidative stress
response, transcription during the G1-S phase, and the two-component pathway, and (iv)
it interacts with Sec3 in polarizad exocitosis (see Sekiya-Kawasaki, Abe, Saka et al. 2002 for
discussion). No wonder then that Rho1p would be involved in several essential functions
of the cell: wall synthesis and integrity, exocytosis, septin organization, actin assembly,
microtubule organization, autophagy, cytokinesis, and in general in cell polarity and mor-
phogenesis (Arellano et al. 1999; Drees et al. 2001; Guo 2001; Pham et al. 2009; Schmidt,
Schmelzle, and Hall 2002). The mechanism through which Rho1 fulfils all these functions
is complex, and of course, as can be deduced from the above data, is not due to its effect on
β-1,3-glucan synthase activity. Similarly complex is Rho1 regulation; it was described that
a number of elements positively affected its function: Wsc1, Wsc3, Mtl1, Rom2, Lre1, Zds1,
and Msb1, not only regarding its effect on β-1,3-glucan synthesis, but also on other of the
pathways in which this small GTPase is involved (Sekiya-Kawasaki, Abe, Saka et al. 2002).
6.4.8 Cytological aspects of the synthesis of β-1,3-glucans

Synthesis of β-1,3-glucans occurs by a simple transglycosylation reaction. As described above,
UDPGlc is the universal substrate; accordingly, the reaction can be described as follows:
nUDPGlc → (Glc)n + nUDP
As occurred with chitin synthesis, initial studies analyzed the possible involvement of a
lipid intermediate (Shematek, Broatz, and Cabib 1980; Quigley and Selitrennikoff 1984).
In these studies, different organic solvents were used to extract possible lipid intermedi-
ates, but negative results were obtained in all those experiments. Additionally, the effect
of inhibitors that block reactions involving dolichyl-sugars was analyzed (Fevre 1983), but
these studies also gave negative results. The conclusion of all these studies, as occurred
with chitin synthesis, was that β-1,3-glucan synthesis did not involve a high-energy lipid
intermediate. Contrasting with these negative results, strong evidence was accumulated
suggesting the existence of a priming reaction involving an acceptor of different chemical
composition, probably of proteinaceous nature. The first evidence pointing in that direc-
tion was the observation described above in the sense that glucan microfibrils synthesized
in vitro by cell-free extracts from S. cerevisiae did not contain glucose at their reducing
ends (Larriba, Morales, and Ruiz-Herrera 1981). These results were later reproduced in
C. albicans. Reduction of the in vitro synthesized glucan with NaB[3H]4 followed by acid
hydrolysis, did not liberate any tritiated sorbitol or glucosaminitol, as would happen if a
glucose or a glucosmine moieties were present at the reducing end of the polysaccharide
confirming that glucan biosynthesis was initiated using an acceptor distinct to a glucan
or GlcNac. (Andaluz, Guillen, and Larriba 1986). Further experiments demonstrated that
cell-free extracts of C. albicans incubated with radiolabeled UDPGlc synthesized a product
that could be extracted with a solution of hot mercatoethanol–containing sodium dodecyl
sulfate (SDS). This product was susceptible to protease and to β-1,3-glucanase digestion
(Andaluz, Guillen, and Larriba 1986; Andaluz et al. 1988). Polyacrylamide gel electropho-
resis demonstrated that this product contained two radioactive compounds, one that was
stainable by Coomassie blue (that stains proteins), and another one that barely moved from
the origin of the gel. The first one was digested by papain, and the second one by β-1,3-
glucanase. Kinetic analysis demonstrated that the SDS-soluble product was synthesized
before the insoluble compound, and pulse and chase experiments revealed that the former
was transferred to the second one with time (Ruiz-Herrera and Larriba 1995).
As occurs with chitin synthases, in silico analyses proved that β-1,3-glucan synthases
lack a signal peptide. Since no mechanism has been described in eukaryotes where pro-
teins are directly inserted into the cell membrane it may be anticipated, and was long
hypothesized, that the enzyme reaches the plasmalemma in vesicles of some sort. However,
these may originate, as indicated for chitin synthases (see Chapter 4), by a pathway dif-
ferent from the normal exocytic route. In relation to this hypothesis it was reported in N.
crassa that GS-1 a protein involved in the synthesis of β-1,3-glucan was transported to the
apex in vesicles different to chitosomes, by a nonconventional mechanism, and accumu-
lated in a distinct region of chitosomes in the Spitzenkörper (Verdin, Bartnicki-García,
and Riquelme 2009). GS-1 is part of a macromolecular complex involved in the synthe-
sis of the polysaccharide, although its exact function is unknown. The authors labeled
the protein with green fluorecent protein (GFP) and observed its accumulation in a zone
that surrounded the Spitzenkörper (they called this the Spitzenring), whereas chitosomes
containing a chitin synthase labeled with cherry fluorescent protein were accumulated
in the center of the Spitzenkörper. In the subapical region, large vesicles measuring 200
to 900 nm containing GS-1-GFP which constantly moved by anterograde and retrograde
displacements were observed, suggesting they were carriers of the protein to the apex.
According to the authors, their data are evidence that the enzymes involved in the syn-
thesis of the two main polysaccharides constituting the fungal cell walls are transported
by nonconventional pathway(s) in different conveyors to the apex, accumulate in different
regions of the Spitzenkörper, and become activated by different mechanisms before or
after being distributed to the growing zone.
6.5 Other glycosyl transferases involved

in the synthesis of β-glucans
A family of glycosyl hydrolases, which are classified in family 72, have been described as
involved in the structural organization of the cell wall (reviewed by Popolo, Gualtieri, and
Ragni 2001). The first examples of these enzymes, reported in C. albicans, were denomi-
nated Phr1p and Phr2p, and were suggested to play a role in the control of cell morphology
by pH; although it was later hypothesized that they played a role in the linking of β-1,6-
and β-1,3-glucans in the wall of the fungus (reviewed by Fonzi, 1999). Deletion of either
gene resulted in a pH-conditional defect in cell morphology and virulence, and despite the
fact that these mutants were not severely affected in the polysaccharide composition of the
wall their distribution was abnormal. Homologues of these enzymes are widely distrib-
uted in fungi, and all are bound to the plasmalemma through a GPI anchor (i.e., they are
GPI-proteins). Some examples are: Phr1p, and Phr2p from Candida dubliniensis; Gasp1 to 5
from S. cerevisiae; Phr1 of Pneumocystis carinii; and a similar enzyme encoded by the GEL1
gene that was described in Aspergillus fumigatus (Mouyna et al. 2000; reviewed by Popolo,
Gualtieri, and Ragni 2001). Mutation in the genes encoding Gas1 in S. cerevisiae (Popolo et
al. 1993), Phr1 (Saporito-Irwin et al. 1995) or Phr2 in C. albicans (Muhlschlegel and Fonzi
1997) induced abnormal morphology, and defects in the cell wall properties and structure.
These alterations are related to a reduction in the incorporation of β-glucans into the cell
wall, and release of GPI-proteins to the medium evidencing their importance in cell wall
organization. The gene GEL1 encoding the transglicosylase isolated from the wall of A.
fumigatus was cloned and sequenced (Mouyna et al. 2000), and the predicted amino acid
sequence was found to be highly homologous to Gas from S. cerevisiae and Phr of C. albi-
cans. Mutation in the gene brought about minor alterations as described above.
The biochemical function of these enzymes is not precisely known, but its mechanism
of action has been demonstrated to involve splitting of an internal bond in β-1,3-glucans
and to transfer the newly generated fragment to the nonreducing end of other β-1,3-glucan
molecules to form a new β-1,3-linkage. Hartland, Emerson, and Sullivan (1991) described
an enzyme that, according to the authors, had a different mode of action than Phr1, Gas1,
and so forth. It was reported that the purified fraction contained no exo- or endo-beta-glu-
canase activity, but had glucanosyl transferase activity. The enzyme specifically cleaved
laminaribiose from the reducing end from a β-1,3-glucan and transferred the remain-
der of the molecule to an acceptor in the form of an oligomeric β-1,3-glucan to form a
branched molecule containing a β-1,3- β-1,6-branchpoint. The authors speculated that this
enzyme was responsible for the formation of branched β-1,3- β-1,6-glucans, an idea that
does not have strong support. Still, a different transglicosylase encoded by BGL2 gene
was described to be present in the cell wall of S. cerevisiae and C. albicans (Goldman et al.
1995). This enzyme possessed glucanase activity, but at a high concentration of a suitable
glucan acceptor, the reaction shifted from hydrolysis to glucosyltransferase. After hydro-
lyzing a β-1,3-bond, the released fragment was transferred to an appropriate β-1,3-glucan
acceptor, to form not a β-1,3-bond, but a β-1,6-linkage at the transfer site, giving rise to
a linear polysaccharide. The reaction appeared specific for β-1,3-glucan as acceptor. The
authors suggested a role of this enzyme in polysaccharide cross-linking and wall repair
(see Figure 6.2). Homozygotic bgl2 mutants from C. albicans showed reduced virulence,
decreased growth rate, formation of aggregates at the stationary phase, and increased sen-
sitivity to nikkomycin Z, but an unchanged content of β-1,3- and β-1,6-glucans. These data
Figure 6.2 Schematic representation of β-1,3-glucan branching by transglycosylases (ENZ in the

scheme).
were interpreted as alterations in the wall structure of the mutant that might have been
attenuated by the presence of other glycosyltranferase(s) in the cell (Sarthy et al. 1997).
What was called the first β-1,3-glucan branching was more recently described in A. fumiga-
tus (Gastebois et al. 2009). The gene encoding this protein (BGT2) is an ortholog of BGT1
encoding a transglycosylase described in the same fungal species, and homologous to S.
cerevisiae and C. albicans BGL2 (Mouyna et al. 1998) (see above), but from which it is phylo-
genetically separated. The enzyme cleaves laminaribiose from the reducing end of a β-1,3-
glucan and transfers the residue to an internal C6 hydroxyl group of another β-1,3-glucan
molecule to produce a branched β-1,3- β-1,6-glucan. Nevertheless the demonstration that
the single bgt2 or the double bgt1/bgt2 mutants had no significant phenotype, demonstrated
that they were not the natural enzymes involved in the synthesis of branched β-glucans
present in the fungal cell wall, or else that a further ortholog of these enzymes is present
in the fungus (Gastebois et al. 2009). The conclusion that may be driven from these studies
is that fungi contain different transglycosylases external to the permeability barrier (the
wall or the so-called periplasmic space), whose function is to remodel or crosslink the wall
glucans to make a more resistant structure.
6.6 Synthesis of β-1,6-glucans

As indicated above, β-1,6-glucans constitute a large group of polysaccharides with dif-
ferent molecular size, structure, chemical characteristics, and cellular location. The most
widely studied and best known β-1,6-glucan corresponds to the polysaccharide present in
the cell wall of S. cerevisiae. Contrasting with the knowledge we have on the synthesis of
β-1,3-glucans, synthesis of β-1,6-glucan has been seldom described. Incubation of mem-
brane fractions from S. cerevisiae with radiolabeled UDPGlc gave rise mostly to radioac-
tive β-1,3-glucan, but small amounts of β-1,6-glucans were also detected (López-Romero
and Ruiz-Herrera 1978). Our knowledge of the enzymatic system involved in β-1,6-glucans
stems almost solely from the work of Bussey and collaborators (Al-Aidroos and Bussey
1978; Boone et al. 1990; Brown et al. 1993; Bussey et al. 1979; reviewed by Shahinian and
Bussey 2000). These authors developed a novel approach to investigate the mechanism of
polymer synthesis using the fact that β-1,6-glucan is the receptor for yeast killer toxin 1
from S. cerevisiae (Bussey et al. 1979; Hutchins and Bussey 1983). Isolation of killer-resistant
mutants, genetic analysis to define the complementation groups, and isolation of the cor-
responding genes, a process that as expected involved a great amount of work and a long
time, led to the identification of 10 genes involved in the synthesis of the polysaccharide
(some with several homologues): KRE1, KRE5, KRE6, KRE9, KRE11, CNE1, CWH41/GLS1,
KNH1, ROT2/GLS2, and SKN1 (reviewed by Shahinian and Bussey 2000) (see Table 6.1).
Location of the encoded proteins and their putative function are only partially known.
Although it is generally accepted that the polysaccharide is synthesized at the cell surface,
only Kre1p, a GPI-protein, is located in the plasma membrane, and Kre9p and Knh1p are
soluble extracellular proteins. On the other hand, some of the proteins have been located
intracellularly: Kre11p is located in the cytoplasm; Kre5p, Cwh4p1, Cne1p, and Rot2p are
located in the endoplasmic reticulum; while Kre6p and Skn1p are located at the Golgi
complex. Mutation of any of these genes led to a reduction in the levels of β-1,6-glucan,
and some of the mutations were synthetically lethal. Interestingly, kre9 mutation in C. albi-
cans was lethal in a glucose medium, and mutants were unable to form mycelium in a
medium containing serum, which is a normal method for inducing filament formation in
C. albicans (Lussier et al. 1998). Unfortunately, the genes encoding the transglycosylases
responsible for the synthesis of the polysaccharide, and the mechanism involved are still
Table 6.1 Proteins Involved in the Synthesis of β-1,6-Glucans

Denomination Location Suggested function
Kre1 Cell wall Structural glycoprotein
Kre5 ER UDPGlc glucosyl transferase
Kre6 Golgi β−Glucan synthase
Kre9 Extracellular Uknown
Kre11 ER and Golgi Transport from ER to Golgi
Cne1 ER Unknown
Cwh4/Gls1 ER Mannosyl glycosidase
Knh1 Cell wall Unknown
Rot2/Gls2 ER α-glucosidase
Skn1 Plasma membrane Glucosidase
unknown. Other questions still unanswered are: why are gene products internally located
in the cytoplasm, ER, or Golgi necessary for the synthesis of the polysaccharide? Are they
involved in the initial biosynthesis of the polysaccharide? Are they responsible for the
maturation or transport of the biosynthetic enzyme(s)? Are they involved in the formation
of a receptor for the initiation of synthesis of β-1,6-glucan? Our in silico analysis and sev-
eral other studies have led to the identification of homologues of all these genes in other
Ascomycota species. It was also observed that Basidiomycota species lacked homologues
of genes KRE1, KRE9, KRE11, and CNE1, that Zygomycota and Chitridiomycota species
besides these genes, also lacked KRE6 and SKN1, whereas Encephalitozoon cuniculi, the only
member of Microsporidia whose genome was available for analysis, did not contain homo-
logues of any of the genes identified in S. cerevisiae (Ruiz-Herrera and Ortiz-Castellanos
2010). It must be recalled that Chitridiomycota and Microsporidia, and at least the vegeta-
tive forms of Zygomycota do not contain β-1,6-glucans. It was thus hypothesized that the
only genes strictly involved in the synthesis of the polysaccharide could be homologues of
KRE5, KRE6, CWH41, KNH2, ROT2, and SKN1, the rest of the genes playing only an indi-
rect role on the process. This hypothesis received experimental evidence from the analy-
sis of kre mutants from C. neoformans (Gilbert et al. 2010). These authors described that
kre5, kre6, and skn1 mutants contained reduced amounts of β-1,6-glucan, and displayed
alteration in the capsule, in the levels of chitosan, and phospholipase B1, data that sug-
gested their role in the synthesis of the polysaccharide. All these results indicate that the
information we have on the genes and proteins involved in β-1,6-glucan synthesis is not
satisfactory, and that our knowledge does not provide an explanation for the mechanism
of synthesis of this important wall polysaccharide. It is possible that the substrate of the
until now unknown transglycosylase involved in chain growth of the β-1,6-glucan chains
is UDPGlc, as suggested by the observation that yeast cell-free extracts incubated solely
with labeled UDPGlc synthesized a branched polymer made of β-1,3 and β-1,6-glucan
(López-Romero, and Ruiz-Herrera 1977); but more in vitro, and also in vivo confirmation of
this hypothesis is necessary. Moreover, unexpectedly, no final evidence exists that one of
the identified KRE genes encodes the transglycosylase responsible for chain growth, and
as revealed by the information discussed above, the enzyme involved in chain branching
is still unknown. Apparently, once in the exterior of the cell, β-1.6-glucan (either before
or after associating with β-1,3-glucan) joins covalently to other components of the wall.
Results obtained in C. albicans by Surarit et al. (1988) showed that β-1,6-glucans and chitin
were covalently bound by a glycosidic linkage at position 6 of N-acetylglucosamine from
chitin, and position 1 of the glucose in the β-1,6-glucan chain, and evidence discussed in
other Chapter 7 of the volume was obtained that GPI proteins are covalently bound to
β-1,6-glucan in the cell wall of species of Ascomycota.
6.7 Synthesis of α-1,3-glucans

Contrasting with the wide distribution of β-glucans in members of the different fungal
phyla, the presence of α-1,3-glucan appears to be more restricted, and most of our knowl-
edge has been derived from a limited number of species. More surprising is the fact that
synthesis of the polysaccharide in vitro has not been reported, and all the information we
have regarding its synthesis has been derived from the analysis of mutants defective in
α-1,3-glucan synthesis, and the identification of the genes involved in their phenotype (see
below). Even the importance of the polysaccharide for the building of the wall structure has
been a matter of discussion. As an example of this last assertion is the fact that Zonneveld
(1972) described a mutant of A. nidulans devoid of α-1,3-glucan whose phenotype was not
very different from one of the wild-type strains suggesting that the polysaccharide was
not an essential component of the cell wall. This result contrasts with the reports described
above that stated the importance of α-1,3-glucans on the virulence of different human
pathogens, such as H. capsulatum, B. dermatitidis, and P. brasiliensis. Regarding the role of
the polysaccharide in cell wall structure, the data obtained in S. pombe are more illustra-
tive. It was described that a thermosensitive mutant in a gene denominated AGS1 (ags1-1ts)
when grown at a low (permissive) temperature of 19oC displayed normal morphology; at
a limiting temperature of 34oC, the cells lost the normal rod shape; and at nonpermissive
temperature (37oC), most of the cells lysed. The levels of α-1,3-glucan in the mutant grown
at 19 oC were almost normal, but at 34 oC they were reduced to a third of these values.
Further results also revealed that the polysaccharide synthesized at 34 oC by the mutant
had a size, degree of polymerization (DP), half of the normal one (Grün et al. 2005). These
data demonstrated the importance of α-1,3-glucans in cell wall structure, and accordingly,
in cell morphology (Hochstenbach et al. 1998). Two homologue genes of AGS1 encod-
ing proteins with a similarity of 51% and 42% respectively to Ags1p were identified, and
denominated AGS2 and AGS3, but their role has not been analyzed. It is important to point
out that S. cerevisiae, which does not contain α-1,3-glucans in its cell wall, does not contain
homologues of these genes.
Analysis of the structure of Ags1p revealed a membrane-bound protein, which the
authors identified as a catalytic subunit of the enzyme responsible for α-1,3-glucan syn-
thase, and proposed a transmembrane structure with three domains, a transmembrane
domain forming a pore-like structure possibly for transfer of the glucan molecule, an
intracellular synthase domain, and an extracellular transglycosylase domain. The putative
functions of these last two domains were proposed based on the analysis of similarities.
Thus, similarities of the amino acid sequence of Agsp were found with those from differ-
ent starch and glycogen synthases, with a strategic lysine involved in catalysis, as well as
a Lys/Arg-X-(Gly)2 motif essential for substrate binding, being conserved at strategic sites
(see Figure 6.3). Regarding the extracellular domain, the authors reported blast analysis
results showing similarities with different types of α-amylases, for example with a char-
acteristic (β/α)8-barrel domain with two aspartate and one glutamate invariant residues
responsible for catalysis. The site of mutation in ags1-1ts was located in the extracellular
domain (Hochstenbach et al. 1998). Considering the results on the study of the nature of the
α-1,3-glucan synthesized by ags1-1ts mutant, it has been suggested that Agsp is involved not
only in the synthesis of the polysaccharide, but also in coupling of the building blocks that
Figure 6.3 Alignment of α-1,3-glucan, glycogen and starch synthases. The region with higher simil-
itude among the three glucosyl transferases is represented. (1) Escherichia coli Glga glycogen syn-
thase. (2) Ags1 α-1,3-glucan synthase from Schizosaccharomyces pombe. (3) Q948H2 starch synthase
from Oryza sativa.
constitute the structure of the polysaccharide (Grün et al. 2005; see above). This last func-
tion would depend on the extracellular domain. It is important to indicate that Ags1p, in
contrast to chitin and β-1,3-glucan synthases does possess a signal peptide, suggesting that
during its movement to the plasma membrane it follows the normal exocytic route.
Peculiarly enough, one year after the isolation and description of gene AGS1, the same
gene was cloned again, almost with the same methodology, and denominated differently.
A collection of temperature-sensitive mutants of S. pombe with abnormal morphology
(round or pear-shaped) were transformed with a genomic library made in vector pDB248,
and transformants able to grow at 36oC (nonpermissive temperature) were selected, and
the gene present in a plasmid that cured thermosensitivity was identified and denomi-
nated MOK1 (Katayama et al. 1999). This was a member of a gene family characterized by
10 loci, but only MOK1 was further analyzed in that work and analysis of the rest has not
been published. Also peculiar is that several research groups continue denominating the
gene putatively encoding α-1,3-glucan synthase as MOK1, instead of the original name of
AGS1 (a designation more informative of the putative function), in contradiction of the rule
of precedence.
Besides morphological alterations, mok1 mutants showed reduced amounts of α-1,3-
glucans, and defects in F-actin localization and cytokinesis. Staining of the wild-type
strain with anti-Mok1 antibodies showed that the protein was accumulated at the tip of the
growing cells, and at anaphase it localized mostly in the medial zone, that is, at the zones
where wall synthesis is supposed to take place; but in the mutants the protein was miss-
localized. Evidence for a possible role of the protein kinase C Pkc2 in the localization of
Ags1/Mok1 was provided by the colocalization of the enzyme and the kinase in the wild-
type cells, and their delocalization in the mutants (Katayama et al. 1999). These results
were later extended with the hypothesis that Rho2 was involved in Ags1/Mok1 activation
probably with Pkc2 serving as an intermediate (Calonge et al. 2000). The apical localization
of the enzyme was later confirmed by immunoelectron microscopy (Konomi et al. 2003).
It is evident that these data confirm, and partially complement the data published previ-
ously by Hochstenbach et al. 1998 (see above), giving rise to an image of the mechanism of
α-1,3-glucan biosynthesis in S. pombe, and probably in the rest of the fungi containing this
polysaccharide in their cell walls.
A study of the phylogenetic relationships of glucan synthases has provided interesting
results (Ruiz-Herrera and Ortiz-Castellanos, in preparation). As indicated above, α-glucan
synthase was specifically present only in Dikarya species, since neither Zygomycota nor
the limited numbers of species of Chitridiomycota and Microsporidia whose genomes
have been sequenced possess the enzyme-encoding genes. In contrast, a great number
of Ascomycota and Basidiomycota species analyzed contain genes encoding α-1,3-glucan
synthases that form two homogenous groups clearly separated (see Figure 6.4). An analy-
sis of the species of Basidiomycota that do and do not contain the enzyme revealed that
they do not form any particular taxonomic group, and they are mixed even at the level of
family. We tentatively interpret these data as suggestive that the Ags1/Mok1 gene appeared
right after Zygomycota separated from the evolutionary line, and before Dikarya sepa-
rated into Ascomycota and Basidiomycota. Considering the peculiar characteristics of the
encoded enzymes, it is likely that the encoding gene possibly derived from modifications
of an existing gene, which encoded another glycosyl transferase. Why some species of
Dikarya lost the encoding genes while others conserve them is possibly related to the eco-
logical niches they occupy, and the importance of the role of the products for their physi-
ological success (see Table 6.2).
Figure 6.4 Evolutionary relationships of alpha-1,3-glucans in Basidiomycota and Ascomycota. The

evolutionary history was inferred using the Neighbor-Joining method. The optimal tree with the
sum of branch length = 4.98550130 is shown. The percentage of replicate trees in which the associated
taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The tree
is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used
to infer the phylogenetic tree. The evolutionary distances were computed using the Dayhoff matrix
based method and are in the units of the number of amino acid substitutions per site. All positions
containing alignment gaps and missing data were eliminated only in pairwise sequence compari-
sons (pairwise deletion option). There were 2715 total positions in the final dataset. Phylogenetic
analyses were conducted in MEGA4. Basidiomycota: Pp, Postia placenta, Fp, Fomitopsis pinicola; Cs,
Ceriporiopsis subvermispora; Scc, Schizophyllum commune; Cc, Coprinopsis cinerea; Lc, Laccaria bicolor;
Cn, Cryptococcus neoformans; Ml, Melampsora larici-populina; Pt, Puccinia triticina. Ascomycota: En;
Emericella nigerpar; Sp, Schizosaccharomyces pombe; Sj, Schizosaccharomyces japonicus; Nc, Neurospora
crasa; Bf, Botryotinia fuckeliana; Pb, Paracoccidioides brasiliensis; Ao, Aspergillus oryzae; Nf, Neosartorya
fischeri; Af, Aspergillus fumigatus.
Table 6.2 Ascomycota Species Containing or Not Containing Genes Encoding

α-1,3-Glucan Synthase
A: With at least one gene encoding α-1,3-glucan synthases
Ajellomyces capsulatus; Aspergillus clavatus; Aspergillus fumigatus; Aspergillus nidulans; Aspergillus
niger; Aspergillus oryzae; Aspergillus terreus; Botryotinia fuckeliana; Botrytis cinerea; Chaetomium
globosum; Coccidioides immitis; Magnaporthe oryzae; Neosartorya fischeri; Neurospora crassa;
Paracoccidioides brasiliensis; Penicillium chrysogenum; Penicillium marneffei; Podospora anserina;
Schizosaccharomyces pombe; Sclerotinia sclerotiorum
B: Without genes encoding α-1,3-glucan synthases

Alternaria brassicicola; Ashbya gossypii; Candida albicans; Cryphonectria parasitica; Debaryomyces
hansenii; Fusarium virguliforme; Gibberella zeae; Hebeloma cylindrosporum; Hypocrea jecorina;
Kzachstania exigua; Kluyveromyces lactis; Lachancea thermotolerans; Lodderomyces elongisporus;
Mycosphaerella graminicola; Nectria haematococca; Phaeosphaeria nodorum; Pichia guilliermondii; Pichia
pastoris; Pichia stipitis; Pneumocystis carinii; Saccharomyces bayanus; Saccharomyces cerevisiae;
Saccharomyces kluyveri; Saccharomyces kudriavzevii; Saccharomyces mikatae; Saccharomyces paradoxus;
Sclerotinia sclerotiorum; Uncinocarpus reesii; Yarrowia lipolytica; Zygosaccharomyces rouxii
References
Abe, M., Nishida, I., Minemura, M., Qadota, H., Seyama, Y., Watanabe, T., and Ohya, Y. 2001. Yeast
β-1,3-glucan synthase activity is inhibited by phytosphingosine localized to the endoplasmic
reticulum. J. Biol. Chem. 276:26923–26930.
Al-Aidroos, K. and Bussey, H. 1978. Chromosomal mutants of Saccharomyces cerevisiae affecting cell
wall binding site for killer factor. Can. J. Microbiol. 24:717–720.
Andaluz, E., Guillen, A., and Larriba, G. 1986. Preliminary evidence for a glucan acceptor in the yeast
Candida albicans. J. Biochem. 240:495–502.
Andaluz, E., Ridruejo, J. C., Ramirez, M., Ruiz-Herrera, J., and Larriba, G. 1988. Initiation of glucan
synthesis in yeast. FEMS Microbiol. Lett. 49:251–255.
Aréchiga-Carvajal, E. T. and Ruiz-Herrera, J. 2005. The Rim101/PacC homologue from the basid-
iomycete Ustilago maydis is functional in multiple pH-sensitive phenomena. Eukaryot. Cell
4:999–1008.
Arvindekar, A. U. and Patil, N. B. 2002. Glycogen, a covalently linked component of the cell wall in
Saccharomyces cerevisiae. Yeast 19:131–139.
Awald, P. D., Frost, D., Drake, R. R., and Selitrennikoff, C. P. 1994. β-1,3-Glucan synthase activ-
ity of Neurospora crassa: Identification of a substrate-binding protein. Biochim. Biophys. Acta
1201:312–320.
Bacon, J. S. D., Jones, D., Farmer, V. C., and Webley, D. M. 1968. The occurrence of (1→3)-α-glucan
in Cryptococcus, Schizosaccharomyces and Polyporus species, and its hydrolysis by a Streptomyces
culture filtrate lysing cell walls of Cryptococcus. Biochim. Biophys. Acta 158:313–315.
Baguley, B. C., Rommele, G., Gruner, J., and Wehrli, W. 1979. Papulacandin B: An inhibitor of glucan
synthesis in yeast spheroplasts. Eur. J. Biochem. 97:345–351.
Balint, S., Farkas, V., and Bauer, S. 1976. Biosynthesis of β-glucans catalyzed by a particulate enzyme
preparation from yeast. FEBS Lett. 64:44–47.
Barker, S. A., Bourne, E. J., O’Mantz, D. M., and Stacey, M. 1957. Studies of A. niger. Part VI. The sepa-
ration and structures of oligosaccharides from nigeran. J. Chem. Soc. 2448–2454.
Beauvais, A., Bruneau, J. M., Mol, P. C., Buitrago, M. J., Legrand, R., and Latge, J. P. 2001. Glucan
synthase complex of Aspergillus fumigatus. J. Bacteriol. 183:2273–2279.
Bluhm, T. L. and Sarko, A. 1977. Packing analysis of carbohydrates and polysaccharides. V. Crystal
structures of two polymorphs of pachyman triacetate. Biopolymers 16:2067–2089.
Boone, C., Sommer, S. S., Hensel, A., and Bussey, H. 1990. Yeast KRE genes provide evidence for a
pathway of cell wall β-1,6-glucan assembly. J. Cell Biol. 110:1833–1843.
Breuel, K. F., Kougias, P., Rice, P. J., Wei, D., De Ponti, K., Wang, J., Laffanm, J. J., Li, C., Kalbfleisch,
J., and Williams, D. L. 2004. Anterior pituitary cells express pattern recognition receptors for
fungal glucans: Implications for neuroendocrine immune involvement in response to fungal
infections. Neuroimmunomodulation 11:1–9.
Brown, J. L., Kossaczka, Z., Jiang, B., and Bussey, H. 1993. A mutational analysis of killer toxin resis-
tance in Saccharomyces cerevisiae identifies new genes involved in cell wall (1–6)-β–glucan syn-
thesis. Genetics 133:837–849.
Bussey, H., Saville, D., Hutchins, K., and Palfree, R. G. E. 1979. Binding of yeast killer toxin to a cell
wall receptor on sensitive Saccharomyces cerevisiae. J. Bacteriol. 140:888–892.
Calonge, T. M., Nakano, K., Arellano, M., Ritsuko Arai, R., Katayama, S., Toda, T., Mabuchi, I., and
Perez, P. 2000. Schizosaccharomyces pombe Rho2p GTPase regulates cell wall α-glucan biosynthe-
sis through the protein kinase Pck2p. Mol. Biol. Cell 11:4393–4401.
Campbell, J. A., Davies, G. J., Bulone, V., and Henrissat, B. 1997. A classification of nucleotide-
diphospho-sugar glycosyltransferases based on amino acid sequence similarities. J. Biochem.
326:929–939.
Castro, C., Ribas, J. C., Valdivieso, M. H., Varona, R., Del Rey, F., and Duran, A. 1995. A papulacandin
B resistance in budding and fission yeasts: Isolation and characterization of a gene involved in
β-1,3-D-glucan synthesis in Saccharomyces cerevisiae. J. Bacteriol. 177:5732–5739.
Cervantes-Chávez, J. A., Ortiz-Castellanos, L, Tejeda-Sartorius, M., Gold, S., and Ruiz-Herrera, J.

2010. Functional analysis of the pH responsive pathway Pal/Rim in the phytopathogenic
basidiomycete Ustilago maydis. Fungal Genet. Biol. 47:446–457.
Chang, Y. C. and Penoyer, L.A. 2000. Properties of various Rho1 mutant alleles of Cryptococcus neofor-
mans. J. Bacteriol. 182:4987–4991.
Cortés, J. C., Carnero, E., Ishiguro, J., Sánchez, Y., Durán, A., and Ribas, J. C. 2005. The novel fission
yeast (1,3)β-D-glucans. J. Cell Sci. 118:157–174.
Daba, A. S. and Ezeronye, O. U. 2003. Anti-cancer effect of polysaccharides isolated from higher
basidiomycetes mushrooms. African J. Biotechnol. 2:672–678.
Davila, T., San-Blas, G., and San-Blas, F. 1986. Effect of papulacandin B on glucan synthesis in
Paracoccidioides brasiliensis. J. Med. Vet. Mycol. 24:193–202.
Denning, D.W. 2002. Echinocandins: A new class of antifungal. J. Antimicrob. Chemother. 49:889–891.
Douglas, C. M. 2001. Fungal β(1,3)-D-glucan synthesis. Med. Mycol. 39(1):55–66.
Douglas, C. M., D’Ippolito, J. A., Shei, G. J., Meinz, M., Onishi, J., Marrinan, J. A., Li, W., Abruzzo,
G. K., Flattery, A., Bartizal, K., Mitchell, A., and Kurtz, M. B. 1997. Identification of the FKS1
gene of Candida albicans as the essential target of 1,3-β-D-glucan synthase inhibitors. Antimicrob.
Agents Chemother. 41:2471–2479.
Douglas, C. M., Foor, F., Marrinan, J. A., Morin, N., Nielsen, J. B., Dahl, A. M., Mazur, P., Baginsky,
W., Li, W., El-Sherbeini, M., Clemas, J. A., Mandala, S. M., Frommer, B. R., and Kurtz M. B. 1994.
The Saccharomyces cerevisiae FKS1 (ETG1) gene encodes an integral membrane protein which is
a subunit of β-1,3-D-glucan synthase. Proc. Natl. Acad. Sci. USA 91:12907–12911.
Drees, B. L., Sundin, B., Brazeau, B., Caviston, J. P. et al. 2001. A protein interaction map for cell polar-
ity in development. J. Cell. Biol. 159:549–571.
El-Sherbeini, M. and Clemas, J. A. 1995. Concomitant nikkomycin Z-supersensitivity of an echino-
candin-resistant mutant of Saccharomyces cerevisiae. Antimicrob. Ag. Chemother. 39:200–207.
Farkas, I., Hardy, T. A., DePaoli-Roach, A. A., and Roach, P. J. 1990. Isolation of the GSY1 gene encod-
ing yeast glycogen synthase and evidence for the existence of a second gene. J. Biol. Chem.
265:20879–20886.
Feather, M. S. and Malek, A. 1972. A highly branched exocellular β-glucan from Monolinia fructicola.
Biochim. Biophys. Acta 264:103–105.
Fevre, M. 1983. Inhibitors of synthesis of lipid-linked saccharides also inhibit β-glucan synthesis by
cell-free extracts of the fungus Saprolegnia monoica. J. Gen. Microbiol. 129:3007–3013.
Fonseca-García, C. 2010. Análisis de los mecanismos de regulación de diversas rutas metabólicas por
medio de la vía Pal/Rim en Ustilago maydis. (M.Sc. Thesis) Irapuato, Mexico: Unidad Irapuato,
Centro de Investigación y de Estudios Avanzados, IPN.
Fonseca-García, C., López, M. G., Aréchiga-Carvajal, A., and Ruiz-Herrera, J. 2011. A novel polysac-
charide secreted by pal/rim mutants of the phytopathogen fungus Ustilago maydis. Carbohyd.
Polymers 86:1646–1650.
Fontaine, T., Simenel, C., Dubreucq G., Adam, O., Delepierre, M., Lemoine, J., Vorgias, C. E., Diaquin,
M., and Latge, J. P. 2000. Molecular organization of the alkali-insoluble fraction of Aspergillus
fumigatus cell wall. J. Biol. Chem. 275:27594–27607.
Fonzi, W. A. 1999. PHR1 and PHR2 of Candida albicans encode putative glycosidases required for
proper cross-linking of β-1,3- and β-1,6-glucans. J. Bacteriol. 181:7070–7079.
Frost, D., Brandt, K., Estill, C., and Goldman, R. 1997. Partial purification of (1,3)-β-glucan synthase
from Candida albicans. FEMS Microbiol. Lett. 146:255–261.
Garrett-Engele, P., Moilanen, B., and Cyert, M. S. 1995. Calcineurin, the Ca2+/calmodulin-dependent
protein phosphatase, is essential in yeast mutants with cell integrity defects and in mutants that
lack a functional vacuolar H(+)-ATPase. Mol. Cell. Biol. 15:4103–4114.
Gastebois, A., Mouyna, I., Simenel, C., Clavaud, C., Coddeville, B., Delepierre, M., Latgé, J. P., and
Fontaine, T. 2009. Characterization of a new β(1-3)glucan branching activity of Aspergillus
fumigatus. J. Biol. Chem. 285:2386–2396.
Gilbert, N. M., Donlin, M. J., Gerik, K. J., Specht, C. A., Djordjevich, J. T., Wilson, C. F., Sorrell, T. C.,
and Lodge, J. K. 2010. KRE genes are required for β-1,6-glucan synthesis, maintenance of cap-
sule architecture and cell wall protein anchoring in Cryptococcus neoformans. Mol. Microbiol.
76:517–534.
Gold, M. H., Larson, S., Segel, I. H., and C. R. 1973. Stocking, Intracellular localization of nigeran in
the wall of Aspergillus aculeatus by autoradiography with the electron microscope. J. Bacteriol.
118:1170–1178.
Goldman, R. C., Sullivan, P. A., Zakula, D., and Capobianco, J. O. 1995. Kinetics of β-1,3 glucan inter-
action at the donor and acceptor sites of the fungal glucosyltransferase encoded by the BGL2
gene. Eur. J. Biochem. 227:372–378.
Gopal, P. K., Shepherd, M. G., and Sullivan, P. A. 1984. Analysis of wall glucans from yeast, hyphal
and germ-tube forming cells of Candida albicans. J. Gen. Microbiol. 12:3295–3301.
Grün, C. H., Hochstenbach, F., Humbel, B. M., Verkleij, A. J., Sietsma, J. H., Klis, F. M., Kamerling,
J. P., and Vliegenthart, J. F. G. 2005. The structure of cell wall α-glucan from fission yeast.
Glycobiology 15:245–257.
Gupta, A. K. and Tomas, E. 2003. New antifungal agents. Dermatol. Clin. 21(3):565–576.
Ha, C. H., Lim, K. H., Kim, Y. T., Lim, S. T., Kim, C. W., and Chang, H. I. 2002. Analysis of alkali-
soluble glucan produced by Saccharomyces cerevisiae wild-type and mutants. Appl. Microbiol.
Biotechnol. 58:370–377.
Harada, T., Koreeda, A., Sato, S., and Kasai, N. 1979. Electron microscopic study on the ultrastructure
of curdlan gel: Assembly and dissociation of fibrils by heating. J. Electron. Microsc. 28:147–153.
Hartland, R. P., Emerson, G. W., and Sullivan, P. A. 1991. A secreted β-glucan-branching enzyme from
Candida albicans. Proc. R. Soc. Lond. B. Biol. Sci. 246:155–160.
Hochstenbach, F., Klis, F. M., Van Den Ende, H., Van Donselaar, E., Peters, P. J., and Klausner, R. D.
1998. Identification of a putative α-glucan synthase essential for cell wall construction and mor-
phogenesis in fission yeast. Proc. Natl. Acad. Sci. USA 95:9161–9166.
Hogan, L. H. and Klein, B. S. 1994. Altered expression of surface α-1,3-glucan in genetically related
strains of Blastomyces dermatitidis that differ in virulence. Infect. Immun. 62:3543–3546.
Humbel, B. M., Konomi, M., Takagi, T., Kamasawa, N., Ishijima, S. A., and Osumi, M. 2001. In situ
localization of β-glucans in the cell wall of Schizosaccharomyces pombe. Yeast 18:433–444.
Hutchins, K. and Bussey, H. 1983. Cell wall receptor for yeast killer toxin: Involvement of (l→6)-β-D-
glucan. J. Bacteriol. 154:161–169.
Ishibashi, K., Yoshida, M., Nakabayashi, I., Shinohara, H., Miura, N. N., Adachi, Y., and Ohno
N. 2005. Role of anti-β-glucan antibody in host defense against fungi. FEMS Immunol. Med.
Microbiol. 44:99-109.
Ishiguro, J., Saitou, A. Duran, A., and Ribas, J. C. 1997. Cps1, a Schizosaccharomyces pombe gene homo-
log of Saccharomyces cerevisiae FKS genes whose mutation confers hypersensitivity to cyclospo-
rin A and papulacandin B. J. Bacteriol. 179:7653–7662.
Ishihara, S., Hirata, A., Nogami, S., Beauvais, A., Latge, J. P., and Ohya, Y. 2007. Homologous sub-
units of 1,3-beta-glucan synthase are important for spore wall assembly in Saccharomyces cere-
visiae. Eukaryot. Cell 6:143–156.
Kanetsuna, F. and Carbonell, L. M. 1970. Cell wall glucans of the yeast and mycelial forms of
Paracoccidioides brasiliensis. J. Bacteriol. 101:675.
Kanetsuna, F. and Carbonell, L. M. 1971. Cell wall composition of the yeast-like and mycelial forms
of Blastomyces dermatitidis. J. Bacteriol. 106:946-–948
Kanetsuna, F., Carbonell, L. M., Gil, F., and Azuma, I. 1974. Chemical and ultrastructural studies on
the cell walls of the yeast-like and mycelial forms of Histoplasma capsulatum. Mycopathol. Mycol.
Appl. 54:1–13.
Kang, M. S. and Cabib, E. 1986. Regulation of fungal cell wall growth: A guanine nucleotide-binding
proteinaceous component required for activity of (1→3)-β-D-glucan synthase. Proc. Natl. Acad.
Sci. USA 83:5808–5812.
Kang, M. S., Szaniazlo, P. J., Notario, V., and Cabib, E. 1986. The effect of papulacandin B on
(1—3)-β-D-glucan synthases. A possible relationship between inhibition and enzyme confor-
mation. Carbohydr. Res. 149:13–21.
Katayama, S., Dai Hirata, D., Arellano, M., Pérez, P., and Toda, T. 1999. Fission yeast α-glucan syn-
thase Mok1 requires the actin cytoskeleton to localize the sites of growth and plays an essential
role in cell morphogenesis downstream of protein kinase c function. J. Cell Biol. 144:1173–1186.
Kondoh, O., Tachibana, Y., Ohya, Y., Arisawa, M., and Watanabe, T. 1997. Cloning of the RHO1 gene
from Candida albicans and its regulation of β-1,3-glucan síntesis. J. Bacteriol. 179:7734–7741.
Konomi, M., Fujimoto, K., Toda, T., and Osumi, M. 2003. Characterization and behaviour of α-glucan
synthase in Schizosaccharomyces pombe as revealed by electron microscopy. Yeast 20:427–438.
Kopecka, M. and Kreger, D. R. 1986. Assembly of microfibrils in vivo and in vitro from (1→3)-β-D-
glucan synthesized by protoplasts of Saccharomyces cerevisiae. Arch. Microbiol. 143:387–395.
Kreger, D. R. and M. Kopecká 1975. On the nature and formation of the fibrillar nets produced by
protoplasts of Saccharomyces cerevisiae in liquid media: An electronmicroscopic, X-ray diffrac-
tion and chemical study. J. Gen. Microbiol. 92:207–220.
Larriba, G., Morales, M., and Ruiz-Herrera, J. 1981. Biosynthesis of β-glucan microfibrils by cell-free
extracts from Saccharomyces cerevisiae. J. Gen. Microbiol. 124:375–383.
Leal, F., Ruiz-Herrera, J., Villanueva, J. R., and Larriba, G. 1984. An examination of factors affecting
the instability of Saccharomyces cerevisiae glucan synthetase in cell free extracts. Arch. Microbiol.
137:209–214.
Leon, M., Jaafar, L., and Zueco, J. 2003. RHO1 (YlRHO1) is a non-essential gene in Yarrowia lipolytica
and complements rho1D lethality in Saccharomyces cerevisiae. Yeast 20:343–350.
Lesage, G., Sdicu, A. M., Menard, P., Shapiro, J., Hussein, S., and Bussey, H. 2004. Analysis of β-1,3-
glucan assembly in Saccharomyces cerevisiae using a synthetic interaction network and altered
sensitivity to caspofungin. Genetics 167:35–49
Liu, J., Tang, X., Wang, H., and Balasubramanian, M. 2000. Bgs2p, a 1,3-β-glucan synthase subunit, is
essential for maturation of ascospore wall in Schizosaccharomyces pombe. FEBS Lett. 478:105–108.
López-Romero, E. and Ruiz-Herrera, J. 1977. Biosynthesis of β-glucans by cell-free extracts from
Saccharomyces cerevisiae. Biochem. Biophys. Acta 500:372–384.
Lopez-Romero, E. and Ruiz-Herrera, J. 1978. Properties of β-glucan synthetase from Saccharomyces
cerevisiae. Ant. v. Leeuwenhoeck 44:329–339.
Lowman, D. W., Ferguson, D. A., and Williams, D. L. 2003. Structural characterization of (1→3)-β-D-
glucans isolated from blastospore and hyphal forms of Candida albicans. Carbohydr. Res.
338(14):1491–1496.
Lussier, M., Sdicu, A. M., Shahinian, S., and Bussey, H. 1998. The Candida albicans KRE9 gene is
required for cell wall β-1,6-glucan synthesis and is essential for growth on glucose. Proc. Natl.
Acad. Sci. USA 95:9825-9830.
Manners, D. J. and Meyer, M. T. 1977. The molecular structure of some glucans from the cell walls of
Schizosaccharomyces pombe. Carbohyd. Res. 57:189–203.
Manners, D. J., Masson, A. J., and Patterson, J. C. 1973. The structure of a β−(1→3)-D-glucan from
yeast cell walls. J. Biochem. 135:19–30.
Martín, V., García, B., Carnero, E., Durán, A., and Sánchez, Y. 2003. Bgs3p, a putative 1,3-β-glucan
synthase subunit, is required for cell wall assembly in Schizosaccharomyces pombe. Eukaryot. Cell
2:159–169.
Mazur, P. and Baginsky, W. 1996. In vitro activity of 1,3-D-glucan synthase requires the GTP-binding
protein Rho1. J. Biol. Chem. 271:14604–14609.
Mazur, P., Morin, N., Baginsky, W., el-Sherbeini, M., Clemas, J. A., Nielsen, J. B., and Foor, F. 1995.
Differential expression and function of two homologous subunits of yeast β-1,3-D-glucan syn-
thase. Mol. Cell Biol. 15:5671–5681.
Mio, T., Adachi-Shimizu, M., Tachibana, Y., Tabuchi, H., Inoue, S. B., Yabe, T., Yamada-Okabe, T.,
Arisawa, M., Watanabe, T., and Yamada-Okabe, H. 1997. Cloning of the Candida albicans homo-
log of Saccharomyces cerevisiae GSC1/FKS1 and its involvement in β-1,3-glucan synthesis. J.
Bacteriol. 179: 4096–4105.
Mishra, N. C. and Tatum, E. L. 1972. Effect of L-sorbose on polysaccharide synthetase of Neurospora
crassa. Proc. Natl. Acad. Sci. USA 69:313–317.
Miyata, M., Kanbe, T., and Tanaka, K. 1985. Morphological alterations of the fission yeast
Schizosaccharomyces pombe in the presence of aculeacin A: Spherical wall formation. J. Gen.
Microbiol. 131:611–621.
Mol, P. C. and Wessels, J. G. H. 1987. Linkages between glucosaminoglycan and glucan determine
alkali-insolubility of the glucan in walls of Saccharomyces cerevisiae. FEMS Microbiol. Lett. 41:95–99.
Mouyna, I., Fontaine, T., Vai, M., Monod, M., Fonzi, W. A., Diaquin, M., Popolo, L., Hartland, R. P.,
and Latgé, J. P. 2000. Glycosylphosphatidylinositol-anchored glucanosyltransferases play an
active role in the biosynthesis of the fungal cell wall. J. Biol. Chem. 275:14882–14889.
Mouyna, I., Hartland, R. P., Fontaine, T., Diaquin, M., Simenel, C., Delepierre, M., Henrissat, B., and
Latgé, J. P. 1998. A 1,3-β-glucanosyltransferase isolated from the cell wall of Aspergillus fumiga-
tus is a homologue of the yeast Bgl2p. Microbiology 144:3171–3180.
Muhlschlegel, F. and Fonzi, W. A. 1997. PHR2 of Candida albicans encodes a functional homolog of
the pH-regulated gene PHR1 with an inverted pattern of pH-dependent expression. Mol. Cell
Biol. 17:5960–5967.
Nakano, K., Arai, R., and Mabuchi, I. 1997. The small GTP-binding protein Rho1 is a multifunctional
protein that regulates actin localization, cell polarity, and septum formation in the fission yeast
Schizosaccharomyces pombe. Genes Cells 2:679–694.
Nakano, K. and Mabuchi, I. 1995. Isolation and sequencing of two cDNA clones encoding Rho pro-
teins from the fission yeast Schizosaccharomyces pombe. Gene 155:119–122.
Notario, V., Kawai, H., and Cabib, E. 1982. Interaction between yeast β-(1→3) glucan synthetase and
activating phosphorylated compounds. A kinetic study. J. Biol. Chem. 25:1902–1905.
Okada, H., Abe, M., Asakawa-Minemura, M., Hirata, A., Qadota, H., Morishita, K., Ohnuki, S.,
Nogami, S., and Ohya, Y. 2010. Multiple functional domains of the yeast l,3-beta-glucan syn-
thase subunit Fks1p revealed by quantitative phenotypic analysis of temperature-sensitive
mutants. Genetics 184:1013–1024.
Parent, S. A., Nielsen, J. B., Morin, N., Chrebet, G., Ramadan, N., Dahl, A. M., Hsu, M. J., Bostian,
K. A., and Foor, F. 1993. Calcineurin-dependent growth of an FK506- and CsA-hypersensitive
mutant of Saccharomyces cerevisiae. J. Gen. Microbiol. 139:2973–2984.
Pham, C. D., Zhanyang Y., Sandrock, B., Bölker, M., Scott, E., Gold, S., and Perlin, M. H. 2009. Ustilago
maydis Rho1 and 14-3-3 homologues participate in pathways controlling cell separation and cell
polarity. Eukaryt. Cell 8:977–989.
Popolo, L., Gualtieri, T., and Ragni, E. 2001. The yeast cell-wall salvage pathway. Med. Mycol.
39:111–121.
Popolo, L., Vai, M., Gatti, E., Porello, S., Bonfante, P., Bellestrinoi, P., and Alberghina, L. 1993.
Physiological analysis of mutants indicates involvement of the Saccharomyces cerevisiae GPI-
anchored protein gp115 in morphogenesis and cell separation. J. Bacteriol. 175:1879–1885.
Qadota, H., Python, C. P., Inoue, S. B., Arisawa, M., Anraku, Y., Zheng, Y., Watanabe, T., Levin, D. E.,
and Ohya, Y. 1996. Identification of yeast Rho1p GTPase as a regulatory subunit of β-1,3-glucan
synthase. Science 272:279–281.
Quigley, D. R., Hrmova, M., and Selitrennikoff, C. P. 1988. β(1–3)Glucan synthase of Neurospora crassa:
Solubilization and partial characterization. Exp. Mycol. 12:141–150.
Quigley, D. R. and. Selitrennikoff, C. P. 1984. β (1–3) Glucan synthase activity of Neurospora crassa:
Kinetic analysis of negative effectors. Exp. Mycol. 8:320–333.
Ram, A. F., Brekelmans, S. S., Oehlen, L. J., and Klis, F. M. 1995. Identification of two cell cycle regu-
lated genes affecting the β1,3-glucan content of cell walls in Saccharomyces cerevisiae. FEBS Lett.
358:165–170.
Rau, U. 2004. Glucans secreted by fungi. Turkish Electr. J. Biotechnol. 2:30–36.
Reverberi, M., Di Mario, F., and Tomati, U. 2004. β-Glucan synthase induction in mushrooms grown
on olive mill wastewaters. Appl. Microbiol. Biotechnol. 66:217–225.
Robledo-Briones, A. M. 2009. “Análisis de la Expresión de las Quitina Sintasas Y β 1,3 Glucana
Sintasa de Ustilago Maydis a Nivel Transcripcional.” (M.Sc. Thesis) Irapuato, Mexico: Unidad
Irapuato, Centro de Investigación y de Estudios Avanzados, IPN.
Rommele, G., Traxler, P., and Papulacandins, W. 1983. The relationship between chemical structure
and effect on glucan synthesis in yeast. J. Antibiot. 36:1539–1542.
Ruiz-Herrera, J. 1991. Bioynthesis of β-glucans in fungi. Ant. v. Leeuwenhoek 60:73–81.
Ruiz-Herrera, J., Elorza, M. V., Alvarez, P. E., and Sentandreu, R. 2004. “Biosynthesis of the Fungal
Cell Wall.” In Pathogenic Fungi: Structural Biology and Taxonomy. Edited by G. San-Blas and R.
Calderone, 41–99. Wymondham, Norfolk, UK: Caister Academic Press.
Ruiz-Herrera, J. and Larriba, G. 1995. High molecular weight precursors of glucans in Saccharomyces
cerevisiae. Ant. v. Leeuwenhoek 68:231–235.
Ruiz-Herrera, J. and Ortiz-Castellanos L. 2010. Analysis of the phylogenetic relationships and evolu-
San-Blas, G. and San-Blas, F. 1977. Paracoccidioides brasiliensis:cell wall structure and virulence. A
review. Mycopathologia 67:77–86.
San-Blas, G. and San-Blas, F. 1982. Effect of detergents on membrane- associated glucan synthase
from Paracoccidioides brasiliensis. J. Bacteriol. 152:563–566.
San-Blas, G. and San-Blas, F. 1986. Effect of nucleotides on glucan synthesis in Paracoccidiodes brasil-
iensis. Med. Vet. Mycol. 24:243–245.
San-Blas, G., San-Blas, F., and Serrano, L. E. 1977. Host–parasite relationships in the yeastlike form of
Paracoccidioides brasiliensis strain IVIC Pb9. Infect. Immun. 15, 343–346.
Sanchez-Hernandez, M. E., Garcia-Mendoza, C., and Novaes-Ledieu, M. 1993. Two different alkali-
soluble alpha-glucans in hyphal walls of the basidiomycete Armillaria mellea. Microbiologia
9:34–42.
Santamaría, F., Reyes, F., and Lahoz, R. 1978. Extracellular glucan containing (1→3)-β and (1-->6)-β
linkages isolated from Monilinia fructigen. J. Gen. Microbiol. 109:287–293.
Saporito-Irwin, S. M., Birse, C. E., Sypherd, P. S., and Fonzi, W. A. 1995. PHR1, a pH-regulated gene
of Candida albicans, is required for morphogenesis. Mol. Cell. Biol. 15:601–613.
Sarthy, A. V., McGonigal, T., Coen, M., Frost, D. J., Meulbroek, J. A., and Goldman, R. C. 1997. Phenotype
in Candida albicans of a disruption of the BGL2 gene encoding a 1,3-β-glucosyltransferase.
Microbiology 143:367–376.
Schaefer, H. P. 1977. An alkali-soluble polysaccharide from the cell walls of Coprinus lagopus. Arch.
Schimoler-O’Rourke, R., Renault, S., Mo, W., and Selitrennikoff, C. P. 2003. Neurospora crassa FKS pro-
tein binds to the (1,3-) β-glucan synthase substrate, UDP-glucose. Curr. Microbiol. 46:408–412.
Schmidt, A., Schmelzle, T., and Hall, M. N. 2002. The RHO1-GAPs SAC7, BEM2 and BAG7 control
distinct Rho1 functions in Saccharomyces cerevisiae. Mol. Microbiol. 45:1433–1441.
Sekiya-Kawasaki, M., Abe, M., Saka, A. et al. 2002. Dissection of upstream regulatory components
of the Rho1p effector, 1,3-β-glucan synthase, in Saccharomyces cerevisiae. Genetics 162:663–676.
Sentandreu, R., Elorza, M. V., and Villanueva, J. R. 1975. Synthesis of yeast wall glucan. J. Gen.
Microbiol. 90, 21–31.
Sentandreu, R., Elorza, M. V., Valentín, E., and Ruiz-Herrera, J. 2004. “The Structure and Composition
Blas and R. Calderone, 3–39. Wymondham, Norfolk, UK: Caister Academic Press.
Shahinian, S. and Bussey, H. 2000. β-1,6-Glucan synthesis in Saccharomyces cerevisiae. Mol Microbiol.
35:477–489.
Shematek, E. M., Broatz, J. A., and Cabib, E. 1980. Biosynthesis of the yeast cell wall. I. Preparation
and properties of β(1→3) glucan synthetas. J. Biol. Chem. 255:888–894.
Shematek, E. M. and Cabib, E. 1980. Biosynthesis of the yeast cell wall. II. Regulation of β-(1, 3)-glu-
can synthetase by ATP and GTP. J. Biol. Chem. 255:895–902.
Smith, S. E., Csank, C., Reyes, G., Ghannoum, M. A., and Berlin, V. 2002. Candida albicans RHO1 is
required for cell viability in vitro and in vivo. FEMS Yeast Res. 2:103–111.
Stagg, C. M. and Feather. 1973. The characterization of a chitin-associated D-glucan from the cell
walls of Aspergillus niger. Biochim. Biophys. Acta 320:64–72.
Surarit, R., Gopal, P. K., and Shepherd, M. G. 1988. Evidence for a glucosidic linkage between chitin
and glucan in the cell wall of Candida albicans. J. Gen. Microbiol. 134:1723–1730.
Tartar, A., Shapiro A. M., Scharf Dancia, W., and Boucias Drion, G. 2005. Differential expression of
chitin synthase (CHS) and glucan synthase (FKS) genes correlates with the formation of a modi-
fied, thinner cell wall in in vivo-produced Beauveria bassiana cells. Mycopathologia 160:303–314.
Tsumuraya, Y., Misaki, A., Takeda, S., and Torii, M. 1978. A new fungal α-D-glucan, elsinan, elabo-
rated by Elsinoe leucospila. Carbohyd. Res. 66:53–65.
Varona, R., Perez, P., and Duran, A. 1983. Effect of papulacandin B on β-glucan synthesis in
Schizosaccharomyces pombe. FEMS Microbiol. Lett. 20:243–247.
Verdin, J., Bartnicki-García, S., and Riquelme, M. 2009. Functional stratification of the Spitzenkörper
of Neurospora crassa. Mol. Microbiol. 74:1044–1053.
Zhao, C., Jung, U. S., Garrett-Engele, P., Roe, T., Cyert, M. S., and Levin, D. E. 1998. Temperature-
induced expression of yeast FKS2 is under the dual control of protein kinase C and calcineurin.
Mol. Cell. Biol. 18:1013–1022.
Zonneveld, B. J. 1972. Morphogenesis in Aspergillus nidulans. The significance of α-1→3-glucan of
the cell wall and α-1→3-glucanase for cleistothecium development. Biochim. Biophys. Acta
273:174–187.
chapter seven
Proteins
7.1 Introduction
As described previously in Chapter 2, initial studies of the cell wall of fungi considered
that the cell walls lacked proteins, and that those detected were in fact contaminants
coming from inadequate methodologies used during their isolation and purification. For
that reason, some early analysis included harsh extraction methods involving hot alkali
treatment and boiling with sodium dodecyl sulfate (SDS) to remove all proteins from the
walls. It took some time before the concept that proteins were bona fide components of
the fungal cell became accepted (for a description of the early evidence for the presence
of proteins in the fungal cell wall and a discussion on this matter, readers may consult
Ruiz-Herrera 1992). We must acknowledge the concepts developed by Sentandreu and col-
laborators (for example, Pastor et al. 1984; Elorza, Murgui, and Sentandreu 1985; Herrero,
Saenz, and Sentandreu 1987; Sentandreu, et al. 2004), who demonstrated the existence in
the fungal cell wall of two different populations of proteins; ones that were joined to the
wall by noncovalent linkages: hydrophobic, hydrogen bonding, or ionic; and others, sur-
prising at that time, that were joined through covalent linkages. Noncovalently bound
proteins can be extracted by hot water, but mainly by ionic detergents or chaotropic agents,
whereas covalently bound proteins are resistant to these treatments and can be extracted
only after digestion of the structural polysaccharides, or by breakage of specific linkages
through which they are bound to the wall polysaccharides. These concepts were slowly
assimilated, but they are now generally accepted by all workers in the field, and identifi-
cation of cell wall proteins has advanced rapidly in recent years due to the introduction
of three novel methodological approaches: (i) sequencing of fungal whole genomes, (ii) in
silico analysis of the genomes, and (iii) sensitive proteomic techniques of analysis, mainly
mass spectrometry.
If we forget for a moment a number of proteins devoid of a carbohydrate moiety known
as atypical or moonlighting (for example, see Pitarch et al. 2002), whose relevance and mech-
anism of retention remain obscure, and which will be discussed later, all true cell wall
proteins, linked by either covalent or noncovalent bonds are glycoproteic in nature; that is,
they are constituted by a protein and a carbohydrate moieties.
It is important to indicate that glycoproteins are not exclusive of eukaryotic cells, as
was considered until recently. This concept was changed by the observation that the bac-
terium Campylobacter jejuni possesses glycoproteins, although their carbohydrate moiety
differs from that of the eukaryotic glycoproteins (see Szymanski et al. 1999; Teerstra et
al. 2001; Thibault et al. 2001; Wacker, Linton, and Hitchen 2002; Young, Brisson, Kelley
et al. 2002). For this reason, the definition of bona fide wall-bound proteins (Sentandreu
et al. 2004) includes those having a secretory motif and N- and/or O-glycosylated mol-
ecules covalently bound to them. Some of them have further modifications as discussed
below. As could be anticipated, the synthesis of the protein moiety occurs by the normal
procedure through which all cellular proteins are made at the surface of the ribosomes
following the sequence dictated by specific mRNAs. However, their modifications by
103
addition of the carbohydrate moiety occur during their transit to the cell surface through
the secretory pathway, as will be discussed in detail in Chapter 8.
7.2 Chemical structure of glycoproteins

The importance of glycoproteins in eukaryotes may be revealed by the observation that
mutations or inhibitors that block early steps in N- or O-glycosylation are lethal. Binding of
carbohydrates to the protein moiety in glycoproteins is a process characteristic of eukary-
otes (although some exceptions, not discussed here, exist) that involves two different types
of linkages: O-glycosylation and N-glycosylation. The nature of the O-glycosidic bond of
glycoproteins was demonstrated by the pioneering work of Sentandreu and Northcote
(1968), whereas most of what we know about the basic structure of the N-glycosidically
bound carbohydrate moiety of glycoproteins in fungi comes from the studies by Ballou
and his coworkers (Ballou and Reschke 1974; Ballou 1976; 1982). O-glycosylation in fungi
is established between a serine or threonine residue and a short oligosaccharide, made
of at most 5 mannose units bound through α-1,2- and α 1,3-linkages, although different
sugars may be found O-linked in different fungal species (see below and the review by
Goto 2007). N-glycosylation occurs between a larger oligosaccharide and an asparagine
residue through a diacetylchitobiose (GlcNAc-β-1,4-GlcNAc) molecule. The asparagine
residue to which the oligosaccharide is bound is normally present in a tripeptide with the
sequence N-Asn-Xaa-Ser/Thr , where X is any amino acid, with the normal exception of
proline (Roitsch and Lehle 1989), known as sequon. According to the chemical nature of
the N-glycosidically bound oligosaccharides, animal glycoproteins have been classified in
three classes: (i) high mannose, which are characterized by the presence of 2 to 6 additional
mannose units joined through α−1,3- and α−1,2-bonds, (ii) complex, which contain external
chains made of different kinds of sugars, and (iii) hybrid, having common characteristics
with the high mannose and complex glycoproteins. Because of their mechanism of syn-
thesis (see below), the fungal wall proteins have been classified into the hybrid class. The
relative amount of the carbohydrate moiety may be extremely variable oscillating from
less than 10% to more than 95% of the mass of the glycoprotein molecule (reviewed by
Orlean 1997), but fungal walls are characterized for having glycoproteins with a large
proportion of carbohydrate constituting the fraction known as mannan. N-linked carbohy-
drate residues are made of two fractions as revealed by Ballou’s studies (see below); a core
made of a limited number of mannose residues joined to diacetylchitobiose, and a large
outer chain containing a backbone of α-1,6-linked mannose residues. It must be indicated
that synthesis of the glycoprotein core (which may be modified later, depending on the
protein, the organism, or both) is a universal process in the eukaryotic organisms. The
outer chains of the N-linked carbohydrate in turn contain a variable number of α-1,2- and
α-1,3-bound mannosyl moieties, and occasionally other sugars, mainly GlcNAc, galactose,
glucose, fucose, and rhamnose and phosphomannosyl residues that confer a net negative
charge to the cell surface (Jigami and Odani 1999). Most of the large Mr of the wall glyco-
proteins is due to the size of the outer chains.
Cell wall glycoproteins in fungi comprise a mixture of heterogeneous populations
with Mr oscillating between 25 to 1000 kDa. Ballou and collaborators (Ballou and Reschke
1974; Ballou 1976; 1982) devised an extremely ingenious method to analyze the carbohy-
drate fraction of these glycoproteins which involved acetolysis with a mixture of acetic
anhydride, and acetic and sulfuric acids with an almost enzymatic specificity for selec-
tively breaking α-1,6-bonds, which released the corresponding α-1,2-bound side branches.
These were then separated by exclusion chromatography revealing the finger printing of the
Chapter seven: Proteins 105
Figure 7.1 Structure of the N-linked polysaccharide moiety from the Saccharomyces cell wall gly-
coproteins. (Slightly modified from Ruiz-Herrera, J., 1992, Fungal Cell Wall: Structure, Synthesis and
Assembly, Boca Raton, FL: CRC Press. With permission from Taylor & Francis.)
molecules present in the walls of the different fungal species. Furthermore, Ballou devised
a technique denominated nearest neighbor to determine whether the order of the side chains
was random or not (Ballou 1976). Using these methods, it was possible to demonstrate that
mannans from different species of yeasts were different, and even strain-specific; and that
they are useful to identify species, and to realize taxonomic studies (for example, Ballou
1976; Kocourek and Ballou 1969).
As indicated above, the methodology used by Ballou allowed the demonstration that
the N-linked carbohydrate fraction of the glycoproteins was made of two moieties: the
inner core and the outer chain (Nakajima and Ballou 1974). During these studies, and
through the use of selective agglutination of mutagenized populations of yeasts by specific
antibodies directed against the different moieties of the mannan outer chain, Ballou and
Reschke (1974) were able to isolate mutants defective in the synthesis of the α−1,2- and the
α−1,3 side chains. Of particular interest was the observation that mutants defective in the
synthesis of the α-1,2-chain, that in theory should contain only the α−1,6-main side chain,
still contained a small fraction of α−1,2- and α-1,3- bound side chains. Digestion of the
mannan from this mutant with an endo α-1,6-mannanase isolated from a soil bacterium
led to the observation that the products were only mannose, plus a resistant fraction that
was chemically identified as what we now know as the inner core. This result constituted
strong evidence that the mechanism and the enzymes involved in the synthesis of the
inner core and the outer chain were different, a hypothesis that was demonstrated subse-
quently (see Figure 7.1).
The carbohydrate moieties bound through O-glycosylation linkages, as indicated
above, are made by chains of up to 5 α-1,2- and α-1,3-bound mannose units linked to Ser
or Thr residues (Ernst and Prill 2001; Girrbach and Strahl 2003; Sentandreu and Norcothe
1968; Strahl-Bolsinger et al. 1999). This type of bond is sensitive to weak alkali treatment
(β-elimination) (Sentandreu and Norcothe 1968; Tanner and Lehle 1987). The proteins rich
in this type of substituent are characterized by their rigid structure, which protrudes from
the wall surface analogous to antennae (Jentoft 1990).
With the development of the new techniques of genome sequencing and protein
identification, it became necessary to determine how well conserved the wall proteins
are in the different fungal taxa, and what are their phylogenetic relationships. The strat-
egy and results obtained from this line of research by the Lipke group was noticeable.
These authors soon realized that the usual in silico methods, which involved protein
comparison according with their amino acid sequence, introduced high levels of error
because wall proteins are what has been called low-complexity proteins that can not be
reliable aligned because of what is known as low-complexity corruption (see Coronado
et al. 2006). As these authors indicated, the main problem of alignment of the wall low-
complexity glycoproteins stems from their high frequency of a limited number of amino
acids: Ser, Thr, Pro, Ala, and Val. The average of Ser and Thr accounts for 35% in wall pro-
teins, and Ser and Thr may constitute the only amino acids in protein segments so large
as to be made of 100 residues. To solve this problem the authors devised scoring matrices
adjusted for the frequency of each amino acid in the query sequence which allowed them
to obtain corrected alignments using BLAST and FASTA searches. With this instrument,
the authors proceeded to analyze a large number of proteins present in the Saccharomyces
cerevisiae wall or involved in the synthesis of the cell wall, and their phylogenetic relation-
ships with proteins present in other fungal species (Coronado et al. 2007b). It was found
that the least conserved wall glycoproteins were the adhesins and invasins; for example,
the adhesins involved in mating were conserved only among the genus Saccharomyces,
whereas mannose binding floculins were conserved in Saccharomyces and some other
yeasts, but not in other fungal groups. On the other hand, different glycoproteins classi-
fied as glycosylases were well conserved in several fungal taxa. Equally important was
the observation that conservation of the analyzed glycoproteins followed a different pat-
tern of conservation according to their function. Included into the group of poorly con-
served proteins (a conservation score of less than 0.4) were, besides adhesins, cell wall
structure proteins. The other group of fairly or well-conserved proteins included those
involved in glycosylphosphatidylinositol (GPI) synthesis (see below), and hydrolases in
general: lipases, proteases, and glycosylases, in addition to chaperones and metabolic
enzymes. In a further study, the same group of authors (Coronado et al. 2007a) proceeded
to examine the degree of homology of 171 wall proteins of S. cerevisiae to determine their
conservation. In that study, they utilized the methodology they had developed previ-
ously (see above) to determine the existence of conserved motifs in the proteins. In that
way they identified 217 motifs made by sequences of at least 8 amino acid residues with
a median value of 22 amino acids, 95% of which were present in two or more wall gly-
coproteins. Importantly, it was found that these motifs covered from 10% to even 100%
of the protein length, with a peak of more than 70 proteins being covered by the motifs
in 60% to 80% of their length. According to the authors, these motifs constitute building
blocks for the assembly of wall glycoproteins.
7.3 Different classes of covalently bound glycoproteins

A large number of studies made mainly with Ascomycota species have led to the conclu-
sion that there are three types of proteins bound covalently to the cell wall: (i) Proteins
bound to β-1,6-glucan through a a residue derived from a GPI moiety (De Nobel and Lipke
1994; Kapteyn et al. 1995; Van der Vaart et al. 1996), (ii) Pir-proteins (proteins with internal
repeats), characterized as containing repetitive sequences (Russo et al. 1992; Toh-e et al.
1993) and being highly O- glycosylated. These are attached to β-1,3-glucan by an alkali-
labile bond (Castillo et al. 2003; Kapteyn et al. 1999a, 1999b; Kollar et al. 1997; Mrsa et al.
1997), and (iii), glycoproteins joined to other cell wall proteins by disulfide bridges that
are released by reducing agents such as β-mercaptoethanol (β-ME) or dithiothreitol (DTT)
(Moukadiri and Zueco 2001; Orlean et al. 1986). Some proteins from this group appear to
be Pir-proteins.
7.3.1 GPI-proteins
GPI-proteins are rich in Ser and Thr residues and are usually highly O-glycosylated.
These proteins are universally present in all eukaryotes and are normally located mainly
in the plasma membrane. They are characterized as containing a GPI anchor, which is
added posttranslationally to a motif located at their C-terminus (the ω site). The motif that
links the protein to the lipid moiety has been conserved in all proteins from the different
organisms analyzed thus far, with the estructure: Man-α-1,2-Man-α-1,6-Man-α-1,4-GlcN-
α-1,6-inositol. This is linked to a phospholipid that binds to the protein through a residue
of ethanolaminephosphate. The structure of the core is branched displaying α-1,3- and
-α-1,2-linked mannose (Sipos, Puoti, and Conzelmann 1995). The number of GPI-proteins
in fungal species is variable. For example, GPI-proteins appear to be the most important
wall proteins in Candida albicans, which possesses 169 proteins, where they account for
about 88% of all covalently linked wall proteins. On the other hand, Schizosaccharomyces
pombe contains only 28 probable GPI-proteins; Neurospora crassa, 87; Aspergillus nidulans, 74;
and, S. cerevisiae, 59 GPI-proteins (Eisenhaber et al. 2004). Analysis of the Ustilago maydis
genome revealed 55 genes in total that encode putative GPI-proteins of probable extracel-
lular location (Ruiz-Herrera et al. 2008).
As indicated before, it has been demonstrated that these proteins are covalently
bound to β-1,6-glucans of the cell wall of Ascomycota species; but preliminary unpub-
lished results (J. Ruiz-Herrera, R. Sentandreu, J. A. Lopez, and E. Calvo, unpublished)
suggest that Basidiomycota do not contain GPI-proteins bound covalently to β-1,6-glucan.
Nevertheless, I must point out that surface GPI-proteins that are not covalently linked to
β-1,6-glucans, but only remain associated to the plasma-lemma by means of the hydropho-
bic GPI moiety, should be considered also as wall proteins, taking into consideration that
the full protein moiety is immersed within the wall itself. Whether these proteins may
establish another type of covalent bond with other wall components remains unknown.
It has been suggested that the sequence of amino acids located upstream of the ω site is
important to determine if a GPI-protein establishes or not a covalent bonding with β-1,6
glucan and localizes to the cell wall or remains associated to the plasma membrane. In S.
cerevisiae, it was demonstrated that this signal consists of a region of hydrophobic amino
acids, followed by another short region of more hydrophylic amino acids and a binding site
formed by three amino acid residues (Frieman and Cormack 2004; Frieman, McCaffery,
and Cormack 2002). Similar results were reported for C. albicans GPI-proteins. Two pro-
teins, one located in the plasma membrane (Ecm331), and one bound to β-1,6-glucans in
the cell wall (Hwp1) were selected and engineered to contain the green fluorescent protein
(GFP) as a reporter, a three-flag tag recognizable by commercial antibodies, and different
modifications at their N- and C-termini. It was observed that a short amino acid sequence
located at the C-terminus of either protein (49 or 66 amino acids in length, respectively)
was sufficient to direct the proteins to their normal location. Even a sequence as short as
five amino acids located upstream of the ω site and the presence or absence of selective
amino acids in this region had a strong effect on their capacity to bind or not to β−1,6-
glucan (Mao et al. 2008).
7.3.2 Proteins with internal repeats

Pir-proteins (proteins with internal repeats) are highly O-glycosylated proteins char-
acterized by possessing repetitive sequences (Russo et al. 1992; Toh-e et al. 1993). They
also contain a signal peptide, a Kex2 cleavage site, and a glutamine-rich domain The
internal repeats have the consensus sequence: Q[IV]XDGQ[IVP]Q) and a C-terminal

domain with four cysteine residues following the structure-C-65/66-C-16-C-12-C. In
the case of mycelial fungi, Pir-proteins enclose an extra cysteine residue in the four-
cysteine domain located at the N-terminal region, after the Kex2 site and before the
internal repeats. Pir-proteins do not contain a GPI anchor motif and are attached to
β-1,3-glucans of the cell wall by an alkali-labile bond. This alkali-sensitive bond has been
identified as an ester linkage between the γ-carboxyl group of glutamic acid of the pro-
tein and hydroxyl groups of glucose from the carbohydrate (Ecker et al. 2006). Originally
described in S. cerevisiae (Toh-e et al. 1993), Pir-proteins were later found to be present
in different Ascomycota species (see Martínez et al. 2004): 10 putative Pir-proteins were
identified in S. cerevisiae, 11 in Candida glabrata, 5 in Yarrowia lipolytica, 5 in Kluyveromyces
lactis, 3 in Eremothecium gossypii, 2 in Debaryomyces hansenii, and 1 in Pichia augusta. But in
silico studies have failed to demonstrate homologues of this class of proteins, not only in
other fungal phyla, but in the rest of living organisms, indicating that Pir-proteins sensu
stricto are specific to Ascomycota.
It is important to indicate that not only Pir-proteins have internal repeats. A number
of other cell surface proteins present in different fungi have been found to contain inter-
nal repeats in their sequence. Among them I may cite Flo1, Flo5, Flo9, Flo10, and Flo11
floculins from S. cerevisiae; Aga1, Tir1, Tir4, and Dan4 (Verstrepen et al. 2005), repellent
Rep1, and the atypical hydrophobin Hum3, which provides hydrophobic properties to
the cell wall and is involved in virulence of U. maydis (Müller, Schreier, and Uhrig 2008;
Teertstra et al. 2006).
7.3.3 Proteins associated with the wall through disulfide bonds

This group is constituted by mannoproteins that are extracted from the walls by treat-
ment with reducing agents such as β-mercaptoethanol or dithiothreitol (Moukadiri and
Zueco 2001), indicating that they are bound to other wall components (proteins) through
S-S bonds. They are probably the most neglected group of wall proteins despite the obser-
vation that the extracts thus obtained reveal a complex protein composition. A possible
explanation for the scant number of studies on these proteins is the absence of specific
algorithms for their in silico analysis. Apparently, these proteins do not have uniform
structure, properties, or function, and the nature of the proteins with which they become
associated in the wall is unknown, but it appears obvious that these proteins are bound to
the wall by other types of covalent bonds. One important observation made by pulse and
chase experiments of wall proteins from the Ascomycota species S. cerevisiae, C. albicans,
and Y. lipolytica is that they constitute a homogeneous population, which is not a precursor
of GPI or Pir-proteins (Ruiz-Herrera, Martínez, and Sentandreu 2002).
7.4 Biosynthesis of glycoproteins

Synthesis of glycoproteins is initiated in the endoplasmic reticulum (ER), and the final gly-
cosylation process is terminated in the Golgi. For sake of simplicity, I will first revise the
synthesis of the basic structure of all glycoproteins and further on what is known about
the special case of GPI- and Pir-proteins. The biosynthetic process of glycoproteins can be
divided into the following aspects: (i) synthesis of the inner core of the glycoproteins, (ii)
transfer of the inner core to the protein moiety, (iii) processing of the inner core, (iv) further
glycosylation of the inner core, and (v) O-glycosylation of the proteins. Each one of these
aspects will be discussed separately.
7.4.1 Synthesis of the inner core, or the “dolichol pathway”

The process of biosynthesis of the inner core (GlcNAc2Man9Glc3) is common to all euk-
arytic cells. This process is initiated in the ER and it involves unique glycosyl donors,
besides the normal nucleotide sugars. These are dolichyl sugars where the monosaccha-
rides are bound to an isoprenoid alcohol denominated dolichol. Dolichols contain between
11 and 21 isoprenol units in animals and between 14 and 24 units in fungi, with the char-
acteristic that their α-isoprene unit is saturated, but this general characteristic may have
variants. For example, in Aspergillus niger, dolichols are a family of species containing 18–24
isoprene units, each one with two trans residues and three saturated residues (α, β, and γ)
instead of one, and an exo-methylene substituent on the β carbon atom of the isoproply
group in the ω-residue (Barr and Hemming 1972a). The work from Leloir´s group that pio-
neered the demonstration of the involvement of nucleotide sugars as glycosyl donors, also
provided the initial evidence of the role of dolichols in the synthesis of glycoproteins by
rat liver (Behrens and Leloir 1970). It is interesting to point out that the initial demonstra-
tion of the mechanism of synthesis of the inner core of the glycoproteins was obtained in
animal systems, and later confirmed in yeasts, mainly S. cerevisiae. After these initial stud-
ies, the isolation of yeast mutants defective in each one of the steps involved in the process
became of utmost importance for our present knowledge of the process.
It is now known that biosynthesis of the glycoprotein inner core occurs through the
step-by-step transfer of glycosyl units from nucleotide sugars or dolichyl sugars to a mem-
brane-bound dolichol unit (see reviews in Burda and Aebi 1999; Ziegler, Gemmill, and
Trimble 1994; Kornfeld and Kornfeld 1985). The important characteristic of dolichols is
their hydrophobic nature that anchors the inner core to the ER membrane playing the role
of transporter. The initial reaction of the synthesis of the inner core occurs by a nongly-
cosylation transfer of a GlcNAc residue from UDPGlc to dolichol phosphate (DolP), with
the formation of Dol-P-P-GlcNc and liberation of uridine monophosphate (UMP). This
reaction is carried out by an enzyme denominated GlcNAc phosphotransferase (GPT or
enzyme I) (Sharma, Lehle, and Tanner 1982), and it is selectively inhibited by tunicamycin.
Tunicamycin is extremely toxic (an evidence of the importance of protein glycosylation in
eukaryotes) and has been extremely useful in the study of the role of glycoproteins, both
specifically and in general. This reaction is followed by the transfer of a second molecule
of GlcNAc from UDPGlcNAc to Dol-P-P-GlcNAC to form Dol-P-P-GlcNac-GcNAc, a reac-
tion catalyzed by enzyme II (Sharma, Lehle, and Tanner 1982), encoded by ALG13 and
ALG14 genes. The following sugars to be transferred to the nascent oligosaccharide are five
molecules of mannose utilizing GDPMan as sugar donor, which cannot be substituted by
Dol-P-Man (Levy et al. 1974), with formation of the molecule Dol-P-P-GlcNAc2Man5. The
first Man unit forms a β−1,4-bond with the second added GlcNAc residue by a reaction
catalyzed by the enzyme encoded by the ALG1 gene. This reaction is followed by addition
of four mannosyl residues at the α-1,3- and α-1,6- positions by Alg2 and Alg11 mannosyl
transglycosidases. The biosynthetic process up to this stage occurs on the cytoplasmic face
of the ER. In the next step, the oligosaccharide is transferred to the lumen of the ER. This
change in the topology of the process was determined by the sensitivity of the oligosac-
charide to glycosyl transferases in intact vesicles, and its recognition by concanavalin A, a
lectin known to bind to mannosyl residues (Abeijon and Hirschberg 1992; Hirschberg and
Snyder 1987). Once in the inner part of the ER, remaining mannosyl and glucosyl residues
are transferred utilizing Dol-P-Man and Dol-P-Glc as substrates (Burda and Aebi 1999). It
is interesting to mention that the process of translocation of the oligosaccharide from the
outer to the inner face of the ER does not appear to be a spontaneous process (Helenius et
al. 2002). The role of Dol-P-Man as donor of the initial five mannose units was originally
demonstrated by the incapacity of a lymphoma line devoid of Dol-P-Man to synthesize the
whole inner core (Chapman, Fujimoto, and Kornfeld 1980; Kornfeld and Kornfeld 1980),
and the observation that mutants alg3 of S. cerevisiae defective in the enzyme involved
in the first reaction using Dol-P-Man as sugar donor accumulated Dol-P-P-GlcNAc2Man5
(Huffaker and Robbins 1983). Synthesis of both dolichyl derivatives is carried out with
GDPMan and UDPGlc as sugar donors, respectively, and the process occurs on the cyto-
plasmic face of the ER, from where the dolichyl sugars are internalized to carry out the gly-
cosylation reaction. The process regarding sugar transfer from the cytoplasmic face of the
ER to its luminal face was difficult to accept considering the polar nature of the nucleotide
sugars was cleverly approached by Haselbeck and Tanner (1982). They demonstrated the
reversible transfer of mannose from internal to external GDP-Man in synthetic liposomes
containing in their membrane the S. cerevisiae mannosyl transferase and Dol-P (see Figure
7.2).
It is important to mention that the genes encoding the enzymes involved in all these
processes responsible for the synthesis of the inner core were initially identified in S. cere-
visiae (ALG genes), and the corresponding mutants obtained through the procedure of
tritium suicide. The role of the Phillips Robbins and Stuart Kornfeld research groups in
the investigation of these aspects of glycoprotein biosynthesis was crucial at this point.
Interestingly, the mutants affected in the genes encoding the enzymes catalyzing late reac-
tions are viable, but mutations affecting early genes are lethal.
5 GDP
5 GDPMan 4 GDP
4GDPMan
UDP DOLPP(GIcNAc)2(Man)5
DOLPP(GIcNAc)2 4DOLPMan
UDPGICNAc
DOLPP(GIcNAc)2(Man)9
UMP
DOLPPGIcNAc
3UDPGlc
UDPGICNAc
3UDP
DOLP 3DOLPGlc
DOLPP(GIcNAc)2(Man)9(Glc)3 3DolP
DOLPP
Pi
PROTEIN-
PROTEIN(GIcNAc)2(Man)9(Glc)3
Figure 7.2 Synthesis of the inner core of glycoproteins through the dolichol pathway. (Modified
from Ruiz-Herrera, J., 1992, Fungal Cell Wall: Structure, Synthesis, and Assembly, Boca Raton, FL: CRC
Press. With permission from Taylor & Francis.)
7.4.2 Transfer of the inner core to proteins

An oligosaccharyl transferase (OST) is responsible for the transfer of the oligosaccharide
GlcNAc2Man9Glc3 joined to Dolichyl-P-P, to the amido group of an asparagine residue pres-
ent in nascent acceptor proteins in the ER. OST is a protein complex made of at least 8 sub-
units. In the case of S. cerevisiae, nine subunits, all containing at least one transmembrane
domain have been identified: Ost1-6p, Wbp1p, Swp1p, and Stt3p, the most highly conserved
ones contain the catalytic site of the enzyme (Karaoglu, Kelleher, and Gilmore 1995; Knauer
and Lehle 1999; Silberstein and Gilmore 1996). The encoding genes and mutants have been
obtained, demonstrating that subunits Stt3p, Ost1p, Ost2p, Swp1p, and Wp1p are essential,
while mutation of the genes encoding the rest of the proteins only reduces efficiency of
transfer in vitro (Kelleher and Gilmore 1994; Knauer and Lehle 1994; Pathak, Parker, and
Imperiali 1995; Silberstein and Gilmore 1996; Reiss et al. 1997). Maximum transfer activity
occurs when the substrate contains the full oligosaccharide, although transfer of incom-
plete oligosaccharides may occur, but with a low efficiency. In vitro assays have demon-
strated that the minimal substrate for transfer activity is Dol-P-P-GlcNAc, and that the
acetyl group appears to be very important for the transfer reaction (Tai and Imperiali 2001).
It has also been suggested that the presence of the three terminal glucosyl residues may
be involved in the recognition of the oligosaccharide by the enzyme, since when these are
absent, transfer rate is reduced 5- to 20-fold (Alvarado et al. 1991). The possibility that the
enzymatic complex contains different catalytic and regulatory sites has been entertained,
hypothesizing the presence of both complete and incomplete oligosaccharides. According
to this idea, the different affinity of the enzyme for them favors the transfer of the com-
plete substrate. Regarding the protein substrates, it is known that, as was indicated above,
oligosaccharides are transferred to the amido group of an asparagine residue. But, this is
not a random process, the motif recognized by the transferase, as indicated before is a tri-
peptide made of Ans-X-Ser/Thr, the sequon, (Lehle and Bause 1984), where X is any amino
acid with the exception of proline. It has also been demonstrated that not all sequons of
the protein are required to be glycosylated; for example, of the 14 sequons existing in yeast
invertase, only eight or five become glycosylated (Reddy et al. 1988). The selectivity of this
process may be due to the accessibility of the corresponding sequons that depends on
protein folding. In addition, alterations of the oligosaccharide structure have a role in this
process; accordingly, invertase from alg mutants of S. cerevisiae contain a minor number of
glycosylated sequons (Verostek, Atkinson, and Trimble 1993a, 1993b).
7.4.3 Processing and maturation of the N-linked oligosaccharides

Once the oligosaccharide has been transferred to an asparagine residue of the protein, it suf-
fers a series of modifications in a process that has been denominated maturation or trimming.
The process involves removal of the three glucose residues and one mannose residue (see
Herscovics 1999). Initially the terminal α-1,2-bound glucose is removed by a glucosidase
denominated GlsI. This step is followed by the removal of the two remaining α-1,3-bound
glucosyl residues by the action of the glucosidase GlsII. Glucosidase II is a heterodimer that
uses different mechanisms for the removal of the second and the third glucose units. The
active site located in the α-subunit is sufficient for removal of the second glucose residue,
but requires the β-subunit for removal of the last residue of glucose (Wilkinson, Purswani,
and Stirling 2006). The final step for processing of the oligosaccharide is the removal of
one α-1,2-mannosyl residue by mannosidase Mns1p. This final process is necessary for the
adequate recognition of the nascent glycoprotein by the quality control system responsible
1 2 3 4
P P P P P
Figure 7.3 Maturation (trimming) of the inner core of glycoproteins. Triangles: GlcNAc; Circles:
mannose; Pentagons: glucose. Step 1 is catalyzed by glucosidase I; Steps 2 and 3 by glucosidase II;
Step 4 by mannosidase I.
for the routing of correctly folded proteins for secretion (see Hebert, Foellmer, and Helenius
1995). This aspect will be more thoroughly reviewed in Chapter 8 (see Figure 7.3).
7.4.4 Further glycosylation of the inner core to form

the outer chain of glycoproteins
After trimming has concluded, those glycoproteins that are correctly folded are trans-
ported to the Golgi apparatus, where they may be further glycosylated. In the yeast, it
was demonstrated that the carbohydrate moiety of the glycoproteins is modified to make
two different structures, one short, and one extremely large, constituting what we know
as mannan. The short modification involves only limited glycosylation, and it is charac-
teristic of intracellular glycoproteins, such as carboxypeptidase. The severe modification
involves the formation of the large chains of mannans, characteristic of glycoproteins pres-
ent in the cell wall or the periplasmic space such as invertase.
Our knowledge of the mechanisms involved in the synthesis of the outer chain of
glycoproteins came initially from the isolation of mutants defective at different steps of
the biosynthetic process. The isolation procedure involved the use of specific fluorescent
antibodies recognized by means of a fluorescent-activated cell sorter. The mutants were
classified into several complementation groups defined by genes MNN7/MNN8, MNN9,
and MNN10 (see Ballou, Cohen, and Ballou 1980), which were summed to the previously
obtained mutants defining genes MNN1, MNN2, MNN5 and MNN6. The outer chain of
mutants mnn2 contained only linear chains made of α-1,6-bound mannosyl residues, indi-
cating that the gene product was involved in the initiation of the branches.
It is important to indicate that in the synthesis of the outer chain, the only mannosyl
donor is GDP-mannose, without the intervention of dolichol derivatives, and that the man-
nosyl transferases are dependent on Mn++. Regarding the synthesis of the short or core
oligosaccharides (see above), it was demonstrated that their synthesis was initiated by the
addition to the trimmed oligosaccharide of an α-1,2-linked mannosyl residue by Och1p, this
process was followed by the binding of an α-1,3-mannose in a reaction catalyzed by Mnn1p.
Studies on the synthesis of the large mannan residue led to the demonstration that
addition of the first α-1,6-mannosyl residue which starts the formation of the mannan
chain is catalyzed by the mannosyl transferase Och1p (Nakayama et al. 1992; Nakanishi-
Shindo et al. 1993). The process of formation of the α-1,6-mannan chain is followed by
the action of two mannosyltransferase complexes, M-Pol I (also called V-complex) and
M-Pol II (also called A-complex) (Jungmann, Rayner, and Munro 1999; Kojima, Hashimoto,
and Yoda 1999). The M-Pol I complex is made of Mnn9 and Van1 proteins, while M-Pol II
complex is made by a larger number of proteins: Mnn9p, Mnn10p, Mnn11p, Anp1p, and
Hoc1p. Their function is also different and they operate in tandem. M-Pol I adds approxi-
mately 10 mannosyl residues to the protein, its activity being followed by that of M-Pol II
which adds approximately 40 more residues.
Branching of the α−1,6-mannose backbone is brought about by addition of manno-
syl units that form α-1,2- and α−1,3-bonds. Initially, the α-1,2-mannosylation is catalyzed
by mannosyltransferases Mnn2 and Mnn5 (Rayner and Munro 1998), confirming the
previous data obtained with mnn2 mutants (see above); and addition of α−1,3-mannosyl
residues is catalyzed by Mnn1. Addition of phosphate to the mannan that occurs in S. cere-
visiae whose cells are characteristically stained by Alcian blue, is achieved by addition of
mannosylphosphate, a reaction that is catalyzed by Mnn6/Ktr6p (Wang et al. 1997).
Although the concepts indicated above refer mainly to S. cerevisiae, they have only
slight differences to other fungi. Thus, it has been demonstrated that the mannan moiety
from C. albicans has a larger size, it contains lesser amounts of α−1,3-bound mannose, and
it contains β−1,3-bound mannose. As indicated above, there are nine proteins involved in
the synthesis of the yeast mannan. In contrast, the number of proteins involved in the syn-
thesis of C. albicans mannans is only five, and some have redundant functions.
7.4.5 O-glycosylation of mannoproteins

It was already indicated that O-glycosylation of proteins occurs by the binding of a short
oligosaccharide to the β-hydroxyl group of the amino acids serine or threonine. The sugars
that are attached by this linkage to glycoproteins depend on the organism, and there is no
specific protein sequence recognized by the enzymes involved in the transfer reactions, as
occurs in the case of N-glycosylation. As occurs with N-glycosylation, O-glycosylation is
an essential process that is involved in a number of properties, modulating the secretion
process, providing stability and the correct structure to the glycoproteins, and so forth.
Furthermore, by use of mutants defective in O-glycosyation, a role of O-glycosylation in
cell morphology and hyphal development and differentiation has been determined (see
Goto 2004, 2007, for reviews).
As occurred with N-glycosylation, O-glycosylation has been mostly studied in S. cere-
visiae, although the process may be somewhat different in other fungi, mostly regarding
the type of sugar bonds. Thus, in the case of yeasts, S. cerevisiae O-linked oligosaccha-
rides are normally pentamannose residues in which the three most internal mannosyl
units are α-1,2-bound, and the outer two are α-1,3-bound, whereas in C. albicans, the five
mannosyl residues of its glycoproteins are α-1,2-bound; in S. pombe, oligosaccharides are
shorter and may have ramifications of galactosyl phosphate, and in Pichia pastoris, the two
outer mannosyl units are β−1,3-bound (Goto 2007). The structure of the O-bound oligosac-
charides in the proteins of filamentous fungi is more complex. Accordingly, the oligosac-
charides present in some glycoproteins of Aspergillus are usually branched and contain
besides mannose, glucose and galactose at the nonreducing end, whereas glycoproteins
from Trichoderma possess mannose, glucose, or galactose phosphate as the most internal
sugar, and phosphate or sulfate bound to the most external sugar residue (reviewed by
Goto 2007).
The initial synthetic reaction occurs, as in the case of N-glycosylation, in the ER and
utilizes Dol-P-Man as the sugar donor (Strahl-Bolsinger, Gentzsch, and Tanner 1999).
This reaction is catalyzed in S. cerevisiae by a family of seven protein mannosyl transfer-
ases (ScPMTs1-7) (for example, Strahl-Bolsinger et al. 1993). Six of these enzymes have a
high level of identity of about 50%, and have a number of transmembrane domains. The
following reactions take place at the Golgi complex using GDP-Man as sugar donor by
three α−1,2-mannosyl transferases dependent on Mn++ (Kre2/Mnt1, Ktr1 and Ktr3) which
are also involved in the synthesis of the N-linked mannan adding the second and third
mannosyl residues to the chain. Addition of the α-1,3-bound mannosyl units is catalyzed
by the mannosyl transferases Mnt1, Mnt2 and Mnt3, with GDP-Man as the sugar donor
for the reactions. As occurs with the α−1,2 mannosyl transferases, these enzymes are also
involved in N-glycosylation (see Goto 2007 for review). C. albicans has some differences
with the yeast system, besides the lack of α−1,3 mannosyl residues, as indicated above. The
fungus contains 5 PMTs involved in the initiation of the synthesis of the O-linked oligosac-
charides, denominated PMT1-2, and PMT4-6 (Prill et al. 2005), and the mannosyltransfer-
ases MNT2 and MNT5 which catalyze the addition of α−1,2 mannose. The first enzyme
adds the second mannosyl unit, MNT2 the third, and the rest are added by MNT5. MNT5
has homology to MNN2 and MNN5 of S. cerevisiae, and it is involved not only in O-, but
also in N-glycosylation. In contrast to S. cerevisae and C. albicans, S. pombe has only three
PMT denominated Oma1/Ogm1, Oma2/Ogm2, and Oma4/Ogm4. Phosphogalactosyl
units are added by enzymes with α−1,2 galactosyl transferase activity, Gma12, and Gth1
which utilize UDP-Gal as the donor.
Our knowledge of the mechanism of O-glycosyation in filamentous fungi is more
restricted than the one we have on yeasts, but, in general, it is similar. In A. nidulans three
genes encoding PMTs have been identified (PmtA, PmtB and PmtC), and also three ortho-
logs of the mannosyl transferases are known: MntA, B, and C. Considering the presence
of galactose in the O-bound oligosaccharides, the presence of galactosyl transferases was
expected; a hypothesis that was confirmed when a homologue of the enzyme from S. pombe
was identified: α-GalpT. The fungus also contains a beta galactosyl transferase, β-GalfT.
7.5 Synthesis of GPI-proteins

GPI-proteins are present in the plasma membrane of all eukaryotes thus far studied.
Originally described in some protozoa and mammals (Ferguson et al. 1988; Homans et
al. 1988), GPI-proteins have been later found in fungi associated to the plasma-lemma and
the cell wall, and have been mostly studied in S. cerevisiae, C. albicans, and other yeasts.
The importance of GPI-proteins can be evaluated from the fact that mutations affecting
their synthesis are lethal. As occurs with all secretory proteins, GPI-proteins possess a
signal peptide at their N-terminal region, but also have a specific recognition signal at
their carboxy terminus. Synthesis of the protein moiety occurs as in the case of all secre-
tory proteins in the ER by membrane-bound ribosomes in a mechanism dependent on
the recognition of the signal peptide by the signal recognition particle (see Chapter 8).
Transfer of the GPI moiety to the terminated protein takes place in the lumen of the ER
and is carried out by a transamidase. This reaction involves cleavage of the carboxy termi-
nal hydrophobic signal with the formation of an amide linkage between an ethanolamine
phosphate of the GPI moiety and the exposed amino group of the now terminal amino
acid, which in yeast is Asn or Gly, although in other organisms may also be Ser, As, or Cys
(see Udenfriend and Kodukula 1995, for review). The anchoring signal involves a hydro-
phobic domain separated from the attachment site by a hydrophylic region (see Nuoffer et
al. 1993; Pittet and Conzelman 2007, for reviews).
The mechanism of synthesis of the GPI moiety is an extremely complex process that
has been amply reviewed by different authors using data from trypanosomes, humans, and
yeast (see Eisenhaber et al. 2003; Ferguson 1999; McConville and Menon 2000; Pittet and
Conzelman 2007). Interestingly, the absence of accumulated intermediates in significant
Protein
CH1 EtNH1
(CH1)1 EtNH1 EtNH1 P
C=O P P 6
2 2 α1Man
2 α1Man2 α1Man2 α1Man
O Ins6 α1G1cNH14 α1Man6 3 α1Man
O=P O
O
lipid
Figure 7.4 Chemical structure of the glycosylphosphatidylinositol residue (GPI).
amounts left as the only mechanism to define the different steps in the biosynthetic process
the isolation of the corresponding enzymes and mutants. In this way, it was established
that biosynthesis of the GPI moiety involves the successive addition of different substitu-
ents to phosphatidylinositol, a process taking place in the ER. Pittet and Conzelman (2007),
in their review of synthesis of what may be called the core of the GPI moiety, described 13
steps and made a comparison of them between S. cerevisiae and humans. The steps con-
sidered were the following: 1, binding of GlcNAc to phosphatidyl inositol; 2, deacetylation
of GlcNAc; 3, acylation of inositol; 4, transfer of a mannosyl residue; 5, transfer of a second
mannosyl unit; 6, addition of an ethanolamine phosphate residue to the first mannose; 7,
transfer of a third mannose residue; 8, addition of a fourth mannose residue; 9, addition
of a second ethanolamine phosphate residue to the third mannose residue; 10, addition of
another ethanolamine phosphate residue to the second mannose; 11, transamidation of
GPI to the acceptor protein; 12, removal of the acyl group from inositol; and 13, remodeling
of the GPI structure (see Figure 7.4).
It has been found that transfer of GlcNAc takes place at the cytoplasmic side of the ER,
and utilizes UDPGlcNAc as the sugar donor. This reaction is catalyzed by a complex made
of 6 different proteins, those from yeast being: Gpi1, Gpi2, Gpi3, Gpi15, Gpi19, and Eri1. This
reaction is followed by deacetylation of the GlcNc molecule by Gpi12 deacetylase and by
the acylation of inositol in the C2 position, a reaction catalyzed by the transacylase Gwt1,
which uses acylCoA as donor. Apparently, this reaction takes place at the luminal side of
the ER, suggesting that the substrate must suffer a flip reaction to the ER lumen. After
this, addition of mannose residues takes place. The first mannosyl residue is transferred
by mannosyltransferase 1 (Mt-1) encoded by gene GPI14. The reaction involves Dol-P-Man
as the sugar donor; on the other hand transfer the second mannosyl residue, a reaction
catalyzed by mannosyltransferase 2 (Gpi18) utilizes GDP-Man. In the next step, Mcd4 adds
an ethanolamine phosphate residue to the first mannose unit, by a reaction whereby phos-
phatidylethanolamine serves as donor of the residue. Step 7 involves the addition of the
third mannosyl residue at C2 of the second mannose by mannosyl transferase 3 (Gpi10), in
a reaction occurring only after the first mannose has been joined to ethanolamine phos-
phate. Transfer of the fourth mannose occurs at the C6 of the third mannosyl residue. The
reaction is catalyzed by the mannosyl transferase (Mt4) Smp3, which utilizes GDP-Man as
substrate, and precedes the addition of ethanolamine phosphate to the third mannossyl
residue in contrast to what occurs in mammals. Addition of the ethanolamine phosphate
residue takes place by the same mechanism as the transfer of the first ethanolamine phos-
phate residue, the reaction being catalyzed by the transferase Etn-P-T2, Gpi13. Finally, a
third ethanolaminephosphate residue is added to mannose 2 by Etn-P-T3 (Gpi7), a reaction

also requiring the activity of Gpi11 in order to take place.
When the inositol lipid has attained this level of complexity, it is transferred to the pro-
tein by a transamidation requiring 5 proteins: Gaa1, Gab1, Gpi16, Gpi17, and Gpi18, the lat-
ter one being possibly the catalytic subunit and having a similarity to cysteine proteases.
Remodeling of GPI-proteins involves changes in the lipid structure. Thus, it appears
that nascent GPI probably contains diacylglycerol with C16 and C18 fatty acids, whereas
the mature GPI-proteins contain small amounts of diacylglycerol with C26 and hydroxy
C26 fatty acids, and most of them contain instead a ceramide moiety containing also C26
fatty acids. Another remodeling reaction taking place in Golgi is the addition of a fifth
side-branching mannose in α−1,2- or α-1,3-linkages to the third Man of the core GPI glycan
by the Smp3 mannosyltransferase. The gene encoding this enzyme has been cloned in C.
albicans and found to be essential for growth, suggesting that C. albicans utilizes the same
pathway as S. cerevisiae for GPI synthesis (Grimme et al. 2004). The mechanism of transfer
of the residue of GPI to β-1.6-glucans is poorly known and the enzymes involved in the
process have not been isolated, nor has the reaction been reproduced in vitro.
7.6 Nonclassical protein secretion

In contrast to the true cell wall proteins, analysis by mass spectrometry has demonstrated
the presence of some proteins that lack a signal peptide and are not glycosylated in the
cell wall of different fungi (for example, see Pitarch et al. 2002). These proteins have been
denominated nonclassical or atypical. Some of these proteins have been recognized to be of
cytoplasmic origin and lack the characteristics of bona fide secreted protein, which places
doubts on their exit mechanisms. Among these proteins we may cite those belonging to
the Hsp70 and Hsp90 families of stress proteins (see Chaffin et al. 1998), enolase and phos-
phoglyceromutase (Angiolella et al. 2002), and phosphoglycerate kinase (Alloush et al.
1997), which have been detected in the cell wall of C. albicans. It is unknown whether these
proteins are trapped in the cell wall during breakage, or are actively secreted and become
associated with the cell wall. Accordingly, it remains to be clearly established whether, or
not, these proteins can be considered bona fide cell wall proteins.
This phenomenon of apparent secretion of proteins lacking the characteristics of secre-
tory proteins, is far from being specific to fungi, and has been reported to take place in a
number of eukaryotic cells (see reviews by Cleves 1997; Hughes 1999; Nickel 2003). In gen-
eral, the characteristics of these proteins, besides lacking a signal peptide, are that they are
not present in the ER or Golgi, they become externalized in mutants deficient in secretion,
and their exit from the cells is not inhibited by brefeldin, a known inhibitor of the classical
secretory pathway. Nevertheless, secretion of these proteins requires metabolic energy, it is
temperature-dependent, and it is regulated by different mechanisms. At this point, it is rel-
evant to cite the data discussed in previous chapters on the absence of a signal peptide in
fungal chitin and β−1,3-glucan synthases, although these proteins must somehow reach the
plasma membrane where they proceed to synthesize the corresponding polysaccharides.
A long time ago, it was described that interleukin 1-β, which lacks a signal peptide
was exported from activated human monocytes by an unknown mechanism (Auron et al.
1987). This mechanism possibly involved the operation of the endocytic system, but not the
classical secretory mechanism, since its secretion was inhibited by methylamine, a com-
pound that blocks endocytosis and was stimulated by brefeldin A. The involvement of an
ABC transporter in secretion has been entertained (Zhou et al. 2002), but the observation
that thioredoxin secretion is stimulated by brefeldin A, inhibited by methylamine, but not
involving ABC transporters has placed doubt on this mechanism. Additional examples of
animal proteins that, lacking a signal peptide, are secreted are galectins, β-galactoside-
specific lectins, which are present in the extracellular matrix and are involved in a num-
ber of physiological processes. These proteins are also devoid of a peptide signal, but are
abundantly secreted. It was shown that they become accumulated below the plasma mem-
brane and appear to be secreted by exosomes, membrane bound vesicles that bleb out
of the membrane. Based on these and a number of further examples, Nickel (2003) has
proposed four basic mechanisms for nonclassical secretion of proteins. These mechanisms
are the following: 1, endosomal recycling, as occurs with interleukin 1-β, En2, and HMGB
1; 2, plasma membrane transporters as putatively occurs with FGF-1 and FGF-2; 3, mem-
brane flip-flop suggested to occur with HASPB; and 4, membrane blebbing as theoretically
occurs with galectins. Whether these mechanisms envisaged for animal and protozoa pro-
teins may be applied to fungi, remains unknown.
References
Abeijon, C. and Hirschberg, C. D. 1992. Topography of the glycosylation reactions in the endoplas-
mic reticulum. Trends Biochem. Sci. 17:32–36.
Alloush, H. M., Lopez-Ribot, J. L., Masten, B. J., and Chaffin, W. L. 1997. 3-phosphoglycerate kinase: A
glycolytic enzyme protein present in the cell wall of Candida albicans. Microbiology 143:321–330.
Alvarado, E., Nukada, T., Ogawa, T., and Ballou, C. E. 1991. Conformation of the glucotriose unit in
the lipid-linked oligosaccharide precursor for protein glycosylation. Biochemistry 30:881–886.
Angiolella, L., Micocci, M. M., D’Alessio, S., Girolamo, A., Maras, B., and Cassone, A. 2002.
Identification of major glucan-associated cell wall proteins of Candida albicans and their role in
fluconazole resistance. Antimicrob. Agents. Chemother. 46:1688–1694.
Auron, P. E., Warner, S. J., Webb, A. C., Cannon, J. G., Bernheim, H. A., McAdam, K. J., Rosenwasser,
L. J., LoPreste, G., Mucci, S. F., and Dinarello, C. A. 1987. Studies on the molecular nature of
human interleukin 1. J. Immunol. 138:1447–1456.
Ballou, C. 1976. Structure and biosynthesis of the mannan component of the yeast envelope. Adv.
Microb. Physiol. 14:93–158.
Ballou, C. E. 1982. “Yeast Cell Wall and Cell Surface.” In Molecular Biology of the Yeast Saccharomyces:
Metabolism and Gene Expression, 335–360. Cold Spring Harbor, NY: Cold Spring Harbor
Laboratory.
Ballou, L., Cohen, R. E., and Ballou, C. E. 1980. Saccharomyces cerevisiae mutants that make mannopro-
teins with a truncated carbohydrate outer chain. J. Biol. Chem. 255:5986–5991.
Ballou, C. E. and Reschke, W. C. 1974. Polymorphism of the somatic antigen of yeast. Science
184:127–134.
Barr, R. M. and Hemming, F. W. 1972. Polyprenols of Aspergillus niger. Their characterization and
subcellular distribution. J. Biochem. 126:1193–1202.
Behrens, N. H. and Leloir, L. F. 1970. Dolichol monophosphate glucose: An intermediate in glucose
transfer in liver. Proc. Natl. Acad. Sci. USA 66:153–159.
Burda, P. and Aebi, M. 1999. The dolichol pathway of N-linked glycosylation. Biochim. Biophys. Acta
1426:239–257.
Castillo, L., Martinez, A. I., Garcerá, A., Elorza, M. V., Valentin, E., and Sentandreu, R. 2003.
Functional analysis of cysteine residues and the repetitive sequences of Sc Pir4: The first repeti-
tive sequence is needed to binding the cell wall β-1,3-glucan. Yeast 20:973–983.
Chaffin, W. L., Lopez-Ribot, J. L., Casanova, M., Gozalbo, D., and Martinez, J. P. 1998. Cell wall and
secreted proteins of Candida albicans: Identification, function, and expression. Microbiol. Mol.
Biol. Rev. 62:130–180.
Chapman, A., Fujimoto, K., and Kornfeld, S. 1980. The primary glycosylation defect in class E Thy-
1-negative mutant mouse lymphoma cells is an unability to synthesize dolichol-P-mannose. J.
Biol. Chem. 225:4441–4446.
Cleves, A. E. 1997. Protein transports: The nonclassical ins and outs. Curr. Biol. 7:R318–R320.
Coronado, J. E., Attie, O., Epstein, S. L., Qiu, W. G., and Lipke, P. N. 2006. Composition-modified
matrices improve identification of homologs of Saccharomyces cerevisiae low-complexity glyco-
proteins. Eukaryot. Cell 5:628–637.
Coronado, J. E., Epstein, S. L. Qiu, W. G., and Lipke, P. N. 2007a. Discovery of recurrent sequence
motifs in Saccharomyces cerevisiae cell wall proteins. MATCH Commun. Math. Comput. Chem.
58:281–299.
Coronado, J. E., Mnimneh, S., Epstein, S. L. Qiu, W. G., and Lipke, P. N. 2007b. Conserved processes
and lineage-specific proteins in fungal cell wall evolution. Eukaryot. Cell 6:2269–2277.
De Nobel, H. and Lipke, P. N. 1994. Is there a role for GPIs in cell wall assembly in yeast? Trends Cell
Biol. 4:42–45.
Ecker, M., Deutzmann, R., Lehle, L., Mrsa, V., and Tanner, W. 2006. Pir-proteins of Saccharomyces
cerevisiae are attached to β-1,3-glucan by a new protein-carbohydrate linkage. J. Biol. Chem.
281:11523–11529.
Eisenhaber, B., Maurer-Stroh, S., Novatchkova, M., Schneider, G., and Eisenhaber, F. 2003. Enzymes
and auxiliary factors for GPI lipid anchor biosyntesis and post-translational transfer to pro-
teins. BioEssays 25:367–385.
Eisenhaber, B., Schneider, G., Wildpaner, M., and Eisenhaber, F. 2004. A sensitive predictor for
potential GPI lipid modification sites in fungal protein sequences and its application to
genome-wide studies for Aspergillus nidulans, Neurospora crassa, Saccharomyces cerevisiae and
Schizosaccharomyces pombe. J. Mol. Biol. 337:243–253.
Elorza, M. V., Murgui, A., and Sentandreu, R. 1985. Dimorphism in Candida albicans: Contribution of
mannoproteins to the architecture of yeast and mycelial walls. J. Gen. Microbiol. 131:2209–2216.
Ernst, J. F. and Prill, S. K. 2001. O-glycosylation. Med. Mycol. 39:67–74.
Ferguson, M. A. 1999. The structure, biosynthesis and functions of glycosylphosphatidylinositol
anchors, and the contributions of trypanosome research. J. Cell Sci. 112 :2799–2809.
Ferguson, M. A., Homans, S. W., Dwek, R. A., and Rademacher, T. W. 1988. Glycosylphosphatidylino
sitol moiety that anchors Trypanosoma brucei variant surface glycoprotein to the membrane.
Science 239(4841):753–759.
Frieman, M. B. and Cormack, B. P. 2004. Multiple sequence signals determine the distribution of glyco-
sylphosphatidylinositol proteins between the plasma membrane and cell wall in Saccharomyces
cerevisiae. Microbiology 150:3105–3114.
Frieman, M. B., McCaffery, J. M., and Cormack, B. P. 2002. Modular domain structure in the Candida
glabrata adhesin Epa1p, a β1,6 glucan-crosslinked cell wall protein. Mol. Microbiol. 46:479–492.
Girrbach, V. and Strahl, S. 2003. Members of the evolutionarily conserved PMT family of protein
O-mannosyltransferases form distinct protein complexes among themselves. J. Biol. Chem.
278:12554–12562.
Goto, M. 2004. Protein O-glycosylation in fungi: Diverse structures and multiple functions. Biosci.
Biotechnol. Biochem. 71:1415–1427.
Grimme, S. J., Colussi, P. A., Taron, C. H., and Orlean, P. 2004. Deficiencies in the essential Smp3 man-
nosyltransferase block glycosylphosphatidylinositol assembly and lead to defects in growth
and cell wall biogenesis in Candida albicans. Microbiology 150:3115–3128.
Haselbeck, A. and Tanner, W. 1982. Dolichyl phosphate-mediated mannosyl transfer through liposo-
mal membranes. Proc. Natl. Acad. Sci. USA 79:1520–1524.
Hebert, D. N., Foellmer, B., and Helenius, A. 1995. Glucose trimming and reglucosylation determine
glycoprotein association with calnexin in the endoplasmic reticulum. Cell 81:425–433.
Helenius, J., Davis, Ng, D. T., Marolda, C. L., Walter, P., Valvano, M. A., and Aebi, M. 2002.
Translocation of lipid-linked oligosaccharides across the ER membrane. Nature 415:227–450.
Herrero, E., Saenz, P., and Sentandreu, R. 1987. Cell wall proteins liberated by zymolyase from sev-
eral ascomycetous and imperfect yeasts. J. Gen. Microbiol. 133:2895–2903.
Herscovics, A. 1999. Processing glycosidases of Saccharomyces cerevisiae. Biochim. Biophys. Acta.
1426:275–285.
Hirschberg, C. D. and Snyder, M. D. 1987. Topography of the glycosylation in the rough endoplasmic
reticulum and golgi apparatus. Annu. Rev. Biochem. 56:63–87.
Homans, S. W., Ferguson, M. A., Dwek, R. A., Rademacher, T. W., Anand, R., and Williams, A. F. 1988.
Complete structure of the glycosyl phosphatidylinositol membrane anchor of rat brain Thy-1
glycoprotein. Nature 333:269–272.
Huffaker, T. C. and Robbins, P. W. 1983. Yeast mutants defficient in protein glycosylation. Proc. Natl.
Acad. Sci. USA 80:7466–7470.
Hughes, R. C. 1999. Secretion of the galectin family of mammalian carbohydrate-binding proteins.
Jentoft, N. 1990. Why are proteins O-glycosylated? Trends Biochem. Sci. 15:291–294.
Jigami, Y. and Odani, T. 1999. Mannosylphosphate transfer to yeast mannan. Biochim. Biophys. Acta
1426:335–345.
Jungmann, J., Rayner, J. C., and Munro, S. 1999. The Saccharomyces cerevisiae protein Mnn10p/Bed1p
is a subunit of a Golgi mannosyltransferase complex. J. Biol. Chem. 274:6579–6585.
Kapteyn, J. C., Montijn, R. C., Dijkgraaf, G. J., Van den Ende, H., and Klis, F. M. 1995. Covalent asso-
ciation of β-1,3-glucan with β−1,6-glucosylated mannoproteins in cell walls of Candida albicans.
J. Bacteriol. 177:3788–3792.
Kapteyn, J. C., Van Egmond, P., Sievi, E., Van Den Ende, H., Makarow, M., and Klis, F. M. 1999a. The
contribution of the O-glycosylated protein Pir2p/Hsp150 to the construction of the yeast cell
wall in wild-type cells and β 1,6-glucan-deficient mutants. Mol. Microbiol. 31:1835–1844.
Kapteyn, J. C., Van den Ende, H., and Klis, F. M. 1999b. The contribution of cell wall proteins to the
organization of the yeast cell wall. Biochim. Biophys. Acta 1426:373–383.
Karaoglu, D., Kelleher, D. J., and Gilmore, R. 1995. Functional characterization of Ost3p. Loss of the
34-kD subunit of the Saccharomyces cerevisiae oligosaccharyltransferase results in biased under-
glycosylation of acceptor substrates. J. Cell. Biol. 130:567–577.
Kelleher, D. J. and Gilmore, R. 1994. The Saccharomyces cerevisiae oligosaccharyltransferase is a pro-
tein complex composed of Wbp1p, Swp1p, and four additional polypeptides. J. Biol. Chem.
269:12908–12917.
Knauer, R. and Lehle, L. 1994. The oligosaccharyltransferase complex from yeast. Biochim. Biophys.
Acta 1426:259–273.
Knauer, R. and Lehle, L. 1999. The oligosaccharyltransferase from Saccharomyces cerevisiae. Isolation
of the OST6 gene, its synthetic interaction with OST3, and analysis of nature complex. J. Biol.
Chem. 2745:17249–17255.
Kocourek, J. and Ballou, C. E. 1969. Method for fingerprinting yeast cell wall mannans. J. Bacteriol.
100:1175–1181.
Kojima, H., Hashimoto, H., and Yoda, K. 1999. Interaction among the subunits of Golgi membrane
mannosyltransferase complexes of the yeast Saccharomyces cerevisiae. Biosci. Biotechnol. Biochem.
63:1970–1976.
Kollar, R., Reinhold, B. B., Petrakova, E., Yeh, H. J., Ashwell, G., Drgonova, J., Kapteyn, J. C., Klis,
F. M., and Cabib, E. 1997. Architecture of the yeast cell wall. β(1,6)-glucan interconnects man-
noprotein, β(1,3)-glucan, and chitin. J. Biol. Chem. 272:17762–17775.
Kornfeld, R. and Kornfeld, S. 1980. “Structure of Glycoproteins and Their Oligosaccharide Units.”
In The Biochemistry of Glycoproteins and Proteoglycans. Edited by W. J. Lennarz, 1–34. New York:
Plenum Press.
Kornfeld, R. and Kornfeld, S. 1985. Assembly of asparagine-linked oligosaccharides. Annu. Rev.
Biochem. 54:631–664.
Lehle, L. and Bause, E. 1984. Primary structural requirements for N- and O-glycosylation of yeast
mannoproteins. Biochem. Biophys. Acta 799:246–251.
Levy, J. A., Carminatti, H., Cantorella, A. I., Behrens, N. H., Leloir, L. F., and Tabora, E. 1974. Mannose
transfer to lipid linked di-N-acetylchitobiose, Biochem. Biophys. Res. Commun. 60:118–125.
Mao, Y., Zhang, Z., Gast, C., and Wong, B. 2008. C-terminal signals regulate targeting of glyco-
sylphosphatidylinositol-anchored proteins to the cell wall or plasma membrane in Candida albi-
cans. Eukaryot. Cell 7:1906–1915.
Martínez, A. I., Castillo, J., Garcerá, A., Elorza, M. V., Valentín, E., and Sentandreu, R. 2004. Role of
Pir1 in the construction of the Candida albicans cell wall. Microbiology 150:3151–3161.
McConville, M. J., Menon, A. K. 2000. Recent developments in the cell biology and biochemistry of
glycosylphosphatidylinositol lipids. Mol. Membr. Biol. 17:1–16.
Moukadiri, I. and Zueco, J. 2001. Evidence for the attachment of Hsp150/Pir2 to the cell wall of
Saccharomyces cerevisiae through disulfide bridges. FEMS Yeast Res. 1:241–245.
Mrsa, V., Seidl, T., Gentzsch, M., and Tanner, W. 1997. Specific labelling of cell wall proteins by bio-
tinylation. Identification of four covalently linked O-mannosylated proteins of Saccharomyces
cerevisiae. Yeast 13:1145–1154.
Müller, O., Schreier, P. H., and Uhrig, J. F. 2008. Identification and characterization of secreted and
pathogenesis-related proteins in Ustilago maydis. Mol. Gen. Genomics 279:27–39.
Nakajima, T. and Ballou, C. E. 1974. Structure of the linkage region betwen the polysaccharide and
protein parts of Saccharomyces cerevisiae mannan. J. Biol. Chem. 249:7685–7694.
Nakanishi-Shindo, Y., Nakayama, K., Tanaka, A., Toda, Y., and Jigami, Y. 1993. Structure of the
N-linked oligosaccharides that show the complete loss of alpha-1,6-polymannose outer chain
from och1, och1 mnn1, and och1 mnn1 alg3 mutants of Saccharomyces cerevisiae. J. Biol. Chem.
268:26338–26345.
Nakayama, K., Nagasu, T., Shimma, Y., Kuromitsu, J., and Jigami, Y. 1992. OCH1 encodes a novel
membrane bound mannosyltransferase: Outer chain elongation of asparagine-linked oligosac-
charides. J. EMBO 11:2511–2519.
Nickel, W. 2003. The mystery of nonclassical protein secretion. A current view on cargo proteins and
potential export routes. Eur. J. Biochem. 270: 2109–2119.
Nuoffer, C., Horvath, A., and Riezman, H. 1993. Analysis of the sequence requirements for gly-
cosylphosphatidylinositol anchoring of Saccharomyces cerevisiae Gas1 protein. J. Biol. Chem.
268:10558–10563.
Orlean, P. 1997. “Biogenesis of Yeast Wall and Surface Components.” In The Molecular and Cellular
Biology of the Yeast Saccharomyces. Edited by J. R. Pringle, J. R. Broach, and E. W. Jones, 229–362.
Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Orlean, P., Ammer, A., Watzele, M., and Tanner, W. 1986. Synthesis of an O-glycosylated cell surface
protein induced in yeast by alpha-factor. Proc. Natl. Acad. Sci. USA 83:6263–6266.
Pastor, F. I. J., Valentin, E., Herrero, E., and Sentandreu, R. 1984. Structure of the Saccharomyces cell
wall:mannoproteins released by zymolyase and their contribution to cell architecture. J. Gen.
Microbiol. 130:1419–1428
Pathak, R., Parker, C. S., and Imperiali, B. 1995. The essential yeast NLT1 gene encodes the 64 kDa
glycoprotein subunit of the oligosaccharyl transferase. FEBS Lett. 362:229–234.
Pitarch, A., Sanchez, M., Nombela, C., and Gil, C. 2002. Sequential fractionation and two-dimen-
sional gel analysis unravels the complexity of the dimorphic fungus Candida albicans cell wall
proteome. Mol. Cell. Proteomics 1:967–982.
Pittet, M. and Conzelmann, A. 2007. Biosynthesis and function of GPI-proteins in the yeast
Saccharomyces cerevisiae. Biochim. Biophys. Acta 1771:405–420.
Prill, S. K., Klinkert, B., Timpel, C. A., Schroppel, K., and Ernst, J. F. 2005. PMT family of Candida albi-
cans: Five protein mannosyltransferase isoforms affect growth, morphogenesis and antifungal
resistance. Mol. Microbiol. 55:546–560.
Rayner, J. C. and Munro, S. 1998. Identification of the MNN2 and MNN5 mannosyltransferases
required for forming and extending the mannose branches of the outer chain mannans of
Saccharomyces cerevisiae. J. Biol. Chem. 273:26836–26843.
Reddy, V. A., Johnson, R. S., Biemann, K., Williams, R. S., Ziegler, F. D., Trimble, R. B., and Maley, F.
1988. Characterization of the glycosylation sites in yeast external invertase. I. N-linked oligo-
saccharide content of the individual sequons. J. Biol. Chem. 263:6978–6985.
Reiss, G., Heesen, S., Gilmore, R., Zufferey, R., and Aebi, M. 1997. A specific screen for oligosacchar-
yltransferase mutations identifies the 9 kDa OST5 protein required for optimal activity in vivo
and in vitro. J. EMBO 16:1164–1172.
Roitsch, T. and Lehle, L. 1989. Structural requirements for protein N-glycosylation. Influence of
acceptor peptides on cotranslational glycosylation of yeast invertase and site-directed muta-
genesis around a sequon sequence. Eur. J. Biochem. 181:525–529.
Ruiz-Herrera, J., Martínez, A. I., and Sentandreu, R. 2002. Determination of the stability of protein
pools from the cell wall of fungi. Res. Microbiol. 153:373–378.
Ruiz-Herrera, J., Ortiz-Castellanos, L., Martínez, A. I., León-Ramírez, C., and Sentandreu, R. 2008.
Analysis of the proteins involved in the structure and synthesis of the cell wall of Ustilago may-
dis. Fungal Gen. Biol. 45:571–576.
Russo, P., Kalkkinen, N., Sareneva, H., Paakkola, J., and Makarow, M. 1992. A heat shock gene
from Saccharomyces cerevisiae encoding a secretory glycoprotein. Proc. Natl. Acad. Sci. USA
89:3671–3675
Sentandreu, R., Elorza, M. V., Valentín, E., and Ruiz-Herrera, J. 2004. “The Structure and Composition
Blas and R. Calderone, 3–39. Wymondham, Norfolk, UK: Caister Academic Press.
Sentandreu, R. and Northcote, D. H. 1968. The structure of a glycopeptide isolated from the yeast cell
wall. J. Biochem. 109:419–432.
Sharma, C. B., Lehle, L., and Tanner, W. 1982. Solubilization of and characterization of the initial
enzymes of the dolichol pathway from yeast. Eur. J. Biochem. 126:319–325.
Silberstein, S. and Gilmore, R. 1996. Biochemistry, molecular biology, and genetics of the oligosac-
charyltransferase. FASEB J. 10:849–858.
Sipos, G., Puoti, A., and Conzelmann, A. 1995. Biosynthesis of the side chain of yeast glycosylphos-
phatidylinositol anchors is operated by novel mannosyltransferases located in the endoplasmic
reticulum and the Golgi apparatus. J. Biol. Chem. 270:19709–19715.
Strahl-Bolsinger, S., Gentzsch, M., and Tanner, W. 1999. Protein O-mannosylation. Biochim. Biophys.
Acta 1426:297–307.
Strahl-Bolsinger, S., Immervoll, T., Deutzmann, R., and Tanner, W. 1993. PMT1, the gene for a
key enzyme of protein O-glycosylation in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA
90:8164–8168.
Szymanski, C. M., Yao, R. J., Ewing, C. P., Trust, T. J., and Guerry, P. 1999. Evidence for a system of
general protein glycosylation in Campylobacter jejuni. Mol. Microbiol. 32:1022–1030.
Tai, V. W. and Imperiali, B. 2001. Substrate specificity of the glycosyl donor for oligosaccharyl trans-
ferase. J. Org. Chem. 66:6217-6228.
Tanner, W. and Lehle, L. 1987. Protein glycosylation in yeast. Biochim. Biophys. Acta 906:81-99.
Teertstra, W. R., Deelstra, H. J., Vranes, M., Bohlmann, R., Kahmann, R., Thibault, P., Logan, S. M.,
Kelly, J. F., Brisson, J. R., Ewing, C. E. Trust, T. J., and Guerry, P. 2001. Identification of the
carbohydrate moieties and glycosylation motifs in Campylobacter jejuni flagellin. J. Biol. Chem.
276:34862–34870.
Teertstra, W. R., Deelstra, H. J., Vranes, M., Bohlmann, R., Regine Kahmann, R., Kämper., J., and
Wösten, H. A. B. 2006. Repellents have functionally replaced hydrophobins in mediating
attachment to a hydrophobic surface and in formation of hydrophobic aerial hyphae in Ustilago
maydis. Microbiology 152:3607–3612.
Toh-e, A., Yasunaga, S., Nisogi, H., Tanaka, K., Oguchi, T., and Matsui, Y. 1993. Three yeast genes,
PIR1, PIR2 and PIR3, containing internal tandem repeats, are related to each other, and PIR1
and PIR2 are required for tolerance to heat shock. Yeast 9:481–494.
Udenfriend, S. and Kodukula, K. 1995. How glycosylphosphatidylinositol-anchored membrane pro-
teins are made. Annu. Rev. Biochem. 64:563–591
Van der Vaart, J. M., Biesebeke, R., Chapman, J. W., Klis, F. M., and Verrips, C. T. 1996. The β-1,6-
glucan containing side-chain of cell wall proteins of Saccharomyces cerevisiae is bound to the
glycan core of the GPI moiety. FEMS Microbiol. Lett. 145:401–407.
Verostek, M. F., Atkinson, P. H., and Trimble, R. B. 1993a. Glycoprotein biosynthesis in the alg3
Saccharomyces cerevisiae mutant. I. Role of glucose in the initial glycosylation of invertase in the
endoplasmic reticulum. J. Biol. Chem. 268:12095–12103.
Verostek, M. F., Atkinson, P. H., and Trimble, R. B. 1993b. Glycoprotein biosynthesis in the alg3
Saccharomyces cerevisiae mutant. II. Structure of novel Man6-10GlcNAc2 processing intermediates
on secreted invertase. J. Biol. Chem. 268:12104–12115.
Verstrepen, K. J., Jansen, A., Lewitter, F., and Fink, G. R. 2005. Intragenic tandem repeats generate
functional variability. Nat. Genet. 37:986–990.
Wacker, M., Linton, D., Hitchen, P. G. et al. 2002. N-linked glycosylation in Campylobacter jejuni and
its functional transfer into E. coli. Science 298:1790–1793.
Wang, X. H., Nakayama, K., Shimma, Y., Tanaka, A., and Jigami, Y. 1997. MNN6, a member of the
KRE2/MNT1 family, is the gene for mannosylphosphate transfer in Saccharomyces cerevisiae. J.
Biol. Chem. 272:18117–18124.
Wilkinson, B. M., Purswani, J., and Stirling, C. J. 2006. Yeast GTB1 encodes a subunit of glucosidase II
required for glycoprotein processing in the endoplasmic reticulum. J. Biol. Chem. 281:6325–6333.
Young, N. M., Brisson, Kelley et al. 2002. Structure of the N-linked glycan present on multiple glyco-
proteins in the Gram-negative bacterium, Campylobacter jejuni. J. Biol. Chem. 277:42530–42539.
Zhou, X., Engel, T., Goepfert, C., Erren, M., Assmann, G., and Von Eckardstein, A. 2002. The ATP
binding cassette transporter A contributes to the secretion of interleukin 1b from macrophages
but not from monocytes. Biochem. Biophys. Res. Commun. 291: 598–604.
Ziegler, F. D., Gemmill, T. R., and Trimble, R. B. 1994. Glycoprotein synthesis in yeast. Early events in
N-linked oligosaccharide processing in Schizosaccharomyces pombe. J. Biol. Chem. 269:12527–12535.
chapter eight
Cytological aspects of cell wall synthesis

8.1 Introduction
As repeatedly stated, the fungal cell wall is a coherent structure made up by the correct
association of different compounds. These compounds are synthesized in different cell
compartments and they finally come together at specific points of the cell surface and
become associated by means of covalent and noncovalent bonds to make the structure
that provides strength and form to the fungal cell. In this chapter, I will discuss different
aspects of the cytological bases of these processes.
The process of synthesis of the cell wall by association of its different components may
be schematically summarized as follows:
1. Cell wall proteins are synthesized by membrane-bound ribosomes and become ini-
tially glycosylated in the endoplasmic reticulum (ER); they are then transported into
vesicles through a series of membrane compartments where their glycosylation is
terminated to finally reach the cell surface.
2. Chitin synthases and glucan synthases are synthesized inside the cell and become
externalized by a process dependent on specific microvesicles that does not involve
the classical secretory mechanism (see previous chapters), eventually reaching the
plasma membrane and proceeding to synthesize their respective products.
3. The synthesized chains of the structural polysaccharides associate among them-
selves and crystallize to form microfibrils.
4. During this last process, chemical modifications may occur altering the characteris-
tics of glucans and chitin.
5. The different secreted compounds associate among themselves by means of nonco-
valent linkages forming the nascent cell wall.
6. Establishment of covalent linkages occurs giving rise to the mature cell wall.
Of course, the schematized process noted above is continuous, permitting the expansion
of the cell wall, which essentially represents the growth of the full organism.
In the following pages of this chapter, I will discuss in detail the processes taking
place inside the cell, whereas the last aspects of the organization of the cell wall will be
examined later.
8.2 Synthesis of secretory and membrane-bound proteins

At the end of the sixties and the beginning of the seventies of the last century, it was dem-
onstrated that secretion of proteins by eukaryotic cells involved their initial transfer to the
endoplasmic reticulum (ER) (reviewed by Palade 1975) and the concept of the endomem-
brane system made by the sequential operation of the ER, Golgi apparatus, and secretory
vesicles was introduced (see Morré and Mollenhauer 1974). It was further demonstrated
that in contrast to intracellular proteins, which are synthesized by cytosolic ribosomes,
extracellular proteins in animals are synthesized by membrane-bound ribosomes. The
123
observation that particulate fractions treated with puromycin, the protein synthesis inhib-
itor, accumulated nascent polypeptides in the lumen of microsome vesicles was taken as
evidence that proteins were translocated into the ER membrane during the biosynthetic
process (Redman and Sabatini 1966). The transfer of proteins into the ER presented the
problem that the molecules of globular proteins are organized in such a way that hydro-
philic and charged amino acids accumulate at the surface, whereas hydrophobic amino
acids are kept internal; accordingly, their transfer through a lipid bilayer represents ther-
modynamic difficulties, which make almost impossible their transit. This problem made
some authors think that proteins might cross the membrane not in their final form, but
in a denatured stage; although this mechanism would also require that the protein were
transferred not through a lipid bilayer, but using an aqueous channel existing in the ER
membrane. The experiment that opened up this problem was provided by Milstein et al.
(1972), who observed that the light chain of immunoglobulin synthesized in vitro by free
ribosomes had a larger Mr than the mature form, exceeding this by 15 amino acids. This
experiment was followed by those performed by the Blobel group, who realized that dur-
ing synthesis of this same protein by membrane-bound ribosomes, there occurred a pro-
teolytic event, which removed a fragment of the protein at the N-terminal region (Blobel
and Dobberstein 1975a). These results were rapidly followed by further experiments con-
firming the observation that during transfer of secreted proteins to the ER lumen, there
occurred the proteolytic removal of a small peptide at the N-terminus of the protein. We
now know this peptide as signal peptide (for example, see Blobel and Dobberstein 1975b).
The demonstration of the presence of a protease present in the ER from animal and yeast
cells that cuts the signal peptide confirmed the validity of the model (Straus et al. 1979;
Julius 1983). To explain the process at the molecular level, Blobel (1980) introduced the idea
that proteins crossed what he called translocationally competent membranes in a denatured
stage, and that the proteins, which achieved this feat, contained specific signals (topogenic
sequences) in their amino acid sequence, which were specifically recognized by the system
catalyzing translocation of the protein. This hypothesis, denominated intracellular protein
topogenesis, considered that proteins to be translocated had a signal sequence (signal pep-
tide) in order to initiate the transfer; stop sequences, which avoided further transfer of the
peptides, the region of the peptide that continued synthesizing under these conditions
and remained in the cytosolic side of the ER secreted sequences that targeted the protein
to their proper location in the cell or out of it.
As may be seen from this model of protein secretion, synthesis of the protein occurs
at the same time as translocation. This type of phenomenon is denominated cotranslational
translocation. This mode of translocation is found in all cells and is used for the transport
of secretory proteins as well as for the integration of most membrane proteins. All these
data crystallized in a model that occurs as follows: the process starts by the recognition of
the mRNA molecules encoding secretory or membrane proteins by normal cytosolic ribo-
somes, which start the synthesis of the polypeptide. Apparently, when the signal peptide
is synthesized, this is recognized by the so-called signal-recognition particle (SRP), which
transfers the nascent polypeptide and the ribosome to a specific membrane receptor in
the ER, where the ribosome interacts with a specific receptor, and the nascent polypeptide
moves to a translocation channel (for reviews see Luirink and Sinning 2004; Halic and
Beckmann 2005). It is known that binding of SRP to the signal peptide stops further syn-
thesis of the polypeptide. This inhibition is important to guarantee that the nascent protein
retains an unfolded structure. SRP is a complex made of six proteins and a small RNA of
300 nucleotides (7SL RNA). The polypeptides are organized in the form of two heterodi-
mers made of two proteins each; respectively, one is made of a 19 and a 54 kDa polypeptide,
Chapter eight: Cytological aspects of cell wall synthesis 125
and the other one is made of 9 kDa and 14 kDa polypeptides, and of two separate polypep-
tides of 68 kDa and 72 kDa. After the association of SRP with the nascent polypeptide, the
complex with the ribosome is led to the SRP receptor (or docking protein) located on the
cytoplasmic surface of the ER. Once docking occurs, SRP is liberated to engage in a new
reaction. The SRP receptor is a hydrophobic 69 kDa protein with two membrane spanning
domains. After docking and release of the SRP, translation of the polypeptide is resumed
with the energy of guanosine triphosphate (GTP) hydrolysis, and the polypeptide becomes
translocated across the ER membrane in a reaction independent of metabolic energy. As
stated by Blobel’s theory, in the case of membrane proteins, translocation is stopped accord-
ing to the corresponding topogenic signal (see above), and the protein comes out from the
translocation channel to form the cytosolic domains, but other parts of the protein remain
in the channel to constitute the transmembrane domains (see Figure 8.1).
8.3 Translocation of proteins to the endoplasmic reticulum

The mechanism of cotranslational translocation in eukaryotes and prokaryotes has been
reviewed by Stirling (1999) and Rapoport (2007), who have indicated the similarities in the
process occurring in both types of organisms. As indicated above, transfer of the proteins to
the lumen of the ER in eukaryotes takes place through an aqueous channel existing in the
ER membrane. This is made by a complex of three subunits named Sec61 in eukaryotic cells
or SecY in the plasma membrane prokaryotes that have a significant level of conservation.
The α subunit of the complex forms the pore of the channel and surrounds the polypeptide
during its translocation. Its 10 helices are divided into two groups, which are maintained
associated through the γ subunit, whereas the β subunit makes contact with the periphery
of the α subunit. The helices are arranged in the form of an hourglass–shaped funnel with
the face looking toward the cytoplasm side empty, and the one looking toward the lumen
closed by a plug constituted by a short helix.
At the start of the translocation step, the synthesizing polypeptide inserts into the
translocation channel as a loop with the signal sequence intercalated into the walls of the
channel and the segment distal to it located in the pore proper. Opening of the channel
for loop insertion probably occurs in two steps. The first step normally involves binding
of the ribosome, which causes an alteration in the plug that obstructs the pore (see above).
In the second step, the hydrophobic segment of the signal sequence is intercalated into the
lateral gate and it causes alteration in the structure of the pore helping to move away the
plug. Maintaining the pore open during translocation will finally depend on the intro-
duction of the nascent polypeptide into the channel. The plug will move back to close the
translocation channel when synthesis of the polypeptide terminates. During the process
of translocation of the polypeptide, protein folding in the lumen of the ER takes place. This
process involves both physical and biological factors. Accordingly, it is known that the
environment in the ER lumen is oxidative, a factor that facilitates the formation of disulfide
bonds in the proteins through a process catalyzed by the protein disulfide isomerase. The
process also requires metabolic energy in the form of hydrolysis of adenosine triphos-
phate (ATP) provided by the ATPase activity of the chaperone BiP and very importantly,
activity of the complex system involving calnexin, calreticulin, and glucosyltransferases
(see below). Finally, the signal peptide is removed by the signal protease and degraded by
other proteases. It has been demonstrated that this process is extremely important for the
final folding of the secreted polypeptide.
Besides this cotranslational process, which is the most important one in eukaryotes
and abundant in prokaryotic cells, some proteins are secreted posttranslationally. This
126
mRNA
+ SRP
NH2
1 2 3 NH2
Fungal cell wall: Structure, synthesis, and assembly

SEC61/SECY DOLP-S DOLP
S
SRP
RECEPTOR 4 5 6 S 7 S 8
S NH2
S
S
Figure 8.1 Schematic representation of the mechanism of cotranslational synthesis of secretory glycoproteins. (1) through (3): Synthesis of the
protein starts in the cytosol. Once the signal peptide is synthesized, the signal recognition particle (SRP) binds to the ribosome, and synthesis is
stopped. (4): SRP binds the ribosome to the receptor present in the ER membrane. (5) and (6): The ribosome binds to the Sec61/SecY complex, pep-
tide synthesis restarts, and it crosses to the ER lumen through a pore. (7): As the peptide elongates, it becomes glycosylated (the scheme represents
N-glycosylation only). (8): Synthesis of the peptide is terminated, the signal peptide is excised, and later it is digested.
process occurs mostly with proteins with a limited level of hydrophobicity, and the mecha-
nism differs between eukaryotic and prokaryotic cells (see review by Rapoport 2007). In
eukaryotes, the process involves, besides the translocation channel described above, the
Sec62/Sec63 complex made by proteins Sec62, Sec63, Sec71, and Sec72, plus the chaperone
BiP, which belongs to the HsP70 family and is localized in the lumen of the ER. In eukary-
otic cells, the process starts by the binding of the unfolded protein to the translocation
channel and its initial penetration to the lumen where it binds BiP in its open stage (ATP-
bound). Interaction of the chaperone with Sec63 causes rapid ATP hydrolysis and closure
of the binding pocket of the chaperone. Continuation of the transfer of the polypeptide
through the translocation channel exposes new segments to other BiP molecules that bind
to the polypeptide following the same mechanism described for the first BiP molecule.
The process is repeated until the whole protein is translocated to the ER lumen. Finally,
adenosine diphosphate (ADP) bound to BiP molecules is exchanged by ATP causing the
liberation of the polypeptide.
8.4 The importance of protein glycosylation in translocation

As previously described, it is a general rule that secreted proteins are glycoproteins, and
evidence was cited that protein glycosylation occurs cotranslationally. Glycosylation of
proteins occurs in serine or threonine residues (O-glycosylation) and in asparagine resi-
dues (N-glycosylation) of the proteins (see Chapter 7 on proteins). Glycosylation of proteins
is important because the carbohydrate moiety helps the protein to reach its final structure,
and protects it from the harsh conditions existing in the environment, mostly changes in
pH, high temperature, dehydration, and proteolysis. Nevertheless, the correct glycosyl-
ation of the proteins is of outmost importance for their secretion. Riederer and Hinnen
(1991) demonstrated that yeast phosphatase should be N-glycosylated in order to be effi-
ciently secreted. These authors proceeded to mutagenize the different sequons present in
the enzyme, and analyzed the phenotype of the corresponding mutants. They observed
that only strains where at most five of the 10 sequons had been mutagenized were able to
secrete the phosphatase, but with an efficiency inversely proportional to the number of
mutations, and always decreased when compared to the wild-type strain. Inhibition of
secretion was not only dependent on the number, but as expected, also on the position of
the mutated sequons: when mutations were located at the N-terminus, secretion was more
sensitive than when mutations were present at the C-terminus. This decrease in secretion
efficiency was independent of the synthesizing capacity of the mutants, and was due to
retention of the underglycolylated phosphatase molecules in the ER. These data are evi-
dence that protein glycosylation serves to monitor the protein folding in the ER.
As described in the previous chapter, N-glycosylation of the proteins in the ER
involves the transfer of an oligosaccharide of the composition –(GlcNAc)2(Man)9(Glc)3,
from dolichol pyrophosphate to an Asn residue of the protein. Synthesis of the donor
lipid-oligosaccharide occurs with interesting topographic changes at the surface of the
ER membrane (see Hirschberg and Snyder 1987), as demonstrated by different strategies
including different sensitivity to nonpermeable inhibitors, effect of proteases, and use of
nonpermeating inhibitors. According to the results obtained, it was concluded that syn-
thesis of Dol-P-P-GlacNAc, Dol-P-Man, Dol-P-, and Dol-P-(GlcNc)2(Man)5 took place at the
cytoplasmic face of the ER membrane, whereas further steps in the synthesis up to the
final oligosaccharide, occurred in the luminal face of the ER. It appears that the nucleotide
sugars involved in this stage of the synthesis, with the exception of GDPMan are trans-
ported across the ER membrane by an active mechanism (Perez and Hirschberg 1985;
1986). Regarding O-mannosylation of glycoproteins, it was concluded that transfer of the

first mannosyl unit, a reaction, which utilizes Dol-P-Man as sugar donor, as well as further
mannose additions using GDP-Man as substrate, all occur in the ER (Larriba et al. 1976;
Zueco, Mormeneo, and Sentandreu 1986).
Once the secreted proteins are synthesized, they are transferred to the Golgi complex.
However, in order that this step is accomplished, proteins must pass a strict inspection
of its structure. This is a process involving the selection of proteins, which leave the ER
in the direction of the Golgi complex, by a process denominated quality control (QC) (see
review by Trombeta and Parodi 2003). Functions of QC have been summarized as follows:
(i) to prevent the deployment of aberrant proteins, (ii) to retain protein precursors in an
environment suitable for their correct folding, (iii) to favor the correct assembly of proteins
made by more than one polypetide by increasing their concentrations, (iv) to reduce the
problems caused by ill folded or assembled proteins, (v) to maintain the homeostasis in the
early secretory system, (vi) to regulate protein secretion, and (vii) to store proteins for their
regulated secretion. Quality control is exerted on proteins that have not attained their
correct folding, that failed to bind different ligands, or that lack posttranslational modi-
fications such as signal sequence cleavage, disulfide bond formation, N-glycosylation, or
binding of the GPI moiety. Among the members involved in QC, we may cite chaperones
such as BiP and Hsp47, protein disulfide isomerase (PDI), and other members of the thio-
redoxin family, which are involved in the establishment of S-S bonds, and UDPglucose
glycoprotein glucosyl transferase (transglucosylase) whose role is of great importance in
this phenomenon. It is known that after transfer of the oligosaccharide to the glycoproteins
(N-glycosylation), this is processed in the lumen of the ER by the removal of the three glu-
cose and one mannose residues (see Chapter 7, Figure 7.3). This trimming is followed by
the addition of a further glucose unit by the ER resident UDPglucose glycoprotein glucosyl
transferase. This process, which at first sight appears odd and of obscure importance, was
later found to play an extremely important function in the selection of the glycoprotein
molecules, which are transported from the ER to the Golgi complex. In this process, a fun-
damental role is played by two lectins, one soluble, calreticulin, and the other membrane
bound, calnexin. The proteins that have been glucosylated are recognized by the lectins; if
correctly folded, they are deglycosylated by glucosidase II, and are transferred to the Golgi
complex. However, in contrast, incorrectly folded proteins are unable to cross to Golgi
(Helenius et al. 1997). This rejection has two consequences; the proteins are retained in the
ER or are degraded. In the first case, the proteins are maintained in the ER under condi-
tions that favor their correct folding, the glucose molecule is further eliminated by glu-
cosidase II, and the glucosylation process is repeated; but if the correct folding cannot be
accomplished, the proteins are degraded. In yeasts, this degradation process may occur in
the ER, in vacuoles, or in the cytosol by the proteasome after ubiquitination (for a review,
see Vitale and Denecke 1999). Binding of calreticulin and calnexin also has the property of
exposing the proteins to the action of thiol oxidoreductase Erp77 (a PDI homologue) facili-
tating the formation of the correct disulfide bonds. As indicated above, if after repeated
cycles of glucosylation and deglucosylation, and exposure to the chaperones, a protein
does not reach its correct folding, it is degraded by a process denominated ER-associated
degradation (ERAD). The process involves transfer of the protein to the translocon, trans-
port back to the cytosol, and as indicated above, ubiquitination, and degradation by the
proteasome (see Tsai, Ye, and Rapoport 2002). One problem associated with the transfer
of the missfolded protein through the translocon is that if it is partially folded, how can it
traverse through the membrane channel back to the cytoplasm? Apparently, this transfer
is facilitated by the action of chaperones BiP, PDI, and ERP57. Degradation is facilitated
by the elimination of the N-linked oligosaccharide by a N-glycanase, the oligosaccharide

being then further hydrolyzed by a chitobiase and mannosidases. When some proteins
not having a clearly abnormal configuration pass to the Golgi, they may be further recog-
nized during their further glycosylation and are conveyed to the lysosomes where they are
degraded. See Figure 8.2 for a schematic representation of QC operation.
Accumulation of missfolded proteins in the ER induces a signal transduction pathway
denominated unfolded protein response (UPR). UPR leads to the upregulation of chaper-
ones and foldases, which accumulate in the ER. In the case of Saccharomyces cerevisiae, the
first sensor of the stress is a membrane-bound Ser/Thr kinase with an RNAase domain
denominated Ire1. Accumulation of unfolded proteins in the ER induces oligomerization
of the kinase, a reaction stimulated by the action of the chaperones Kar2/Bip. Inactivation
of Ire1 is brought about by the protein phosphatase Ptc2. It has been demonstrated that
the active form of Ire1 causes the splicing of HCA1 mRNA producing the mature and
active form of this transcriptional factor, which activates the expression of the compo-
nents responsible for UPR.
8.5 Transfer of proteins along the endomembrane system

Once proteins are approved in the ER by the QC system, they initiate their transit to their
final destination utilizing the endomembrane system, whose first component is the Golgi
complex. From there they are distributed to their different target organelles: vacuoles or
lysosomes, storage granules, mitochondria, or chloroplasts by a process denominated pro-
tein targeting, which, as previously indicated, depends on the topogenic signals possessed
by the different proteins. Since this volume is a review on the fungal cell wall, I will concen-
trate my discussion on the mechanisms that finally lead to secretion of the proteins; those
that may become associated with the plasma membrane; those kept in the so-called peri-
plasmic space, secreted to the medium; and, those bona fide bound to the wall by means of
covalent or noncovalent bonds. The animal cell was the system through which the mecha-
nism of protein secretion was originally established at the level of the steps followed by pro-
teins in their transit, masterly developed by the Palade group (see Siekevitz and Palade 1960;
Palade 1975). Fungi, represented by S. cerevisiae entered into scene later, mostly through the
investigations of Randy Schekman and his collaborators (see reviews by Novick, Ferro, and
Schekman 1981; Novick, Field, and Schekman 1980; Schekman 1985). These authors isolated
a great number of mutants denominated sec, which identified the components involved in
the several steps of protein secretion. In their work, they proceeded to isolate thermosensi-
tive mutants able to grow at the permissive temperature of 28oC, but unable to do so at the
restrictive temperature of 37oC, on the grounds that the process of protein secretion was
vital for the cells, and on the observation that mutants suffered an increase in buoyant den-
sity during a period of 1–3 hours after a shift to the restrictive temperature. Accordingly,
the mutagenized cells were subjected to density centrifugation in Ludol, searching for the
heaviest cells (Novick, Field, and Schekman 1980). In this way, the authors were initially
able to isolate mutants, which defined 23 SEC genes. Phenotypic characterization of the
mutants was mainly done by electron microscopic observations, demonstrating that there
were three classes according to the types of membranous organelles that they accumulated:
vesicles (of two sizes), ER, and Golgi stacks. Epistatic analysis of double mutants led the
authors to conclude that the secretory process in S. cerevisiae followed the same pattern
described by Palade in animal cells (Novick, Ferro, and Schekman 1981), basically:
ER → Golgi → Secretory Vesicles → External Medium

CYTOSOL
130
ER
GLUCOSIDASES I and II
UDP
CNX/CRT
CYCLE
Fungal cell wall: Structure, synthesis, and assembly

UGGT1 CHAPERONS
GLUCOSIDASE II
MANNOSIDASE I
UDPG
SPECIFIC
LECTINS
BULK FLOW
TO GOLGI OR TRANSPORTERS
ERAD
Figure 8.2 Scheme for the quality control (QC) operation for the correct synthesis of secretory glycoproteins. Once in the lumen of the ER, the
terminated proteins reach their proper folding by the assistance of chaperones, and their N-glycan maturation by the action of glucosidases I and
II, and mannosidase I, and follow their transit to the Golgi complex. If folding is incorrect, they are glucosylated again by the UDP-glucose: protein
glucosyl transferase (UGGT1) to reinitiate the folding process again. If this fails, the proteins bind to specific lectins and are taken to the proteasome
to be degraded by the ERAD (ER associated degradation) process.
Later, it was observed that transfer from ER to Golgi also involved vesicles as vehicles
for the proteins, and that special membranous organelles originally described as Berkeley
bodies represented accumulation of misfolded proteins in vacuoles. The next (and more
laborious) aspects of these studies involved the determination of the function of the SEC
gene products in the secretory process.
8.5.1 From ER to Golgi

As indicated above, proteins that have acquired their proper folding and are substrates
for the secretory pathway become separated from the proteins that are normal ER resi-
dents (see above). It has been described that there are some particular exit sites, known
as transitional ER distributed over the surface of the ER. These are organized membrane
domains that appear to close tubular clusters denominated pre-Golgi intermediates, vesicular-
tubular clusters (VTCs), or the ER-Golgi-intermediate compartment (ERGIC) (Hauri et al. 2000).
In these sites, multiple budding of vesicles may be observed, representing the devise of
protein mobilization to the Golgi complex, although the possibility of existence of conti-
nuity between the ER and Golgi for protein transfer cannot be completely dismissed. To
explain the transfer by vesicles between both compartments, two mechanisms have been
proposed: active transport and bulk-flow. In the case of active transport, the presence of
specific receptor molecules that bind cargo proteins with the formation of a vesicle coat has
been entertained. The proteins that normally reside in the ER do not bind to the receptors.
These proteins are recognized for having a specific motif in the form of a tetrapetide pres-
ent at the C-terminus of the proteins: KDEL and HDEL. In the case of bulk-flow, it is sug-
gested that vesicles are spontaneously formed by budding. These vesicles would have some
membrane-spanning proteins interacting with the vesicle coat. During budding all proteins
in the vicinity would be trapped, including ER resident proteins also. These ER resident
proteins possibly would be brought back to ER from the Golgi by a retrograde movement
utilizing as a retrieve mechanism the above-mentioned topogenic signals that would be
recognized by specific receptors in the Golgi (Pelham 1988). The specific receptor ERD2
was originally identified in S. cerevisiae (Lewis and Pelham 1992), and later, a homologue
was described in humans and in other organisms. Originally, the bulk-flow model was
preferred for its simplicity, for the results obtained using as cargo protein a small peptide
without an ER retention signal (Wieland et al. 1987), and for the observation that removal of
the ER retention signal from BiP made the protein enter into the secretory pathway (Munro
and Pelham 1987). However, further work demonstrated that the bulk-flow model cannot
explain a series of experimental pieces of evidence. Now it is considered that both pathways
are operative in the cell (for reviews on the subject see Vitale and Denecke 1999). This rather
simple image may be more complicated than had been anticipated. Thus, in an investiga-
tion of the secretory processes in the dimorphic fungus Yarrowia lipolytica with the use of
specific mutants in the genes involved in secretion, Titorenko, Ogrydziak, and Rachubinski
(1997) obtained evidence suggesting the existence of four distinct mechanisms for protein
secretion. According to these authors, one pathway diverging from the main route at the
ER level was involved in cell surface growth, and two further pathways diverged from the
main route at the level of the Golgi complex, one involved in the specific surface growth
dependent on the dimorphic transition, and the other one involved in peroxisome biogen-
esis. In this sense, it may be opportune to recall that solid evidence has been presented that
chitin synthases, and perhaps the enzymatic system involved in β−1,3-glucan synthesis are
transported in the cell by an unorthodox mechanism (see Chapters 4 and 6, this volume;
Riquelme et al. 2007; Verdin, Bartnicki-García, and Riquelme 2009).
The mechanism of protein transport by means of vesicles from ER to Golgi is a varia-

tion of the same theme of vesicle organization in the donor membrane and binding to the
receptor membrane; a model found to be conserved in human cells and yeasts, and by
extension, in all eukaryotes. In a few words, it has been demonstrated that vesicles are
formed in a donor membrane by its association with coat proteins (COPs), which cover the
vesicle. These vesicles are transported to the receptor membrane, and bind to it through
the recognition of specific protein receptors (SNAREs). Upon docking, the cage formed by
the COP proteins becomes disorganized, the membranes from the vesicles and the target
compartment fuse, and the cargo proteins are discharged inside this latter compartment.
The specific details of this process, also known as the SNARE system were worked
out by studies in animal cells and yeast, and summarized by Rothman (1994), using a sys-
tem of transfer in Golgi stacks of vesicles containing the vesicular stomatitis virus (VSV),
which becomes modified by GlcNAc addition (VSVG) in the Golgi complex. According
to this concept, proteins are transported across the membrane system in vesicles with a
diameter of about 100 nm covered by a characteristic coating. Maturation of these vesicles
was found to be selectively inhibited by the GTP nonhydrolyzable analog GTP-γS and
the S-alkylating compound N-ethylmaleimide (NEM). The order of inhibitor effect indi-
cated that GTP hydrolysis preceded the NEM-sensitive step with accumulation of coated
vesicles, whereas NEM addition gave rise to uncoated vesicles. It was hypothesized that
GTP was necessary for the binding of proteins that covered the vesicles (coating proteins
or coatomers), and that these had to be removed to allow vesicle fusion to the target mem-
brane. Analysis of COPs revealed the presence of eight proteins, one an ADP-ribosylation
factor (ARF), and the rest structural proteins. Taking into consideration that ARF in its
soluble forms binds guanosine diphosphate (GDP), but when bound to GTP is membrane-
bound in a reaction dependent on myristic acid, led to a model suggesting that the asso-
ciation of ARF in the GTP form by the action of a GDP-GTP exchanger, served to recruit
the COP proteins thus inducing vesicle budding. As indicated above, removal of the coat
is a step necessary for vesicle fusion. The process would involve exchange of ATP by ADP,
elimination of the myristoyl residue causing release of ARF, and disorganization of the
coatomer structure. As indicated above, in the presence of NEM, naked vesicles accumu-
late in the trans-Golgi membrane, indicating that the inhibitor blocked membrane fusion
by a reaction with a NEM-sensitive fusion protein (NSF) that binds ATP and has a low
ATPase activity. This protein is equivalent to yeast Sec18, providing further evidence of
the conservation of the secretory process. Binding of NSF to the acceptor Golgi membrane
requires specific factors, three of which have been identified (α, β, γ), purified and named
soluble NSF attachment proteins (SNAPs). While α and γ SNAPS are widely distributed, β
SNAP appears to be specific to the brain. Sec17 is the yeast homologue of α SNAP. SNAP
binds to the membrane before ARF, recognizing a receptor denominated SNARE. After
binding, NSF is released through ATP hydrolysis, and this step is followed by the elimina-
tion of SNAP, and occurrence of membrane fusion. It was anticipated that the specificity
of membrane fusion was given by specific SNAREs present in the vesicle and in the tar-
get membrane, whose recognition allowed specifically directing the vesicles to the correct
acceptor membrane: SNAREs localized in distinct membrane compartments, which are
competent to form complexes only with appropriate cognate SNAREs, providing a lock-
and-key specificity to membrane fusion.
This hypothesis was confirmed by the analysis of membrane fusion occurring in brain
particulate extracts, showing that after release of NSF, three proteins forming complexes
with SNAP were released. These proteins were recovered and purified. The putative
SNAREs present in the brain synaptic membranes were those denominated syntaxin
(HPC-1) and SNAP-25, localized in the presynaptic membrane, whereas the other one, a
vesicle associated membrane protein (VAMP) or synaptobrevin was associated with the ves-
icle membrane. The first two correspond to what is known as target SNAREs (tSNAREs),
and the last one to a vesicular SNARE, (vSNAREs). The generality of this mechanism in
protein trafficking and targeting in the cell has since been demonstrated in all eukary-
otic cells analyzed thus far, where a number of SNAREs have been identified. Sequence
alignment of many SNAREs has revealed the relative conservation of several motifs. Thus,
their central part reveals an ionic nature, which may be critical for complex formation.
Glutamine residues are highly conserved in this ionic central layer in relatives of SNAP-
25 and syntaxin 1A, and arginine residues are conserved at the corresponding positions
in VAMP homologues. This difference has led to a change in the taxonomy of SNAREs,
naming as R-SNAREs those having arginine, and Q-SNAREs the ones having a glutamine
at the critical site (Fasshauer et al. 1998). The Rothman group made further experiments
to analyze whether the sole interaction of the cognate SNAREs was enough to permit the
fusion of the membranes. In their experiments, they used artificial membranes under
different conditions containing only the corresponding SNAREs and demonstrated that
membrane fusion was indeed specific, although the rate of fusion was well below the one
occurring in vivo. These results have led to the consideration that other components of the
fusion machinery are involved in the process. The number of proteins that are able to bind
to SNAREs forms a large list, and some of them have been shown to be required for or to
facilitate the action of SNAREs in membrane binding. Accordingly, they might be required
in vivo for the fusion process. Among the most studied ones, we may cite SM proteins,
which are known to regulate membrane fusion under some conditions, synaptogamins,
which are able to bind Ca++ and phospholipids, and complexins, which are involved in
membrane fusion in the synapse (see Duman and Forte 2003, for a review).
The mechanism of transfer of proteins from the ER to Golgi has been reviewed by
Lippincoat-Schwartz, Roberts, and Hirschberg (2000). It was shown that according to the
SNARE hypothesis, the first indication that vesicles will be formed at the ER exit sites is a
change in the structure of the membrane by activity of the GTPase Sar1, a member of the Rab
family of small GTPases. It is now known that Rab proteins exist in a large number, about 10
in yeast and 60 in mammal systems (Pereira-Leal and Seabra 2001). Their role in their active
state (GTP-bound) is to recruit a number of proteins involved in vesicle formation. Their
membrane attachment is regulated by geranylgeraniol residues joined at their C-termini.
At a further stage, binding of the coatomer proteins (COPI and COPII) occurs. These
are heterodimers made of Sec23/Sec24 and Sec13/Sec31 proteins, which become assem-
bled with the help of Sec12, a GTP-GDP exchange factor acting on Sar1. It has been sug-
gested that COPIs are involved in both anterograde and retrogade vesicle transport, and
COPII in anterograde displacements only. It has been observed using GFP-labeled proteins
that the vesicles are transferred to the pre-Golgi intermediates, which move to the cis face
of the Golgi complex, probably being the precursors of Golgi components. As anticipated
by the SNARE hypothesis, before membrane fusion occurs, the coatomers are removed
from the vesicles by hydrolysis of the ATP bound to Sar1, a reaction catalyzed by Sec23
(Yoshihisa, Barlowe, and Schekman 1993). Fusion of the vesicles derived from ER with the
cis Golgi, is controlled by one of the best-characterized SNARE complexes made by Bet1,
ERS24, membrin, and syntaxin 5. It was shown that in general vSNAREs and tSNAREs
became accumulated at the rim of the Golgi stacks, whereas resident proteins, such as
galactosyl transferase were accumulated at the center of the stacks (Cosson et al. 2005). It
was speculated that these results were related to the behavior of the proteins present in the
Golgi, since resident proteins are retained in a passive way, whereas SNAREs, which are
continuously coming in and out of Golgi are temporarily retained by a dynamic equilib-
rium of the transport mechanism. The export of membrane proteins is regulated by spe-
cific sequences recognized by COPIII, one of them located at the C-terminus constituted by
aromatic and hydrophobic amino acids (Iodice, Sarnataro, and Bonatti 2001), and the other
by the internal sequence Glu-X-Asp (Nishimura and Balch 1997). Other proteins require
protein adaptors such as Erv14 in order to be transported. Soluble proteins are transported
as mentioned above by bulk-flow and active transport.
8.5.2 Out from Golgi

The Golgi complex has an extremely important role in targeting proteins to the differ-
ent cell compartments, including the cell surface in the case of plasma membrane and
extracellular proteins. Additionally, the Golgi complex is a place where membrane differ-
entiation takes place. In a classical paper, Grove, Bracker, and Morré (1968) made a careful
determination of the structure and width of the Golgi membranes in the fungus-relative
Oomycete Pythium ultimum, demonstrating that cis-Golgi membranes were thinner and
with staining characteristics different to those present in trans-Golgi. This change was
not abrupt but occurred slowly going along the Golgi stacks, from 3.7 to 9.4 nm when fixed
with glutaraldehyde, osmium tetroxide, and permanganate. The relative proportion of
proteins and their glycosylation, and the changes in lipid composition may be responsible
for these changes in membrane width. Similar observations have been made with Golgi
complex from plant, fungal and animal cells.
It is important to point out that the Golgi complex has different morphologies depend-
ing on the organisms, but they are typically made of stacks of flat cisternae, alternating
with regions rich in vesicles and tubules. The number and complexity of the cisternae
is also variable and appears to be directly related to the secretory capacity of the cells. It
has been demonstrated that the Golgi complex occupies a central position in the secretory
pathway. The role of the Golgi apparatus is to mature different lipids and proteins, for
example, terminate the glycosylation of proteins, as they move through the stacks to the
trans Golgi where they are packaged into vesicles, and targeted to different cell compart-
ments or to the exterior, according to their topogenic signals. Budding of vesicles carrying
proteins directed to the exterior of the cell from trans Golgi has been observed labeling
the proteins with fluorescent markers such as green fluorescent protein (GFP). Movement
of the proteins along the different Golgi stacks involves formation and fusion of vesicles.
Several SNAREs involved in membrane recognition during this transit have been recog-
nized, among these Gs15, Gos28, syntaxin 5, and Ykt6 may be cited.
It has been observed that transit of the proteins out of Golgi, initially involves the
formation of tubules, which later pinch off producing vesicles that move to the cell surface
where they fuse with the plasma membrane through the interaction of the corresponding
SNAREs described above. In the case of yeast, three SNAREs have been identified corre-
sponding to the neuronal SNAREs Vamp-2, Snap-25, and syntaxin (see above): Snc2, Sec9,
and Ssop respectively. In both cases, during vesicle fusion, SNAREs form a bundle made of
four twisted helices with grooves and parallel with their C-termini directed to the recep-
tor membrane, one of the SNAREs corresponding to the vesicular membrane (vSNARE),
whereas the other three are present in the target membrane (tSNARE). The formation of
the tetrameric SNARE complex brings the two membranes together and allows the spe-
cific fusion of the vesicle with the compartment containing the corresponding tSNAREs.
Apparently, the grooves in the bundle offer binding sites for attachment of other proteins
involved in the process. After attachment of vesicles to the plasma membrane, vesicle fusion
occurs, with the release of their cargo to the medium (the cell wall in the case of fungi),
whereas the membrane-bound proteins remain associated to the plasma membrane.
8.6 The secretion mechanism in filamentous fungi

The concept that hypha grow at the apex (see Bartnicki-García and Lippman 1969) led to
the idea that protein secretion should occur in filamentous fungi also at the apical zone.
This hypothesis was verified originally in Aspergillus niger by Wösten et al. (1991). These
authors utilized an ingenious method to grow the fungus over perforated membranes
and they determined growth and glucoamylase secretion simultaneously. Initially, they
observed that both processes were restricted to the periphery of the colonies. Glucoamylase
secretion exclusively at the hyphal tip was confirmed by immunogold labeling. These data
were confirmed later by use of a strain of A. niger carrying a fusion of the genes encoding
glucoamylase and the green fluorescent protein (GFP-glucoamylase) (Gordon et al. 2000).
Similar data have been reported in Trichoderma reesei. Although the secretory process in fil-
amentous fungi is not as well known as the yeast system is, the data obtained with differ-
ent systems suggest that it follows the basic principles described in S. cerevisiae (see above).
Thus, genes encoding different members of the secretory pathway have been isolated from
several filamentous fungi, among them the homologue of gene SAR1 involved in the trans-
fer of vesicles from ER to Golgi; that was isolated from A. niger and T. reesei (Veldhuisen
et al. 1997), several genes encoding Rab proteins from A. niger (Punt et al. 2001), and the
gene encoding the tSNARE Yup1 from Ustilago maydis, which appears to be localized in
the late section of the secretory pathway (Wedlich-Söldner et al. 2000). With the advent of
sequenced genomes, it has been possible to make in silico searches to determine the pres-
ence of members of the secretory pathway in a number of fungi. These analyses have led to
the identification of Rab and SNARE homologues, a result that is evidence of the conserva-
tion of the pathway in filamentous fungi.
8.7 The Spitzenkörper

In the case of filamentous fungi, a special organelle called the Spitzenkörper appears to
be the final station in the intracellular transit of secretory vesicles, before they fuse with
the plasmalema. The name Spitzenkörper (apical body) was given by Brunswick (1924)
to a basophilic body that stained intensely with iron-hematoxylin in the apical region
of Coprinus starquilinus and Coprinus narcoticus. The author hypothesized that this body
was somehow involved in cell growth, although no evidence for it was obtained at that
time. Later, by use of electron microscopy, an apical body was found in a number of spe-
cies belonging to Ascomycota and Basidiomycota, which corresponded to the corpuscle
identified by Brunswick (McClure, Park, and Robinson 1968), and a detailed study of the
structure was made by Grove and Bracker (1970) in the hyphae of species belonging to
the different fungal phyla. The experiments by Girbardt (1955, 1957, 1969) in the last cen-
tury were the ones that demonstrated the role of Spitzenkörper in the apical growth of
fungi, and its structure. Girbardt utilized for his experiments phase contrast microscopy,
time-lapse photomicrography, and electron microscopy. His classical time-lapse photomi-
crographic studies were particularly illustrative showing how changes in the relative posi-
tion of the Spitzenkörper at the hyphal apex preceded changes in the direction of growth,
and how the sudden disappearance of the corpuscle by a strong illumination brought
about a halt in hyphal elongation, which was only reassumed after formation of a new
Spitzenkörper, characteristically in a different position than the previous one. These data
Figure 8.3 Stratification of the Spitzenkörper in a hyphal apex of N. crassa by laser scanning confo-
cal microscopy. Left panel: GS-1-GFP localized at the outer layer of the Spitzenkörper (Spitzenring).
Center panel: Chs1-mChFP localized at the core of the Spitzenkörper. Right panel: Merged image.
Scale bar: 10 µm. (Courtesy of E. Sánchez-León, J. Verdín, and M. Riquelme.) (See color insert.)
were later confirmed in experiments with video microscopy and image analysis of manip-
ulation of the Spitzenkörper with laser tweezers (Bracker, Murphy, and López-Franco 1997).
Regarding the structure of the Spitzenkörper, by analysis of a series of sections of the hyhal
apex, Girbardt (1969) obtained a tridimensional image of the Spitzenkörper that appeared
as a body made by the accumulation of vesicles of two sizes with other structures similar
to microfilaments. Further work has shown that the Spitzenkörper appears as a zone local-
ized normally in the central part of the hyphal apex containing secretory vesicles mea-
suring 100–400 nm, microvesicles of a diameter of 30–100 nm, which were identified as
chitosomes (Riquelme et al. 2007), actin filaments, and microtubules (see Bartnicki-García
2003). According to Verdin, Bartnicki-García, and Riquelme (2009), the Spitzenkörper dis-
plays a functional stratification with the accumulation of microvesicles (chitosomes) in the
central part of the organelle, and an external zone containing a different type of vesicles
that was named the Spitzenring (see Figure 8.3).
It has been hypothesized that the Spitzenkörper targets secretory vesicles and
microvesicles to the cell surface. The strongest evidence of this role involves experiments
from Bartnicki-García, Hergert, and Gierz (1989a; 1989b), who devised a computer simu-
lation program to analyze hyphal growth. Their data suggested that the hyphal shape
is attained by the random fusion with the plasma-lemma of vesicles and microvesicles
that originated from a specific region present in the apical zone of the hyphae, which the
authors designated as vesicle supply center (VSC). Most important was the observation
that the position of VSC in the hypha corresponded to the location of the Spitzenkörper
(Bartnicki-García, Hergert, and Gierz 1989a; 1989b; Bartnicki-García 2003) (see Chapter 9,
Figure 9.3). These data have given rise to the hypothesis that vesicles and microvesicles
originating from trans Golgi or other membranous structures, move to the apical zone,
probably transported by microfilaments, and accumulate forming, with other cytoskeletal
components, and some ill known proteins the Spitzenkörper, from where they are tar-
geted to the plasma membrane. Another hypothesis suggests that the Spitzenkörper may
be involved in the maturation of vesicles before being directed to the cell surface.
8.8 Vesicles and chitosomes in fungal cell wall growth

As described above, fungi are not an exception of the general mechanism that involves
vesicles as the conveyors of extracellular and plasma membrane-bound proteins. It has
been demonstrated that the glycoproteins, which form the cell wall are secreted by ves-
icles, and that the transport of at least the enzyme responsible for the synthesis of the
main structural polysaccharide, chitin, reaches the surface inside a specialized type of
microvesicles, which were named as chitosomes by Bracker, Ruiz-Herrera, and Bartnicki-
García. (1976) (see Ruiz-Herrera and Ruiz-Medrano 2004). This was done on the basis that
they have no other activity but chitin synthase only and are devoid of the enzymatic mark-
ers characteristic of vacuoles, ER, mitochondria, plasmalemma, or secretory vesicles; and,
in opposition, that no other intracellular structure smaller than chitosomes contains chi-
tin synthase activity (see reviews on chitosomes in Bartnicki-García, Ruiz-Herrera, and
Bracker 1979; Ruiz-Herrera 1984; Ruiz-Herrera 1992; Ruiz-Herrera and Martinez-Espinoza
1999) (see Figure 8.4).
Chitosomes were originally isolated from yeast cells of Mucor rouxii, but later they
have been found in all fungal species analyzed thus far (for example, see Barnicki-García
et al. 1978). Chitosomes from M. rouxii contain more than 80% of the total chitin synthase
present in cell-free extracts, although this value may be an underestimate, since chito-
somes have the tendency to bind to larger organelles. Important were the observations
that, although the initial breakage method used to isolate chitosomes was very harsh, use
later of milder breakage methods, including cytoplasmic extraction with a microsyringe or
squeezing sporangiophores of Phycomyces blakesleeanus (Herrera-Estrella et al. 1982) gave
essentially the same results. It was even possible to isolate chitosomes from the wall-less
N. crassa slime variant that surprisingly is unable to synthesize chitin in vivo, but contains
active chitin synthase in vitro (Bartnicki-García et al. 1984; Ruiz-Herrera et al. 1987).
As was described in Chapter 4 devoted to chitin, chitosomes have been thoroughly
analyzed, and found to display unique chemical and physical properties (Ruiz-Herrera
1992; Ruiz-Herrera and Ruiz-Medrano 2004). Chitosomes have a diameter of about 40–70
nm, a density of ca. 1.12–1.14, and a sedimentation coefficient of 95–105 depending on the
fungal species. By negative staining they appear mostly spheroidal and with a character-
istic central depression. In sections, chitosomes are shown to possess a very thin double
membrane 6.5–7.0 nm thick that stains poorly by the usual methods utilized in electron
microscopy. Chitosomes are made of two parts protein of a unique composition, and one
part lipids, with a composition different from the rest of the cell membranes. Analyses of
polypeptides in purified preparations of chitosomes gave peculiar results, since they were
found to contain only low Mr polypeptides, well below the theoretical size of the active
polypeptides expected from the sequence of genes encoding chitin synthases, which are
normally above 100 kDa. Thus, chitosomes from M. rouxii displayed in gel electrophoresis
bands with Mr of 16.5, 18, 28, 30, 44, 47, and 55 kDa, which was the most abundant (Flores-
Martínez et al. 1990). Similarly, absence of large polypeptides whose Mr did not exceed 50
kDa were observed when 16S subunits (obtained by digiton solubilization of chitosomes,
see below) from M. rouxii were analyzed (Lending et al. 1991). In the case of chitosomes
from the slime variant of N. crassa, four major polypeptides with Mr of 22, 30, 38, and 53
kDa were separated by polyacrylamide gel electrophoresis. Interestingly, after trypsin pro-
teolysis, a treatment known to activate chitin synthase (see Chapter 4 on chitin), the 38 kDa
protein disappeared with the appearance of two new polypeptides with molecular masses
of 18 and 21 kDa. That this result is indicative of the activation process of chitin synthase
seems unlikely, taking into consideration the low Mr of all these peptides. Regarding lipid
composition, the most abundant neutral lipids in chitosomes from M. rouxii are sterols,
and the most abundant polar lipids are phosphatidyl choline and phosphatidyl ethanol-
amine; the levels of glycolipids are also high, but they do not contain phosphatidylserine
(Hernández et al. 1981). The lipid composition from chitosomes from Agaricus bisporus is
A
Figure 8.4 Electron microscopy of vesicles and microvesicles in the apical region of a stage I
Phycomyces blakesleeanus sporangiophore. (A) Osmium ferricyanide staining. (B) Thiery stain-
ing. Arrowheads: vesicles; Arrows: microvesicles. Notice their difference in diameter and stain-
ing. Magnification bar: 200 nm. (Modified from Morales, M. and Ruiz-Herrera, J., 1993, Critical
Evaluation of the Ultrastructure of Sporangiophores from Phycomyces blakesleeanus [Zygomycetes] at
Stages I and IV, Cryptogamic Botany 3:273–282.)
Table 8.1 Properties of Chitosomes

Type of property Parameter Characteristics
Physical Location Intracellular
Size 40–70 nm in diameter
Mr ca. 5 × 103 kDa
Sedimentation coefficient 95–105 S
Specific gravity 1.125–1.145 g/cm3
Chemical Protein content 58%–66%
Polypeptide composition Unique, polypeptides with Mr 20–66 kDa
Lipid content 33%–42%
Characteristics of lipid Distinct from membranes
Biochemical Enzymatic activities Only chitin synthase
Activation Normally requires controlled proteolysis
Biological Distribution Fungi
Function Chitin synthesis
Source: Slightly modified from Ruiz-Herrera, J., 1992, Fungal Cell Wall: Structure, Synthesis, and
Assembly, Boca Raton, FL: CRC Press. With permission from Taylor & Francis.
slightly different from the one of M. rouxii (Weete et al. 1985), a result that is not strange,
taking into consideration that one species is a Basidiomycota and the other a Zygomycota,
respectively (see Table 8.1).
When incubated in vitro with the chitin synthase substrate, UDPGlcNAc, and activators
(a protease and GlcNAc), chitosomes synthesize chitin microfibrils essentially identically
to those existing in the fungal cell wall. This analysis, a rare example of a process where
the biochemical and structural events of a single biosynthetic reaction could be followed
simultaneously, occurs very fast, and in a matter of seconds, the appearance of delicate
microfibrils inside chitosomes could be observed by negative staining under the electron
microscope. At a later period, thick, coiled microfibrils were formed into the chitosomes,
and in a more advanced stage, microfibrils broke the chitosomal membrane giving rise to
the appearance of long, straight or kinked chitin microfibrils (Bracker, Ruiz-Herrera, and
Bartnicki-García 1976). Chitosomes could be dissociated by treatment with digitonin into
500 kDa (16 S) subunits, which retained enzymatic activity and reassembled into chitosome-
like aggregates when digitonin was removed (Ruiz-Herrera, Bartnicki-García, and Bracker
1980; Ruiz-Herrera 1984). These subunits gave rise to the formation of chitin microfibrils,
but these appeared much shorter and robust than those synthesized by intact chitosomes.
Regarding the origin of chitosomes and their whereabouts in the cell, most studies
have referred to the mechanisms through which chitin synthases are intracellularly mobi-
lized, probably because of the difficulties in identifying chitosomes in vivo. In these studies,
tagging of Chs with commercial epitopes or fluorescent proteins by directed mutagen-
esis has been extremely useful. Use of antibodies directed against Chsps has been rarer,
but the data obtained with them are extremely useful because they eliminate the pos-
sibility of watching artifacts caused by abnormal behavior of the mutagenized proteins.
Studies with S. cerevisiae led to the identification of a number of genes, whose products
were described as being involved in the intracellular transport of chitin synthases, mainly
Chs3. These genes denominated CHS4 to CHS7 do not encode catalytic polypeptides as
are the case with CHS1 to CHS3, but proteins with different functions (see Roncero et al.
2001). For example, Chs5p was described as a Golgi protein involved in the transport not
only of Chs3, but also of other proteins (Santos and Snyder 1997), playing a role in the
attachment of Chs3 (DeMarini et al. 1997) and phosphatase Glc7 (Kozubowski et al. 2003)
to the site of bud emergence by interaction with Bin4. In addition, independently of the
Chs5 pathway, the products of genes SBE2 and SBE22 were described as involved in the
mobilization of cell wall components from Golgi to the cell surface (Santos and Snyder
2000). In contrast, Chs6 was described as being involved in the transport of Chs3 from the
chitosomes to the plasma membrane (Ziman et al. 1998), whereas Chs7, a protein resident
in the ER, was suggested to operate as a chaperone for Chs3 (Trilla, Duran, and Roncero.
1999). Nevertheless, the absence of a signal peptide in the catalytic polypeptides of chitin
synthases (J. Ruiz-Herrera and L. Ortiz-Castellanos, unpublished), speak against the pos-
sibility that the enzyme (and the microvesicles) follow the classical ER-Golgi pathway for
protein secretion. In this context, the results obtained by Riquelme et al. (2007) in the study
of transport of chitin synthases Chs3 belonging to division 1, and Chs6 belonging to divi-
sion 2 (see Chapter 4 on chitin) from N. crassa are very illustrative. These authors fused the
GFP to the carboxy termini of the Chs–coding regions, and followed the fate of the hybrid
polypeptides by high-resolution confocal laser scanning microscopy (CLSM). The most
conspicuous fluorescence in cells containing either construction was observed in the api-
cal zone corresponding to the most internal region of the Spitzenkörper, localized by use
of fluorescence staining with FM4-64, a membrane-staining fluorescent dye. To analyze
the fluorescence distribution corresponding to Chs in the subapical zone of the hyphae,
this was divided into three regions away from the apical pole: apical (2–5 μm), proximal
(5–20 μm), and distal (20–40 μm). It was observed that fluorescence in the distal region
appeared in the lumen of a network of tubular and globular compartments, whereas in
the proximal region, fluorescence appeared in the form of punctate structures originat-
ing from the disintegration of the above-described compartments, and finely dispersed
fluorescence in the apical region corresponding to microvesicles (chitosomes). Movies of
the growing hyphae revealed that most of the punctate fluorescence moved toward the
apical zone. To determine the origin of the fluorescence (chitosomes containing Chs-GFP)
in the Spitzenkörper, the authors used inhibitors and fluorescence recovery after photo-
bleaching (FRAP) with high intensity laser radiation. It was observed that brefeldin A (a
classical inhibitor of secretion) failed to stop the vesicular movement, and FRAP experi-
ments revealed that fluorescence in the Spitzenkörper was provided by the continuously
advancing microvesicles (chitosomes). Additionally, it was demonstrated that the fluores-
cently labelled endomembrane system did not correlate to specifically stained ER or Golgi.
All these results were interpreted as indicative that chitosomes do not follow the classical
secretory pathway involving the ER and Golgi complex, but a different one whose charac-
teristics will be extremely important to unravel.
As it was indicated earlier, the putative active polypeptide of β-1,3-glucan synthases
lacks a signal peptide (J. Ruiz-Herrera and L. Ortiz-Castellanos, unpublished), suggesting
that it is secreted through a nonconventional mechanism. In this sense Verdin, Bartnicki-
García, and Riquelme (2009) described the localization and possible secretion mechanism
of a protein related to the synthesis of β-1,3-glucans, Gs-1, (Enderlin and Selitrennikof
1994), which provided interesting data on the ultrastructure of the Spitzenkörper (Verdin,
Bartnicki-García, and Riquelme 2009) (see above). The authors obtained a cotranslational
fusion of Gs-1 and GFP. By use of fluorescence laser scanning confocal microscopy, the
authors observed that the fluorescent protein appeared in vesicles larger than chitosomes
of a diameter ca. 330 nm concentrated in an annular zone surrounding the Spitzenkörper
(they baptized this Spitzenring), which displayed dynamic arrangements (see Figure 8.3).
Use of total internal reflection fluorescence microscopy allowed the observation of a popu-
lation of vesicles along the hyphae measuring between 200 and 900 nm, which constantly
moved with anterograde and retrograde displacements. The authors warned that the func-
tion of Gs-1 in β−1,3-glucan synthesis is unknown, but pointed out that sucrose density
centrifugation demonstrated that it forms part of a multiproteic complex involved in the
synthesis of the polysaccharide since it sediments into vesicles that express β−1,3-glucan
synthase activity. The conclusion of the authors, in light of these experiments, is that the
synthases of the two most important structural polysaccharides of the fungal cell wall, chi-
tin and β-1,3-glucans, are transported to the cell surface by unconventional mechanisms
into two different types of vesicles that end in two distinct regions of the Spitzenkörper by
still unknown mechanisms and with a significance that may be relevant to the organiza-
tion and shape of the cell wall.
Regarding the larger vesicles observed in the fungal cells, a long history has revealed
that they are involved in the transport of glycoproteins, determined either by specific
staining at the electron microscopy level or by enzymatic determination after their isola-
tion (see Figure 8.4). These glycoproteins are secreted to the medium or become associ-
ated into the cell wall (reviewed in Ruiz-Herrera 1992). Early experiments revealed that
the content of these vesicles was different from that of microvesicles since, in contrast to
these, they stained by silver hexosamine (Grove and Bracker 1970). These observations
were confirmed and extended during the analysis of the effect of asymmetric light on P.
blakesleeanus sporangiophores that bend toward the light source by a phototrophic reaction.
The data obtained showed that the rate of transport of macrovesicles and microvesicles
(chitosomes), recognized by the use of Thiery’s procedure on electron microscopy sections,
revealed the presence of carbohydrates inside large vesicles, but not in microvesicles, and
was accelerated towards the distal side of the sporangiophore, thus inducing its curvature.
(Morales and Ruiz-Herrera 1990). (See Chapter 9 and Figure 9.2.) Other types of evidence
was obtained by the isolation of vesicles from different fungal species and the study of
their characteristics. Vesicles containing high amounts of glucanase were isolated from
S. cerevisiae and purified by density gradient centrifugation. These vesicles had a higher
density and diameter as compared to chitosomes (Matile et al. 1971). Similar vesicles con-
taining phosphatase (Holcomb et al. 1987) or invertase (Walworth and Novick 1987) were
isolated from yeast sec mutants, which accumulate them in the cytoplasm at the restrictive
temperature (see above).
It is unlikely that in general, these vesicles are specific, only carrying a certain cargo
protein; it is more feasible that their enrichment of different proteins depends on the physi-
ological condition that favor the secretion of some of them. This hypothesis was confirmed
by the isolation of a homogeneous population of vesicles from the slime variant of N. crassa
that contained both phosphatase and invertase (Ruiz-Herrera et al. 1987). It is important
to stress that the cargo transported by the vesicles is not made solely of soluble proteins,
but as was indicated above, they also transport proteins targeted to the plasma membrane.
Accordingly, using S. cerevisiae sec mutants again, it was possible to isolate vesicles contain-
ing membrane ATPase (Holcomb et al. 1988).
Furthermore, large amounts of evidence demonstrate the role of these vesicles in cell
wall synthesis. These roles may be indirect taking into consideration the cargo carried
by these vesicles or by the establishment of direct correlations between their formation
or disappearance with wall formation or inhibition, respectively. Examples of the lat-
ter are the accumulation of vesicles in the zones of active growth other than the apex,
such as during branching or septation, and the intracellular accumulation of vesicles in
yeast sec mutants maintained at the restrictive temperature, and their decrease when
growth is reassumed at the permissive temperature (see above). The rapid rate of vesicle
discharge during wall formation in the encystment process of zoospores from different
Chitridiomycota such as Allomyces arbuscula (Kirby, Kroh, and Sassen 1974) and the obser-
vation that the immediate growth inhibition caused by the calcium ionophore A23187 on
germlings of P. blakesleanus was accompanied by a sudden halt in invertase secretion and
a decrease in the number of apical vesicles (Ruiz-Herrera et al. 1989) strongly support the
role of vescicles in apical growth.
As discussed extensively in Chapter 7 on proteins, some proteins without the charac-
teristic features of the bona fide extracellular proteins, mainly the signal peptides (moon-
light proteins) appear to be secreted under some conditions, and even integrated into the
cell wall by an unknown mechanism different from the classical one. This subject is at
present a matter of discussion.
8.9 The porosome

The mechanism of fusion of vesicles with the plasma membrane was always considered at
the physicochemical level with the idea of determining how two different lipid bilayers can
mix with each other. It was always also understood, so much that it was never mentioned,
that the site of vesicle fusion with the plasma membrane occurred at random, without spe-
cial or preestablished sites involved. Perhaps the main reasons behind this attitude was the
fact that the light microscope has a resolution that does not permit the discovery of very
small structures in the cell surface and that the electron microscope, which has a nanomet-
ric resolution permitting the observation of very small structures, normally only permits
the observation of dead cells fixed and stained by complex procedures, which might alter
the small structures making them unrecognizable. For these reasons, the description of
a novel structure in the cell membrane of some animal cells, the porosome, by B. Jena and
his colleagues was a surprise. They observed it with the help of a different microscope,
the atomic force microscope (AFM) with resolution at the level of the electron microscope,
able to observe living cells without any treatment that might introduce artifacts (see Jena
2002, 2004, 2006; Anderson 2004 for reviews). Jena has pointed out that electrophysiologi-
cal studies of mast cells had anticipated the possible existence of pores in the cell mem-
brane (Monck et al. 1995). Using AFM, it was possible to discern the existence of a novel
membrane structure in living pancreatic acinar cells in the form of circular pits with a
diameter of 0.4–1.2 μm containing smaller circular depressions that corresponded to the
structures now known as porosomes, which displayed structural changes during the secre-
tion process (Schneider et al. 1997). Similarly, fusion pores were observed by the same AFM
technique in nerve cells and in exocrine and neuroendocrine cells (reviewed by Jena 2004).
Porosomes with the same structure of those present in the plasma membrane of pancre-
atic acinar cells were observed in secreting cells of the pituitary and chromaffin cells of
the adrenal medulla (Cho et al. 2002a, 2002b). Enlargement of the porosome diameter was
observed by stimulation of secretion; and, by use of gold-labeled antibodies directed to
secretory proteins, it was possible to determine that these accumulated in the porosomes.
On the cytosolic side, porosomes appeared as inverted cups with a height of 10–15 nm
sometimes attached to vesicles (zymogen granules) and located in a circular structure that
corresponded to the pit observed in the cell surface. The demonstration that these images
corresponded to fusion of zymogen granules came from the observation that antibodies
directed against the tSNARE SNAP23 recognized the inverted cups. The above-described
porosome structure was confirmed by transmission electronic microscopy. Porosomes could
be isolated from pancreatic plasma membranes by a technique involving tissue homogeniza-
tion, differential centrifugation to separate plasma membranes, homogenization, and sucrose
density centrifugation (Jena 2006). Reconstitution of the porosomes into lipid bilayers was
used to determine their functionality by means of electrophysiological techniques (Jeremic

et al. 2003). Addition of zymogen granules to one side of the system led to their binding to
porosomes as observed by an increase in capacitance and conductance, and most important
to transfer of amylase from cis to trans compartments separated by the bilayer. These data
demonstrate the functionality of the reconstituted porosomes, but as can be seen from this
condensed review of porosomes, all data corresponded to cells from mammals. Whether
or not similar structures to the porosome exist in fungal cells and are involved in secretion
remains unknown since similar experiments have not been performed in fungi.
References
Anderson, L. L. 2004. Discovery of a new cellular structure—The porosome: Elucidation of the
molecular mechanism of secretion. Cell. Biol. Int. 28:3–5.
Bartnicki-García, S. 2003. “Hyphal Tip Growth: Outstanding Questions.” In Molecular Biology of
Fungal Development. Edited by H. D. Osiewacz, 29–58. New York: Marcel Dekker.
Bartnicki-García, S., Bracker, C. E., Reyes, E., and Ruiz-Herrera, J. 1978. Isolation of chitosomes from
taxonomically diverse fungi and synthesis of chitin microfibrils in vitro. Exp. Mycol. 2:173–192
Bartnicki-García, S., Bracker, C. E., Lippman, E., and Ruiz-Herrera, J. 1984. Chitosomes from the wall-
less slime mutant of Neurospora crassa. Arch. Microbiol. 139:105–112.
Bartnicki-García, S. Hergert, F., and Gierz, G. 1989a. Computer simulation of fungal morphogenesis
and the mathematical basis for hyphal (tip) growth. Protoplasma 153:46–57.
Bartnicki-García, S., Hergert, F., and Gierz, G. 1989b. “A Novel Computer Model for Generating
Cell Shape: Application to Fungal Morphogenesis.” In Biochemistry of Cell Walls and Membranes.
Edited by P. Kuhn et al., 43–60. Heidelberg: Springer-Verlag.
Bartnicki-García S., Ruiz-Herrera, J., and Bracker, C. E. 1979. “Chitosomes and Chitin Synthesis.” In
Fungal Walls and Hyphal Growth. Edited by J. H. Burnett and A. P. J. Trinci, 149–168. Cambridge:
Cambridge University Press.
Blobel, G. 1980. Intracellular protein topogenesis. Proc. Natl. Acad. Sci. USA 77:1495–1500.
Blobel, G. and Dobberstein, B. 1975a. Transfer of proteins across membranes. I. Presence of proteoliti-
cally processed and unprocessed nascent immunoglobulin light chains on membrane-bound
ribosomes of murine myeloma. J. Cell Biol. 67:835–851.
Blobel, G. and Dobberstein, B. 1975b. Transfer of proteins across membranes II. Reconstitution of
functional rough microsomes from heterologous components. J. Cell Biol. 67:852–862.
Bracker, C .E., Murphy, D. J., and López-Franco, L. 1997. “Laser Microbeam Manipulation of Cell
Morphogenesis in Growing Fungal Hyphae.” In Functional Imaging and Optical Manipulation
of Living Cells. Edited by D. L. Farkas and D. J. Tromberg. Proceedings of SPIE, Vol. 2893.
International Society for Optical Engineering, Bellingham, WA.
Bracker, C. E., Ruiz-Herrera, J., and Bartnicki-García, S. 1976. Structure and transformation of chitin
synthetase particles (chitosomes) during mirofibril synthesis in vitro. Proc. Natl. Acad. Sci. USA
73:4570–4574.
Bruswick, H. 1924. Untersuchungen uber Geschlechts und Kernverhaltnisse bei der
Hymenomyzetengattung Coprinus. In Botanische Abhandlungen, Vol. 5. Edited by K. Goebel,
1–152. Jena: Gustav Fisher.
Cho, S. J., Jeftinija, K., Glavaski, A., Jeftinija, S., Jena, B. P., and Anderson, L. L. 2002a. Structure and
dynamics of the fusion pores in live GH-secreting cells revealed using atomic force microscopy.
Endocrinology 143:1144–1148.
Cho, S. J., Wakade, A., Pappas, G. D., and Jena, B. P. 2002b. New structure involved in transient mem-
brane fusion and exocytosis. New York Acad. Sci. 971:254–256.
Cosson, O., Ravazzola, M., Varlamov, O., Söllner, T. H., Di Liberto, M., Volchuk, A., Rothman, J. E.,
and Orc, L. 2005. Dynamic transport of SNARE proteins in the Golgi apparatus. Proc. Natl.
Acad. Sci. USA 102:14647–14652.
De Marini, D. J., Adams, A. E. M., Fares, H., De Virgilio, C., Valle, G., Chuang, J. S., and
Pringle, J. R. 1997. A septin-base hierarchy of proteins required for localized deposition of chitin
in the Saccharomyces cerevisiae cell wall. J. Cell Biol. 139:75–93.
Duman, J. G. and Forte, J. G. 2003. What is the role of SNARE proteins in membrane fusion? Am. J.
Physiol. Cell Physiol. 285:C237–C249.
Enderlin, C. S. and Selitrennikoff, C. P. 1994. Cloning and characterization of a Neurospora crassa gene
required for (1,3) β-glucan synthase activity and cell wall formation. Proc. Natl. Acad. Sci. USA
91:9500–9504.
Fasshauer, D., Sutton, R. B., Brunger, A. T., and Jahn, R. 1998. Conserved structural features of the
synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs. Proc. Natl. Acad.
Sci. USA 95:15781–15786.
Flores-Martinez, A., Lopez-Romero, E., Martinez, J. P., Bracker, C. E., Ruiz-Herrera, J., and Bartnicki-
García, S. 1990. Protein composition of purified chitosomes of Mucor rouxii. Exp. Mycol.
14:160–168.
Girbardt, M. 1955. Lebendbeobachtungen on Polystictus versicolor (L.). Flora 142:540–563.
Girbardt, M. 1957. Der Spitzenkörper von Polystictus versicolor (L.). Planta 50:47–59.
Girbardt, M. 1969. Die Ultrastruktur der Apikal region von Pilzhyphen. Protoplasma 67:413–441.
Gordon, C. L., Archer, D. B., Jeenes, D. J., Donan, J. H., Wells, B., Trinci, A. P., and Robson, G. D. 2000.
A glucoamylase: GFP gene fusion to study protein secretion by individual hyphae of Aspergillus
niger. J. Microbiol. Meth. 42:39–48.
Grove, S. M., Bracker, C. E., and Morré, J. D. 1968. Cytomembrane differentiation in the endoplasmic
reticulum-Golgi apparatus-vesicle complex. Science 161:171–173.
Grove, S. N. and Bracker, C. E. 1970. Protoplasmic organization of hyphal tips among fungi: Vesicles
and Spitzenkörper. J. Bacteriol. 104:989–1009.
Halic, M. and Beckmann, R. 2005. The signal recognition particle and its interactions during protein
targeting. Curr. Opin. Struct. Biol. 15:116–125.
Hauri, H. P., Kappeler, F., Andersson, H., and Appenzeller, C. 2000. ERGIC-53 and traffic in the secre-
tory pathway. J. Cell Sci. 113:587–596.
Helenius, A., Trombetta, E. S., Herbert, D. N., and Simons, J. F. 1997. Calnexin, calreticulin and the
folding of glycoproteins. Trends Cell Biol. 7:193–200.
Hernandez, J., Lopez-Romero, E., Cerbon, J., and Ruiz-Herrera, J. 1981. Lipid analysis of chitosomes,
chitin-synthesizing microvesicles from Mucor rouxii. Exp. Mycol. 5:349–356.
Herrera-Estrella, L., Chavez, B., and Ruiz-Herrera, J. 1982. Presence of chitosomes in the cytoplasm of
Phycomyces blakesleeanus and the synthesis of chitin microfibrils. Exp. Mycol. 6:385–388.
Hirschberg, C. B. and Snider, M. D. 1987. Topography of glycosylation in the rough endoplasmic
reticulum and Golgi apparatus. Annu. Rev. Biochem. 56:63–87.
Holcomb, C. L., Etcheverry, T., and Schekman, R. 1987. Isolation of secretory vesicles from
Saccharomyces cerevisiae. Anal. Biochem. 166:328–334.
Holcomb, C. L., Hansen, W. J., Etcheverry, T., and Schekman, R. 1988. Secretory vesicles externalize
the major plasma membrane ATPase in yeast. J. Cell Biol. 106:641–648.
Iodice, L., Sarnataro, S., and Bonatti, D. 2001. The carboxyl-terminal valine is required for transport
of glycoprotein CD8 alpha from the endoplasmic reticulum to the intermediate compartment.
J. Biol. Chem. 276:28920–28926.
Jena, B. P. 2002. Fusion pores in living cells. News Physiol. Sci. 17:219–222.
Jena, B. P. 2004. Discovery of the porosome: Revealing the molecular mechanism of secretion and
membrane fusion in cells. J. Cell. Mol. Med. 8:1–21.
Jena, B. P. 2006. “Porosome: The Universal Secretory Machinery in Cells.” In Force Microscopy:
Applications in Biology and Medicine. Edited by B. P. Jena and J. K. H. Hörber, 1–23. New York:
John Wiley & Sons.
Jeremic, A. M., Kelly, M., Cho, S. J., Stromer, M. H., and Jena, B. P. 2003. Reconstituted fusion pore. J.
Biophys. 85:2035–2043.
Julius, D., Blair, L., Brake, A., Sprague, G., and Thorner, J. 1983. Yeast α-factor is processed from a
larger precursor polypeptide: the essential role of membrane-bound dipeptidyl aminopepti-
dase. Cell 32:839–852.
Kirby, E. G., Kroh, M., and Sassen, M. M. A. 1974. Cell wall formation in zoospores of Allomyces
arbuscula. I. Fine structural observations on encysting haploid zooospores. Arch. Microbiol.
98:147–158.
Kozubowski, L., Panek, H., Rosenthal, A., Bloecher, A., DeMarini, D. J., and Tatchell, K. A. 2003. bni4-
glc7 phosphatase complex that recruits chitin synthase to the site of bud emergence. Mol. Biol.
Cell 14:26–39.
Larriba, G., Elorza, M. V., Villanueva, J. R., and Sentandreu, R. 1976. Participation of dolichol phos-
pho-mannose in the glycosylation of yeast wall mannoprotein at the polysomal level. FEBS
Lett. 71:316–320.
Lending, C., Leal-Morales, C. A., Flores-Martinez, A., Bracker, C. E., and Bartnicki-García, S. 1991.
Purification and characterization of 16S chitin synthetase particles from cell walls of Mucor
rouxii. Exp. Mycol. 15:11–25.
Lewis, M. J. and Pelham, H. R. B. 1992. Ligand-induced redistribution of a human KDEL receptor
from the Golgi complex to the endoplasmic reticulum. Cell 68:353–364.
Lippincott-Schwartz, J., Roberts, T. H., and Hirschberg, K. 2000. Secretory protein trafficking and
organelle dynamics in living cells. Annu. Rev. Cell Dev. Biol. 16:557–589.
Luirink, J. and Sinning, I. 2004. SRP-mediated protein targeting: Structure and function revisited.
Matile, P., Cortat, M., Wiemken, A., and Frey-Wyssling, A. 1971. Isolation of glucanase-containing
particles from budding Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 68:634–640.
McClure, W. K., Park, D., and Robinson, P. M. 1968. Apical organization in the somatic hyphae of
fungi. J. Gen. Microbiol. 50:177–182.
Milstein, C., Bromlee, G. G., Harrison, T. M., and Mathews, M. B. 1972. A possible precursor of immu-
noglobulin light chains. Nature New Biol. 239:117–120.
Monck, J. R., Oberhauser, A. F., and Fernandez, J. M. 1995. The exocytic fusion pore interface: A
model of the site of neurotransmitter release. Mol. Memb. Biol. 12:151–156.
Morales, M. and Ruiz-Herrera, J. 1990. Light-induced changes in the vesicular apparatus of
Phycomyces sporangiophores during the phototropic response. Photochem. Photobiol. 52:223–227.
Morales, M. and Ruiz-Herrera, J. 1993. Critical evaluation of the ultrastructure of sporangiophores
from Phycomyces blakesleeanus (Zygomycetes) at stages I and IV. Cryptogamic Botany 3:273–282.
Morré, D. J. and Mollenhauer, H. H. 1974. “The Endomembrane Concept: A Functional Integration
of Endoplasmic Reticulum and Golgi Apparatus.” In Dynamic Aspects of Plant Ultrastructure.
Edited by A. W. Robards, 84–137. London: McGraw-Hill.
Munro, S. and Pelham, H. R. B. 1987. A C-terminal signal prevents secretion of luminal ER proteins.
Cell 48:899–907.
Nishimura, N. and Balch, W. W. 1997. A di-acidic signal required for selective export from the endo-
plasmic reticulum. Science 277:556–558.
Novick, P., Ferro, S., and Schekman, R. 1981. Order of events in the yeast secretory pathway. Cell 25:
461–469.
Novick, P., Field, C., and Schekman, R. 1980. Identification of 23 complementation groups required
for post-translational events in the yeast secretory pathway. Cell 21:205–215.
Palade, G. E. 1975. Intracellular aspects of the process of protein synthesis. Science 189:347–358.
Pelham, H. R. B. 1988. Evidence that luminal ER proteins are sorted from secreted proteins in a post-
ER compartment. J. EMBO 7:913–918.
Pereira-Leal, J. B. and Seabra, M. C. 2001. Evolution of the Rab family of small GTP-binding proteins.
J. Mol. Biol. 313:889–901.
Perez, M. and Hirschberg, C. A. 1985. Translocation of UDP-N-acetylglucosamine into vesicles derived
from rat liver rough endoplasmic reticulum and Golgi apparatus. J. Biol. Chem. 260:4671–4678.
Perez, M. and Hirschberg, C. B. 1986. Topography of glycosylation reactions in the rough endoplas-
mic reticulum membrane. J. Biol. Chem. 261:6822–6830.
Punt, P. J., Seiboth, B., Weenink, X. O., Lenders, M., Konetschny, C., Ram. A. F., Montijn, R., Kubicek,
C. P., and Van den Hondel, C. A. 2001. Identification and characterization of a family of secre-
tion-related small GTPases-encoding genes from the filamentous fungus Aspergillus niger: A
putative SEC4 homologue is not essential for growth. Mol. Microbiol. 41:513–525.
Rapoport, T. A. 2007. Protein translocation across the eukaryotic endoplasmic reticulum and bacte-
rial plasma membranes. Nature 450:663–669.
Redman, C. M. and Sabatini, D. D. 1966. Vectorial discharge of peptides released by puromycin from
attached ribosomes. Proc. Natl. Sci. USA 56: 608–615.
Riederer, M. A. and Hinnen, A. 1991. Removal of N-glycosylation sites of the yeast acid phosphatase
severely affects protein folding. J. Bacteriol. 173:3539–3546.
Riquelme, M., Bartnicki-García, S., González-Prieto, J. M., Sánchez-León, E., Verdín-Ramos, J. A.,
Beltrán-Aguilar, A., and Freitag, M. 2007. Spitzenkörper localization and intracellular traffic
of green fluorescente protein-labeled CHS-3 and CHS-6 chitin synthases in living hyphae of
Neurospora crassa. Eukaryot. Cell 6:1853–1864.
Roncero, C., Rodriguez-García, L. J., Sanz, M., Castrejon, F., Trilla, J. A., and Duran, A. 2001. “The
Control of Chitin Synthesis in Saccharomyces cerevisiae.” In Chitin Enzymology. Edited by R. A. A.
Muzzarelli, 485–492. Grottammare: Atec Edizioni.
Rothman, J. E. 1994. Mechanisms of intracellular protein transport. Nature 372:55–63.
Ruiz-Herrera, J. 1984. “The Role of Chitosomes in the Apical Growth of Fungi.” In Microbial Cell Wall
Synthesis and Assembly. Edited by C. Nombela, 113–120. Amsterdam, Holland: Elsevier.
Ruiz-Herrera, J., Bartnicki-García, S., and Bracker, C. E. 1980. Dissociation of chitosomes by digitonin
into 16 S subunits with chitin synthetase activity. Biochim. Biophys. Acta 629:201–206.
Ruiz-Herrera, J., Martinez, J. P., Casanova, M., Gil, M. L., and Sentandreu, R. 1987. Separation of
chitosomes and secretory vesicles from the “slime” variant of Neurospora crassa. Arch. Microbiol.
149:156–162.
Ruiz-Herrera, J. and Martinez-Espinoza, D. 1999. “Chitin Biosynthesis and Structural Organization
in vivo.” In Chitin and Chitinases. Edited by P. Jolles and R. A. A. Muzzarelli, 39–56. Basel:
Birkhäuser.
Ruiz-Herrera, J., Valenzuela, C., Martinez-Cadena, G., and Obregon A. 1989. Alterations in the
vesicular pattern and wall growth of Phycomyces induced by the calcium ionophore A23187.
Santos, B. and Snyder, M. 1997. Targeting of chitin synthase 3 to polarized growth sites in yeast
requires Chs5p and Myo2p. J. Cell Biol. 136:95–110.
Santos, B. and Snyder, M. 2000. Sbe2p and sbe22p, two homologous Golgi proteins involved in yeast
cell wall formation. Mol. Biol. Cell 11:435–452.
Schekman, R. 1985. Protein localization and membrane traffic in yeast. Annu. Rev. Cell Biol. 1:115–143.
Schneider, S. W., Sritharan, K. C., Geibel, J. P., Oberleithner, H., and Jena, B. P. 1997. Surface dynamics
in living acinar cells imaged by atomic force microscopy: Identification of plasma membrane
structures involved in exocytosis. Proc. Natl. Acad. Sci. USA 94:316–321.
Siekevitz, P. and Palade, G. E. 1960. A cytochemical study on the pancreas of the guinea pig. J. Cell
Biol. 4:631–644.
Stirling, C. J. 1999. Protein targeting to the endoplasmic reticulum in yeast. Microbiology 145:991–998
Straus, A. W., Zimmerman, M., Boime, I., Ashe, B., Mumford, R. A., and Alberts, A. W. 1979.
Characterization of an endopeptidase involved in pre-protein processing. Proc. Natl. Acad. Sci.
USA 76:4225–4229.
Titorenko, V. I., Ogrydziak, D. M., and Rachubinski, R. A. 1997. Four distinct secretory pathways
serve protein secretion, cell surface growth, and peroxisome biogenesis in the yeast Yarrowia
lipolytica. Mol. Cell. Biol. 17:5210–5226.
Trilla, J. A., Duran, A., and Roncero, C. 1999. Chs7p, a new protein involved in the control of protein
export from the endoplasmic reticulum that is specifically engaged in the regulation of chitin
synthesis in in Saccharomyces cerevisiae. J. Cell Biol. 145:1153–1163.
Trombeta, E. S. and Parodi, A. J. 2003. Quality control and protein folding in the secretory pathway.
Annu. Rev. Cell Dev. Biol. 19:649–676.
Tsai, B., Ye, Y., and Rapoport, T. A. 2002. Retrotranslocation of proteins from the endoplasmic reticu-
lum into the cytosol. Nat. Rev. Mol. Cell Biol. 3:246–255.
Veldhuisen, G., Saloheimo, M., Fiers, M. A., Punt, P. J., Contreras, R., Penttilä, M., and Van den
Hondel, C. A. 1997. Isolation and analysis of functional homologues of the secretion-related
SAR1 gene of Sacchromyces cerevisiae from Aspergillus niger and Trichoderma reesei. Mol. Gen.
Genet. 256:446–455.
Verdin, J., Bartnicki-García, S., and Riquelme, M. 2009. Functional stratification of the Spitzenkörper
of Neurospora crassa. Mol. Microbiol. 74:1044–1053.
Vitale, A. and Denecke, J. 1999. The endoplasmic reticulum—Gateway of the secretory pathway.
Plant Cell 11:615–628.
Walworth, N. C. and Novick, P. N. 1987. Purification and characterization of constitutive secretory
vesicles from yeast. J. Cell Biol. 105:163–174.
Wedlich-Söldner, R., Bölker, M., Kahmann, R., and Steinberg, G. 2000. A putative endosomal
t-SNARE links exo- and endocytosis in the phytopathogenic fungus Ustilago maydis. EMBO J.
19:1974–1986.
Weete, J. D., Further, R., Hanseler, E., and Rast, D. M. 1985. Cellular and chitosomal lipids of Agaricus
bisporus and Mucor rouxii. Can. J. Microbiol. 31:1120–1126.
Wieland, F. T., Gleason, M. L., Serafini, T., and Rothman, A. 1987. The rate of bulk-flow from endo-
plasmic reticulum to the cell surface. Cell 50:289–300.
Wösten, H. A. B., Moukha, S. M., Sietsma, J., and Wessels, J. G. H. 1991. Localization of growth and
secretion of proteins in Aspergillus niger. J. Gen. Microbiol. 137:2017–2023.
Yoshihisa, T., Barlowe, C., and Schekman, R. 1993. Requirement of a GTPase-activating protein in
vesicle budding from the endoplasmic reticulum. Science 259:1466–1468.
Ziman, M., Chuang, J. S., Tsung, M., Hamamoto, S., and Scheckman, R. 1998. Chs6p-dependent
anterograde transport of Chs3p from the chitosome to the plasma membrane in Saccharomyces
cerevisiae. Mol. Biol. Cell 9:1565–1576
Zueco, J., Mormeneo, S., and Sentandreu, R. 1986. Temporal aspects of the O-glycosylation of
Saccharomyces cerevisiae proteins. Biochim. Biophys. Acta 884:93–100.
chapter nine
Cell wall growth and expansion

9.1 Introduction
Fungi are encased in a rigid cell wall that, as indicated in previous chapters, provides
protection against the different harmful effects of the medium, provides the cell its mor-
phology, and regulates its interactions with inert or biological surfaces. This situation
originates an apparent contradiction; the wall is rigid, but somehow must expand to per-
mit growth of the organism. Accordingly, the expansion process of the cell wall must be
extremely well regulated to guarantee that the cell does not lyse during the process. How
the synthesis of the different components of the fungal cell wall occurs (and the process
coordinated) was discussed in previous chapters (for reviews see Fuchs and Mylonakis
2009; Lesage and Bussey 2006; Levin 2005; Pruyne and Bretscher 2000a, 2000b).
The disastrous results that occur when the system fails is exemplified by the lysis
occurring to Saccharomyces cerevisie cells carrying a mutated MID2 allele (this gene
encodes one of the sensors involved in the Cell Wall Integrity pathway) (CWI), that fail
to adapt cell wall growth from the yeast to the shmoo forms (Hutzler et al. 2008). (The
shmoo forms are formed during mating when two yeast strains of opposite mating type
find themselves at a short distance and become stimulated each one by the pheromone
produced by the member of the opposite mating type. Budding is then inhibited, and the
cells form characteristic gross mating tubes that give them a bizarre form, which eventu-
ally lead to their mating).
Since the wall provides their characteristic shape to the different fungal species, or the
shapes at their different developmental stages, it is necessary to consider that not only its
composition and structure must be carefully regulated, but also its growth pattern. This
assertion stems from the premise that cell wall shape does not depend on its chemical
composition but on the pattern of synthesis. This in turn depends on internal factors, such
as the location of the sites of active growth, growth kinetics, the duration of the plastic
stage of the cell wall, how the different components become organized, and so forth, and
of external stimuli, such as nutrient availability, light, pH, temperature, and others.
For simplicity, we may consider that cell wall growth displays only two different pat-
terns of expansion: isotropic or spherical, and polarized, that is, occurring over the whole
cell surface, or restricted to a selected region of the cell periphery, respectively. These two
patterns may be absolute or vary between the two extremes. Time is also an important
factor in the operation of either pattern, that is, both patterns may alternate giving rise to
peculiar cell shapes. Examples of these aspects may be illustrative: an absolute isotropic
pattern of growth provides a perfect spherical cell, like that shown by M. rouxii yeasts
(Bartnicki-García and Nickerson 1962), whereas the continuous operation of a polarized
growth produces a tubular structure whose width will depend mostly on how narrow the
expanding region is, as occurs in the fungal mycelium. Simultaneous operation of both
patterns gives rise to a spheroidal shape observed in a number of yeasts, whose geometric
treatment was approached by Merson-Davies and Odds 1989) with their definition of the
morphology index. Alternative operation of the two patterns may give rise to shapes like
those of sterigmata.
149
9.2 Expansion of the cell wall

9.2.1 Introduction
Although unnecessary, it is perhaps convenient to insist on the concept that the fungal cell
wall is a complex structure that in general maintains an approximately constant composi-
tion during growth, which may be altered by changes in the environmental conditions,
or during the different stages in the development of the organism, such as phorogenesis,
sporulation, spore germination, dimorphic transition, and so forth. Considering the wide
number of taxonomic groups and species, on first sight it would appear difficult to accept
that their cell walls follow the same rules for their organization. Nevertheless, this is a
fact if we only consider the general aspects of the cell wall structure, just as it is a fact that
vertebrates have the same organization of their skeletons. Accordingly, the fungal cell wall
can be envisaged as a coherent structure with a scaffold made of structural polysaccha-
rides embedded in a mixture of glycoproteins and amorphous polysaccharides.
It is difficult to accept that such a complex structure is a product of chance; on the
contrary, it has to be deduced that it has been selected through evolution. In Chapter 2, I
presented a model to explain the evolution of the fungal cell wall, pointing out that if it is
true that their biosynthetic enzymes have evolved and differentiated, this does not apply
for wall construction where groups of the kingdom Fungi have followed the same logics.
Emphasis was placed then (and repeated now) on the fact that the chemical composition
and the fine details of joining some of their components are different in the several fungal
taxa. Accordingly, to consider that a single model may be representative of all fungi is an
overexaggeration, and its acceptance may lead to erroneous concepts. In the case of the
mechanisms involved in the organization of the cell wall, most of the published studies
and reviews take Saccharomyces cerevisiae (and only eventually some other yeasts) as the
paradigm of the phenomenon. In this chapter, I have tried to avoid this attitude (when pos-
sible), and I discuss published data of different fungi.
9.2.2 The physical stages of nascent and mature wall and their

relation to cell wall expansion and fungal growth
Different types of analyses support the concept that the cell wall of fungi exists in two
different physical stages: (i) viscoelastic, having both elastic (reversible) and viscous (irre-
versible) mechanical components, and (ii) elastic, which in general terms may be con-
sidered rigid, as was discussed in Chapter 3 on cell wall structure. Among the evidence
supporting this concept are experiments that showed the different behavior of nascent
and consolidated cell wall and their components. Thus nascent chitin in the cell wall can
be subjected to different modification reactions, such as deacetylation to form chitosan
or partial lysis by chitinase (see Davis and Bartnicki-García 1984a, 1984b; Calvo-Mendez
and Ruiz-Herrera 1987), whereas the mature crystalline chitin is resistant to these treat-
ments. The case of β-1,3-glucans is similar, existing in its native stage in a form soluble
in alkali, which becomes insoluble through binding to chitin as was originally reported
by Siestma and Wessels 1979, 1981). In a growing hypha, these two physical stages of the
wall are clearly separated; the viscoelastic form being located at the apex, and the rigid
one, up to five times thicker than the apical one, located at the subapical region (Burnett
1979; Trinci and Collinge 1975). In agreement with this, it was observed as long ago as the
19th century that hyphal tips were much more sensitive than the subapical region to lysis
by osmotic shock (Reinhardt 1892), observations later confirmed by Bartnicki-García and
Chapter nine: Cell wall growth and expansion 151
Lippman (1972a). They are also more sensitive to the lytic action of chitinases (López-
Romero, Ruiz-Herrera, and Bartnicki-García 1982; Calvo-Mendez and Ruiz-Herrera 1987),
or to low concentrations of inhibitory substances such as the chitin synthase inhibitor,
polyoxin (Bartnicki-García and Lippman 1972b). However, probably the most convincing
and direct demonstration for these two mechanically different stages of the cell wall was
obtained, not with hyphae because of their small size, but with the giant sporangiophore
of Phycomyces blakesleeanus (Ahlquist and Gamow 1973; Ortega et al. 1975). As described
previously in Chapter 3, these authors, in a classical experiment, measured the degree
of deformation suffered by the sporangiophores when subjected to stretching with an
Instron machine, demonstrating that deformability of the sporangiophore measured as
engineering stress was maximal at the growing region and rapidly decreased with dis-
tance to this zone. These data agreed with results obtained in plant and algal walls where
it was suggested that wall expansion could be considered a biochemically controlled creep
(the deformation suffered by a viscoelastic material when subjected to a load) resulting
from the loss of the viscous properties of the wall (Sellen 1980).
In previous chapters, I described how the different components making the cell wall
are synthesized, and if relevant, how they are secreted. It became obvious that the amor-
phous components are synthesized into the cell and then are secreted, whereas the gen-
eral concept is that the structural polysaccharides are synthesized and polymerized at
the plasma, membrane-cell wall interface, although as described below, exceptions are
known. Once the different components are present in the exterior of the cell, they interact
to make up the wall through the establishment of connections among them.
One aspect on which I must insist is that, with the expected differences due to
changes in the environment or to cell differentiation, the formation of the cell wall of an
individual species or strain follows the same pattern and utilizes the same components
dictated by their genetic repertoire. This means that the different components of the cell
wall have the imprinted characteristics to recognize them among themselves, even if the
cell wall is made de novo, as occurs with natural or artificial protoplasts, as discussed
below. To describe the final results of wall formation, I will repeat here, with slight
changes, a concept previously stated (see Ruiz-Herrera 1992): Wall formation involves
the mechanisms of maturation that transform a plastic structure into a rigid composite
by an exquisite process of self-assembly, which has been conserved through the evolu-
tion of fungi, and which illustrates how it has fit the necessity for the specialized pattern
of growth of these organisms.
In a growing fungal cell, wall expansion is restricted to the zone existing in a visco-
elastic stage. After a long debate, most authors now consider that the force responsible for
fungal expansion is the turgor pressure, as demonstrated by a series of experimental evi-
dence (Money 1990; Money and Harold 1992). For example, it has been shown that growth
of mycelium stops when submerged in a solution with an osmotic potential superior to
its turgor pressure, but probably the most convincing pieces of evidence come from data
obtained with the giant sporangiophore of P. blakesleeanus. Accordingly, in experiments
similar to the one described above where sporangiophores were subjected to stress with
an Instron machine, it was demonstrated that photomecism (the transient increase in spo-
rangiophore expansion in response to a homogeneous light stimulus) was limited to the
viscoelastic region of the wall (Ortega et al. 1975), and that the light effect was due only
to the change in the mechanical properties of the growing zone, since the turgor pres-
sure remained constant during the whole experiment (Ortega et al. 1988). In a further
work, Ortega et al. (1989) measured the creep relaxation of P. blakesleeanus sporangiophores
when injected with small amounts of inert silicon oil into the vacuole providing data only
compatible with its relationship to the turgor pressure of the cell. To these experiments, we
must add the three-dimensional analysis of video imaging of Rhizoctonia solani (Bartnicki-
García et al. 2000; see below) that demonstrated that expansion of the wall surface at the
hyphal apex followed an orthogonal pattern, a property that agrees only with a uniform
driving force, that is, the turgor pressure.
Obviously, the phenomenon of wall expansion is different to wall growth, the latter
being considered as the ordered addition of all the components that make up the wall in
rather constant proportions if the environmental conditions remain the same. If expansion
of the wall continued in the absence of growth, it would become thinner until cell lysis
occurred. Accordingly, as described above, wall expansion and growth must be simulta-
neous processes to guarantee cell survival. As already described in previous chapters, the
enzymes involved in cell wall growth and in some cases the precursors of the wall are
directed to the growing regions within vesicles and microvesicles through the operation
of directional mechanisms that are described below. Once outside the permeability barrier
of the cell and immersed in the wall, the components that constitute this structure orga-
nize themselves into a coherent form by a self-assembly process and undergo maturation
through the action of different remodeling enzymes as described in previous chapters,
giving as a result, the nascent, and later the mature wall (see Figure 9.1).
9.2.3 Intermittent growth of the cell wall

Contrary to what is usually assumed, the growth of the cell wall is not a constant pro-
cess, but occurs intermittently in the form of extremely rapid pulses. This phenomenon
was originally described a long time ago by Castle (1940) in the P. blakesleenus sporan-
giophore and later rediscovered by López-Franco, Bartnicki-García, and Bracker (1994).
One characteristic of the sporangiophore of Phycomyces is that it responds to different
stimuli, including light, and that it rotates as it grows (Oort 1931). In his ingenious experi-
ments, Castle maintained the sporangiophores in a horizonal position by continuous illu-
mination at the tip, thus avoiding the negative geotropic effect. The author then glued a
thin glass fiber to the sporangium and recorded its rotation by photographing it under
a microscope with stroboscopic light. Measurement of the rotation of the long fiber in
enlarged successive prints, and transformation of the rotational speed to a linear one
provided an extreme sensitivity in his measurements of the growth rate of the sporangio-
phore. He thus observed that growth of the sporangiophore was not a constant process,
but oscillated in pulses of about 200 msec. Using a different experimental system (high
speed video microscopy), López-Franco, Bartnicki-García, and Bracker (1994) confirmed
the pulsed nature of growth of hyphae from fungal species representative of the main
fungal taxa with a frequency of nearly 3 to 13 seconds, concluding that this phenomenon
is universal. The interpretation given by these last authors to their results was that pulse
growth was due to the sudden discharge of vesicles and microvesicles, followed by a
short rest until a new discharge occurred. Not knowing at that time the role of the vesicu-
lar apparatus in fungal growth, Castle estimated that the oscillations in growth rate were
related to periods of expansion of the viscoelastic wall. He also argued that the size of
the stretch in the wall occurring during periods of acceleration of the growth rate could
not be due to intercalation of macromolecules. He concluded that the wall grows not by
intussusception as is still considered by some authors, but by apposition of new material,
a concept that he extended in a further publication (Castle 1958), and that was also sus-
tained by Saunders and Trinci (1979).
Figure 9.1 Scheme of fungal wall growth. (A) The different amorphous wall components are
secreted in the most apical region of a hypha by the discharge or vesicles, and structural polysac-
charides are synthesized at the interface plasma membrane-cell wall. (B) Structural polysaccharides
synthesized at the cell surface start making contact with some of the cell wall proteins. (C) The
chains of structural polysaccharides associate to form microfibrils. (D) There occur noncovalent
associations between structural polysaccharides and proteins. (E) The wall components establish
covalent associations to form a mature wall. During the process, the newly made wall moves down
along the subapical region and there occurs a change in its physical stage from viscoelastic to elastic.
9.2.4 Mobilization of microvesicles and vesicles involved in wall growth

Mobilization of the enzymes and precursors involved in cell wall synthesis to the sites
of the cell surface where wall growth occurs, takes place mostly within vesicles and
microvesicles, following the exocytic routes as is described in previous chapters. The
analysis of the effect of light on vesicle and microvesicle migration during the photo-
trophic response of P. blakesleeanus is an example of this phenomenon (Morales and Ruiz-
Herrera 1990) (see Figure 9.2). This process may be very rapid; for example, the speed at
which vesicles arrive at the growing zone in Neurospora crasa has been calculated be as
high as about 38,000 vesicles per minute (Collinge and Trinci 1974). Most studies on the
mechanism of vesicle migration indicate a role of the cytoskeleton in yeasts and mycelial
D P
A B
D
3
C
VESICLES (per mµ2)
2.5 PROXIMAL
DISTAL
2
1.5
1
0.5
0
2.5 5 7.5 10 12.5
DISTANCE (FROM THE APEX) (mµ)
Figure 9.2 The role of vesicles and microvesicles in wall synthesis during the phototropic response
of Phycomyces blakesleeanus stage I sporangiophores. (A) After asymmetric illumination, the spo-
rangiophore, which behaves as a convergent lens, bends and grows towards the light source. (B)
Section of the apical (growing) zone of the sporangiophore used to determine the number of micro
and macrovesicles in the proximal (P) and distal (D) zones. (C) Data reveal a decrease in the number
of vesicles in the distal side, indicating its faster rate of mobilization to increase the growth rate
of this side in comparison to the proximal one. (Modified from Morales, M. and Ruiz-Herrera, J.,
1990, Light-Induced Changes in the Vesicular Apparatus of Phycomyces Sporangiophores during the
Phototropic Response, Photochem. Photobiol. 52:223–227.) (See color insert.)
fungi, (reviewed by Steinberg 2007a,b; Chang and Martin 2009). Additional data were
obtained by the determination of the effect of agents that inhibit microtubule polym-
erization, for example, the effect of benomyl in N. crassa (That et al. 1988) and methyl
benzimidazole-3-yl carbamate in Fusarium acuminatum (Howard and Aist 1977, 1980). In
mycelial fungi, actin is oriented by the accumulation of formin molecules at the hyphal
tip providing the rails through which vesicles travel. Vesicles have no mechanisms to dis-
place, therefore their displacement depends on motor molecules that provide the energy
for their movement. Most authors have suggested that vesicles are driven by myosins as
revealed in different systems; for example, in S. cerevisiae, myosin-2 seems to be the most
important one (Govindan et al. 1995), whereas in hyphal fungi myosin-5 appears to be
the motor for vesicle displacement (reviewed by Steinberg 2007a,b). Contrary to these
accepted concepts, Regalado et al. (1995), based on mathematical analysis of the physical
stage and behavior of the apical region of hyphae, actin included, came to the conclusion
that a direct association of vesicles with actin was not necessary for their displacement,
and argued that this was a result of the viscoelatic properties of the hyphal tip. The role
of microtubules in the process of growth polarization including Spitzenkörper organiza-
tion (see below) has been sustained by results obtained by use of inhibitors that affect
their function, and by data obtained with mutants affected in kinesin (the anterograde
motors of microtubules) showing defects in polarization in several fungi (see for example,
Horio and Oakley 2005; Howard and Aist 1980; Riquelme et al. 2000). However, their role
in the movement of vesicles to the growing zones is less well understood, although some
authors support the idea for their role in the transport of vesicles along large distances
(see Steinberg 2007a,b, for reviews).
Although most of the evidence suggests the role of the cytoskeleton proteins in the
mobilization of vesicles and microvesicles to the sites where cell wall synthesis occurs,
evidence also exists that ion or electrical currents are also somehow involved in the pro-
cess, as evidenced by now almost forgotten, but solid experimental results. In N. crasssa,
Slayman and Slayman (1962) registered an electric potential along the hyphae, the tip
being more electropositive, a phenomenon existing in other fungi as demonstrated with
the use of a vibrating electrode (Gow 1984). It was evidenced that these electrical currents
were somehow involved in the directionality of wall growth by the observation that when
Schizophylum commune protoplasts were regenerated in a medium subjected to a weak elec-
tric field, most of the germ tubes produced, oriented parallel to the field with the growing
region directed towards the anode (De Vries and Wessels 1982). A similar orientation was
induced for germination of spores and blastospores of different fungi by an electric cur-
rent, with the characteristic that in some species, the growth was directed to the anode and
in others to the cathode (Crombie et al. 1990; McGillivrary and Gooday 1986). Nevertheless,
during the eternal discussion on cause or effect, it was concluded that the electrical cur-
rents are the product and not the cause for establishing the directionality of growth. The
favored hypothesis was that the currents were the result of the operation of transporters
involved in nutrient uptake, although it is difficult to coincide the experiments of growth
and polarization cited above with this concept.
Besides electrical currents, the existence of ion gradients with a possible association
to growth directionality has been noticed in different organisms. In this sense, the case
of Ca2+ is relevant. For example, it was observed that treatment of sporangiospores of P.
blakesleeanus with the calcium ionophore A23987 gave rise to an immediate halt in the syn-
thesis of chitin, and therefore in growth (Ruiz-Herrera et al. 1989). Analysis of the mode
of action of the ionophore discarded different possibilities, such as depletion of substrate,
inhibition of substrate synthesis, a role of calcium in chitin synthase activity, and others.
Finally, the observation that the ionophore also inhibited the secretion of invertase (trans-
ported in vesicles), and induced a reduction in the number of vesicles and chitosomes in
the apical zone of the hyphae, led to the conclusion that the ionophore was inhibiting the
normal process of vesicle and microvesicle mobilization to the hyphal tip. In a further
work, it was observed that not only the Ca2+ ionophore A23187, but also nigericin, a K+/H+
exchanger, had similar effects, in this case on N. crassa germlings (Alcántara-Sánchez et al.
2004). In addition, both compounds induced apical bursting and also branching, and aerial
growth of hyphae (escaping reactions). Interestingly, neither substance affected energy con-
servation in mitochondria, or the membrane potential of the fungus. According to these
data, it was concluded that the effect of both compounds was due to dissipation of a proton
gradient involved in the establishment of growth directionality that is brought about by
the mobilization of chitosomes and vesicles. What then would be the possible relationship
between the cytoskeleton and ion gradients? Data have been published on the existence
of a transhyphal ion current in N. crassa dependent on the presence of subapically located
membrane ATPases that expulse protons from the cell that reenter through the action of
H+/K+ antiporters or H+/PO43- symporters located at the apical region (Takeuchi et al. 1988).
Accordingly, it is feasible that these ion currents may be involved in the orientation of actin
cables and/or microtubules involved in vesicle and microvesicle migration, thus joining
both types of evidence. A further hypothesis discussed by Virag and Harris (2006) consid-
ers that calcium gradients may be involved in vesicle fusion with the plasma membrane
facilitating the process in such a way that the highest fusion process takes place at the sites
containing the highest calcium concentration.
9.2.5 Reinitiation of wall growth

The data discussed above are all related to growing fungi, where the new material would
be added to the expanding viscoelastic wall. It must be anticipated that a different process
should occur in the mechanism involved in wall synthesis in the case of either wall-less cells
or cells whose wall growth would have stopped expanding, and had already suffered a con-
solidation process. The first case applies to naked zoospores during the encystment process
that precedes their development of a mycelium, or to the process of wall regeneration in
artificially prepared protoplasts, and the second one to spore germination and branching.
The data obtained with two Chitridiomycota species, Blastocladiella emersonii and
Allomyces macrogynus are illustrative of the mechanism of wall synthesis by naked zoo-
spores. In B. emersonii, it was demonstrated that the whole primary cell wall was shortly
made (in a matter of 10 to 15 minutes) after the zoospore lost their flagella responding to
an inward flux of a monovalent cation (Lovett 1975). The process involved mobilization
towards the whole cell surface of a great number of vesicles and microvesicles carrying
cell wall precursors and synthesizing enzymes that fused with the plasma-lemma and
discharged their contents to the cell exterior or synthesized the corresponding polysaccha-
rides responsible for wall rigidity, mainly chitin, respectively. This process, representing
the sudden formation of a de novo made cell wall is probably the extreme example dem-
onstrating that fungal cell wall synthesis occurs by a self-assembly process. The origin of
the vesicles was a peculiar organelle with the appearance of a cup surrounded by a double
membrane (gamma particles), that is characteristic of this group of fungi and that consti-
tutes the reservoirs of chitin synthase (Cantino and Mills 1983). An almost exact process
occurred in A. macrogynus as described by Røne and Bøve (1977), who observed that after
only 6 minutes of initiation the process of encystment, blebbing of microvesicles (probably
chitosomes) from the gamma particles that fused with the plasma membrane, was already
visible. The exocytic process accelerated to a maximum after 15 minutes and decreased 20
minutes after encystment initiation. Chitin accumulation in the nascent wall, evidenced by
staining with calcofluor white that started early and reached a maximum after 15 minutes,
was also noticed during zoospore encystment in this fungus (Barstow and Pommerville
1980). As can be seen in these two examples, the cell wall synthesized during encystment
is an organized structure made of different chemical components in balanced proportions.
This phenomenon does not occur during regeneration of the cell wall by artificial pro-
toplasts, although this process is also an example of the de novo synthesis of a cell wall.
Instead of the deposition of a balanced mixture of the normal wall components, initially
there occurs the formation of an abnormal wall that will become organized long after
being deployed. Occurrence of the process of regeneration of protoplasts in almost all fun-
gal species analyzed requires that they be placed in hypertonic solid or semi-solid media
that probably avoids the diffusion of macromolecules necessary for the assembly of the
cell wall (Necas and Svoboda 1961; Necas et al. 1981). As was already described in a previ-
ous chapter, an exception to this rule is C. albicans whose protoplasts can be regenerated in
hypertonic liquid medium. A study of regeneration of the cell wall in this species revealed
that deposition of components of the wall followed a defined pattern, chitin being the first
component associated with what could be described as a nascent wall. Although several
cell wall proteins are synthesized at this stage, they are released to the medium, probably
because the absence of a support that would maintain them associated with the grow-
ing wall (Rico et al. 1997). Chitin deposition was followed by the addition of β-glucans
and manoproteins, whose role was considered important in the further development of
the cell wall since they corresponded to those present in mature yeast or mycelial walls,
depending on whether protoplasts regenerated at 28 or 37oC, conditions that induce yeasts
or mycelial growth, respectively (Elorza et al. 1987; 1994; Murgui, Elorza, and Sentandreu
1986). These proteins formed complexes with other ones at more advanced periods of pro-
toplast regeneration (Castillo et al. 2003). Analysis of the transcriptomic events that occur
during regeneration of C. albicans protoplasts revealed that genes belonging to the cat-
egory of cell wall organization and biogenesis became activated to the highest level in an order
reminiscent of the one followed by the deposition or their products in the nascent cell wall
(Castillo et al. 2006).
Finally, in the third mechanism of de novo wall growth, the one occurring in a cell
whose wall has become rigidified, the phenomenon may be divided into two processes:
controlled partial breakage of the mature cell wall, and reinititiation of wall growth. As
an example of this type of wall growth, we may cite branching. The rate of branching
depends on external factors, probably the most important of which is substrate concen-
tration, since the phenomenon of branching is important to colonize a niche containing
utilizable food material. For this reason, in poor media, branching is reduced, leaving
to the leading hyphae the foraging process, whereas in rich media, it is increased prob-
ably to exhaust all the nutritive material. Branching is also important for hyphal fusion,
an important event for the exchange of nutrients and signals among hypha of the same
colony (reviewed by Harris 2008). Other important factors are pH and temperature, and
specific factors as revealed by the increased branching produced in the mycorrhizal fungi
Gigaspora and Glomus by exudates from the roots of their hosts, or occurring in the forma-
tion of lichens as a response to the photobiont (reviewed by Harris 2008). The presence of
inhibitors also stimulates branching as an escape mechanism, as revealed by the effect of
ionophores described above (Alcántara-Sánchez et al. 2004). Branching also depends on
intrinsic factors, for example the phenomenon of apical dominance, whose mechanism is
poorly known, but probably depends on the accumulation of branching inhibitors in the
apical region of the leading hyphae (Schmid and Harold 1988; Semighini and Harris 2008).
Even apparently unrelated mechanisms may affect branch formation, as revealed by the
behavior of C. albicans mutants in the gene VAC8, involved in vacuole formation, whose
phenotype included besides a reduction in vacuole formation, an increased branching
frequency (Barelle et al. 2006). Apparently this effect is due to a reduction of the vacuole
volume in each fungal article that becomes filled with cytoplasm, a process that regulates
entry into S phase, suggesting a relation between branching and the cell cycle (Barelle et
al. 2003; see also Harris 2008).
The process of branching is complex and involves a great number of players. The first
event leading to the formation of a branch involves the selection of the site where it will be
formed, a phenomenon that depends on the factors mentioned above. The next step would
include the recruitment of the members of the complex responsible for the formation of a
new growth center; this process being followed by the polarization of growth (revised by
Harris 2008). Here, I should include as indispensable the activation or accumulation of lytic
enzymes that should produce a controlled breakdown of the cell wall to a stage that could
yield to the turgor pressure of the cell in order to initiate growth of cell wall of the branch.
Genetic data from several species have demonstrated the role of GTPases, both mono-
(Cdc42) and hetero-trimeric (FadA). Formins and septins involved in cytoskeleton polarity
have also been considered important members of the initiation process, which should be
followed by recruitment of synthetic enzymes such as glucan and chitin synthases. After
an initial period of branch growth, a new Spitzenkörper is formed almost simultaneously
with the appearance of microtubules parallel to the branch axis accompanied by the prod-
ucts of genes related to the Spitzenkörper and microtubules function, and which appear
to be specific to this process (Pod4, Pod5, and Pod8) (Seiler and Plamann 2003) leading to
the further growth of the branch (reviewed by Harris 2008).
Spores are the products of asexual or sexual reproductive processes of fungi, and
constitute the mechanism of resistance and/or fungal dispersal. Spores are in a dormant
state that may last for a short or long period of time until adequate conditions give rise to
their germination. The shape of the fungal spores is extremely variable, depending on the
species, although the ellipsoidal shape predominates. They may be unicellular or multi-
cellular, and contain one or several nuclei. Breakage of dormancy of the spores depends
on the organism; some only require an available carbon source, whereas others have com-
plex requirements, heating to a certain temperature, a previous cooling period, presence
of specific compounds, and even transit through the intestinal tract of an animal, prob-
ably to permit partial digestion of the spore coat. In the germination of S. cerevisiae asco-
spores, the presence of glucose alone is enough to break the dormancy (Herman and Rine
1997). Glucose acts on the GPa1 receptor that transfers the signal via a PKA transducing
pathway, that is not strictly linear, to activate protein synthesis, water entrance, trehalose
mobilization, and glycerol synthesis (see below). Homologues of the genes involved in
this process are known to exist in filamentous fungi (reviewed in Wendland 2001). It
is characteristic of a great number of fungal spores to contain significant amounts of
trehalose, probably involved in resistance to desiccation, and as a stored energy source
for germination. Trehalose is rapidly mobilized and metabolized during germination by
the glucosidase trehalase that is activated by phosphorylation with a PKA kinase (see
d’Enfert 1997 for review).
After activation, the germination process involves spore swelling, a process that not
merely constitutes the entrance of water into the spore, but is an active process of isotro-
pic growth (also denominated isodiametric or spherical growth) involving macromolecule
(proteins and RNA; DNA synthesis is normally delayed) biosynthesis, and germ tube
emergence. As an example, in Mucor spp. as a model of Zygomycota, three steps in spo-
rangiospore germination were noticed: IA, involving spherical growth that transforms the
ellipsoidal spore into a spherical structure without an increase in the length of the major
axis; Ib, involving spherical growth to attain the largest size of the spore; and II, character-
ized by the appearance of the germ tube (or bud if the environmental conditions favored
yeast growth). During spore germination, synthesis of proteins and RNA started without
any log phase or change in the rate, whereas DNA synthesis initiated about 30 minutes
before the beginning of stage II (Cano and Ruiz-Herrera 1988).
Regarding the behavior of the cell wall, during isotropic growth there is a cross-
talk between the PKA kinase pathway involved in spore activation (see above) and the
cell wall integrity pathway that acts as a safety mechanism to avoid cell wall breakage.
In Aspergillus niger as an example of Ascomycota, the initial step of spore germination
involved shedding of the outer layer of the spore (Tsukahara 1968). By use of atomic force
microscopy in the related species Aspergillus nidulans, it was demonstrated that the pro-
cess involved the removal of the hydrophobin (rodlet) layer that covers the spore in many
Ascomycota fungi (Ma et al. 2006) (see Chapter 3). It is possible that removal of the rodlet
layer occurs through a hydrolytic process, as revealed in the germination of macroconidia
from Microsporum gypseum. In this system, the presence of a protease in the spores that
was able to degrade the spore wall and was inhibited by PMSF was observed. Addition of
PMSF inhibited spore germination, whereas addition of the protease stimulated the pro-
cess. These data were taken as evidence that removal of the protein coat (rodlets) that cov-
ers the spores was necessary for spore germination (Leighton and Stock 1970). Following
this step, the outer spore layer is ruptured at the sites of germ emergence, the inner layer of
the spore wall is stretched, and the wall of the emerging germ tube developed, apparently
being continuous with it (Tsukahara 1968). In Fusarium sambucinus var. coeruleum electron
microscopic observations also revealed that the wall of the germ tube was an extension of
the original inner layer of the spore (Stålhammar-Carlemalm 1976), the same as occurred
with Aspergillus fumigatus spores (Campbell 1971), and Neurospora tetrasperma ascospores
(Lowry and Sussman 1968). A similar, but slightly different phenomenon occurs in the
case of Penicillium megasporum conidia, where the inner layer of the wall is thickened dur-
ing germination giving rise to splitting of the wall outer layers, and the thick inner layer
extends at a later stage to become the germling wall (Remsen et al. 1967). Examples of the
second type of wall formation during germination are less common, and most examples
come from Zygomycota species, as demonstrated originally in Rhizopus spp. (Hawker and
Abbott 1963). The general picture is the rupture of the spore wall during spore swell-
ing with formation of a new wall that starts the germinating process (Bartnicki-García,
Nelson, and Cota-Robles 1968; reviewed by Akai et al. 1976), the process following as
described above.
At this time, it is relevant to strongly insist in the existence of two main differences
between wall synthesis in branching and spore germination, both of which start with the
softening of a previously made mature wall. One of them is the change in the directionality
of cell wall growth, and the second one is the chemical nature of the hyphal wall resulting
from either process. In the case of branching, starting from the beginning of the process,
the cell wall grows apically, whereas in spore germination, there occurs spherical growth
in the first stages (swelling of the spores), and in later ones, its directionality changes to
be apical as clearly demonstrated in the germination of M. rouxii spores by use of pulses
of radioactive GlcNAc followed by autoradiography to detect the sites of chitin deposi-
tion (Bartnicki-García and Lippman 1977). Regarding the second difference, branching
involves the extension of the old wall, maintaining the same chemical composition as the
rest of the mycelial wall, whereas in the case of spore germination, the chemical composi-
tion of the wall from the vegetative forms is different from that of the spores. This indicates
that the germ tube cell wall is not a mere extension of the spore wall, even in those spores
germinating by the first procedure indicated above, but a chemically and structurally new
wall which is the product of a developmental program different from the one in progress
when the spore wall was synthesized. For example, regarding chitin, its proportions in the
cell walls of spores versus mycelium showed the following changes in the indicated spe-
cies: M. rouxii: 2.1%:9.4%; Aspergillus phoenices: 36.2%:23.7%; N. crassa: 7.4%:8.0%; Penicillium
chrysogenum: 11.4%:42%; and, Trichoderma viride: 12%:22%. (reviewed in Ruiz-Herrera 1992).
An interesting quantitative difference in the composition of the wall during germination
of spores corresponds to Penicillium notatum where an increasing content of glucosamine,
galactosamine, and glucose, took place; but a decrease in the concentration of galactose
was observed during spore swelling and germination (Martin, Nicolas, and Villanueva
1973). The best studied system is the one of M. rouxii, where quantitative analysis revealed
different amounts in all the components of the spore and vegetative forms of the cell walls
(Bartnicki-García 1968); but more interesting perhaps were the noticeable qualitative dif-
ferences observed; for example, spore walls, but not the wall from vegetative forms con-
tained melanins and glucans.
What is the mechanism of spore wall breakage that permits the development of the
germination tube and its further continuous growth? Considering the solid structure
of the cell wall, it is not surprising that a number of authors have concluded that lytic
enzymes must be involved in the initial stages of the germination process. With the use of
allosamidine, an inhibitor of chitinases, it was demonstrated that in the case of M. rouxii,
the substance inhibited spore germination (Gooday et al. 1992).
Another strong candidate of the process is turgor pressure, which in fungi may reach
very high values as demonstrated by the ones reached in appresoria from phytopatho-
genic fungi during host penetration. During the initial stage of spore germination, water
uptake occurs and a high turgor pressure develops at the expense of the high amounts of
solutes existing in a dried stage within the spores and glycerol synthesized from the exter-
nal carbon source, as occurs in sporangiospores from P. blakesleeanus (Van Schaftingen
and Van Laere 1985). In ungerminated conidia of a nonmelanized strain of Magnaporthe
grisea incubated for 2 hours (that is, in the swelling stage), the turgor pressure reached a
value of 1.4 MPa (Money and Howard 1996). For this reason, some authors are inclined to
think that breakage of the spore wall during germination results from the extreme force
exerted by the turgor pressure. This idea is perhaps partially true, and the data described
with a mutant of Glomerella graminicola are illustrative of the problem (Epstein et al. 2001).
The authors of this work observed that spores of this genetically tagged T30 mutant suf-
fered a sudden lysis during germination. Microscopy of the falcate-shaped conidia of the
fungus during breakage revealed that this always occurred in the median part of the
conidia where the highest force is applied by the turgor pressure, and parallel to conid-
ial main axis. No increase in lytic enzymes of the mutant as compared to the wild-type
strain of the fungus was observed, suggesting that they were not involved in breakage.
Nevertheless, the authors recalled that germ tube emergence during germination of nor-
mal conidia occurred apically, not in the middle of the spores as should happen if turgor
pressure were the only factor responsible for the normal process of wall breakage during
germination. This result suggests that it is the mixture of a high turgor pressure operat-
ing over a weakened cell wall that is responsible for the onset of germ tube formation,
and that in this and other fungal species, weakening of the wall is probably the result of
the action of lytic enzymes operating at specific parts of the cell surface, whose selection
may be random, or depend on internal or external factors; for example, the vicinity of
other spores, gradients of nutrients or certain ions, and even ionic or electrical currents
as described above. The behavior of the G. graminicola mutant resembles the phenotype
of the S. cerevisiae fragile mutants. These mutants isolated by Venkov et al. (1974) can only
grow in hypertonic media, and when transferred to normal medium they suddenly lyse
releasing their cytoplasm. Observation of the walls from lysed cells revealed that they
showed only breakage points at selected sites of the surface. Chemical composition of the
cell walls from the mutants was normal, same as their structure and width (Mateeva et al.
1976). Analysis of the relative amounts of wall per cell, and the amount of neutral polysac-
charide in the wall showed normal values, and they even revealed larger amounts of chitin
when compared to the wild-type (Ruiz-Herrera et al. 1998). As a final test, an increase in
carbohydrate reducing ends, as an indication of polysaccharide hydrolysis was measured
with a method described by Herrera- Estrella and Ruiz-Herrera (1983) that involves label-
ing with radioactive borohydride. Wild-type and mutant cells were collected and washed
with either a hypertonic sorbitol solution or water, in the latter case to induce cell breakage;
cell walls were isolated and subjected to analysis. Data showed that the relative amounts
of reducing ends (osmotic shock/nonosmotic shock) in the wild-type remained without
change, whereas those from the mutant increased to the double (Ruiz-Herrera et al. 1998).
These data were interpreted as an increased amount of lytic enzymes on the grounds of
the theories developed by Koch (1983; 1985; 1988) to explain growth of bacteria without
lysing, the same dilemma confronted by all walled organisms.
Analysis of different mutants affected in spore germination has revealed the involve-
ment of a number of factors in the initial stages of germ tube formation. Among these is
the progression of the cell cycle as observed with mutants bimE and nimM from A. nidu-
lans defective at the S phase, Ca2+-calmodulin dependent reactions, protein phosphatase
BimG11, and more interestingly for the process analyzed here, correct cell wall synthesis
(reviewed by d’Enfert 1997).
Polyamines that play a key role in cell differentiation (reviewed in Ruiz-Herrera 1994)
are also involved in this process, as demonstrated by the observation that inhibitors of the
first ezyme involved in their synthesis (ornithine decarboxylase, ODC) by diamino buta-
none (DAB) did not affect swelling of spores of different Mucor species, which attained
large sizes, but failed to form germ tubes (Obregon et al. 1990). The engrossed cell wall
had an abnormal structure and showed important quantitative and qualitative differences
with the normal spore wall (Obregon et al. 1990) (see Figure 2.1, Chapter 2). Elimination
of DAB led to spore germination with formation of an engrossed germ tube that later
acquired the normal width. The wall of the germ tube appeared to originate de novo, not
being continuous with any layer of the abnormal swollen spore.
Bud emergence in yeast and germ tube formation in filamentous fungi have mecha-
nisms in common, and the latter one has been analyzed as a model of bud emergence.
However, as indicated above, the budding processes although similar to spore germina-
tion in mycelial fungi, display important differences. Momany (2002) has called attention
to the fact that due to the difference in terminology used by different authors to define
when a spore has germinated, some confusion exists regarding the role of all the factors
described to be involved in polarization, some only indirectly related to the establish-
ment of polarization during spore germination. In general, it is accepted that localization
of the Rho GTPase Cdc42 in yeast, as well as in filamentous fungi, define the position of
bud emergence in yeast, or germinating tube in filamentous fungi, and is correlated with
growth polarization as shown by several methods including the use of negative mutants of
Ashbya gossypii, whose polarity is lost (Wendland and Philippsen 2001). The selection of the
site of origin of buds or germ tubes is followed by the organization of the morphogenetic
machinery with the help of formins and septins (reviewed by Harris and Momany 2004).
In the Aspergilli species and other fungi, this information is relayed with the help of its
GTP exchange factor (GEF) Cdc24 and the GTPase activating proteins (GAPs) Rga, Bem2,
and Bem3 that operate downstream to the protein activated kinases Ste20 and Cla4. Actin,
tubulins, and components of the polarisome necessary for the organization of actin and
the exocyst (a complex involved in secretion) are involved in the process, similarly to the
process that occurs in S. cerevisiae (reviewed by Harris and Momany 2004).
During the transition of isotropic to polarized growth, there is a direct intervention of
the cytoskeleton. As demonstrated during germination of M. rouxii sporangiospores, the
distribution of actin suffers a drastic change, from a uniform distribution in the swollen
spores to a preferred localization at the site of emergence of the germination tube and to the
apex of the nascent hypha (Hasek and Bartnicki-García 1994). These authors also observed
a change in the directionality of the microtubules that in the swollen spores were oriented
in all directions, whereas in the germ tube they appeared parallel to the main axis. Despite
these data, to conclude that initiation of the oriented growth is due to a rearrangement of
the cytoskeletal elements is hard to decide, especially considering that these changes in ori-
entation of the cytoskeletal elements appear to follow a previous signal as indicated above.
The complexity of the process is indicated by the observation that, as described above,
analysis of different mutants affected in spore germination has revealed the involvement,
not of one, but of a number of factors in the initial stages of germ tube formation.
9.3 Polarization of cell wall synthesis and apical growth

9.3.1 Introduction
A characteristic of the mycelial fungi is that growth occurs at the apex, a process known
as apical growth. That apical growth is the canonical habit of hyphal expansion was dem-
onstrated by the classical experiment from Bartnicki-García and Lippman (1969) who gave
short pulses of tritiated GlcNAc to Mucor rouxii growing in a N2 atmosphere (to force the
formation of a single germination tube). At short periods of time, the cells were extracted
with hot alkali to eliminate most of the cell components leaving mainly chitin, and this
was followed by autoradiography of the residual cell ghosts. The authors observed that
almost all the radioactivity, seen as silver grains, appeared at the most apical region of
the hyphae. These results were confirmed in other fungal species (Gooday 1971; Galun
1972; Galun, Malki, and Galun 1981), leaving the question settled. The contrasting growth
pattern occurs in yeast cells that grow all over their surface, as indicated before when we
referred to cell wall expansion. This so-called spherical or isodiametric growth pattern
was initially demonstrated in the work by Bartnicki-García and Lippman (1969) using the
same autoradiographic technique described above using yeast cells of M. rouxii that are
almost spherical and where the silver grains appeared homogeneously distributed all over
the cell surface.
But what happens when a cell is not perfectly spherical, as occurs with most yeasts?
Interestingly, it was demonstrated by different techniques that both growth patterns
occurred simultaneously giving rise to a cell shape in the form of a spheroidal of revolution
approaching a sphere more or less depending on the prevalence of one or the other pat-
terns of growth. This process was demonstrated by two types of experimental approaches;
one method relied on the measurement of the cell wall width of S. cerevisiae cells subjected
to macromolecule inhibition (Rodriguez, Laborda, and Sentandreu 1979). The other one
was a very ingenuous experiment that involved the measurement through videorecord-
ing of the displacement of polystyrene beads attached to the surface of growing C. albicans
cells (Staebell and Soll 1985).
9.3.2 Establishment of polarity

Apical growth is due to the establishment of a property of growing hyphae denomi-
nated polarity. Polarity is not a specific characteristic of fungal cells; it appears to be a
widely distributed property, and clear examples of the process may be observed in the
growth of bacilli, pollen tubes, neurons, roots, and as suggested by Baluska et al. (2003),
by almost any cell that departs from the default spherical shape. These authors also cau-
tion differentiating true polarity from unidirectional growth and consider that in the
establishment of polarity, both ends of a cell are as opposite as north and south, and as
indicated above came to the conclusion that polarity depends in walled organisms, fungi
included, on the action of turgor pressure operating on a wall with different mechanical
characteristics, one that is expandable, whereas the other has been rigidified in an irre-
versible way (unless lytic enzymes soften the cell wall, again, I should add). An additional
characteristic of polarized growth is that it depends on targeting vesicles through an
actin cytoskeleton (see for example, Drubin and Nelson 1986; Pruyne and Bretscher 2000a;
2000b), without eliminating the possible intervention of microtubules (Sawin and Nurse
1998). For a revision of the whole polarity process in yeasts and fungi, and the actors
involved see Sudbery (2008).
As repeatedly mentioned, cell polarity is established by the distribution of the small
monomeric GTPase Cdc42 on the plasma membrane. The active form of Cdc42 is bound
to GTP, and its turning on and off depends on GTP binding and hydrolysis, respectively.
These reactions are catalyzed by GTPase activating proteins (GAPs) or the guanine nucleo-
tides-exchange-factors (GEFs), respectively. The selective localization process is followed by
a rearrangement of the cytokeleton (see Pruyne and Bretscher 2000a for a review). Evidence
for a role of actin in the initial period of spore germination, that is, at the onset of polarized
growth, was described above. Regarding the maintenance of polarization in further hyphal
growth, the role played by the cytoskeleton has also received important experimental evi-
dence, as indicated above. The observation of the behavior of actin during the establish-
ment of polarity in yeasts is illustrative. Actin exists mainly in two forms, cortical patches
and actin cables in the form of long F-actin bundles. Their localization and orientation are
random during the growth of the cell, but when budding is initiated, actin patches accumu-
late at the selected site of bud initial, forming a ring, whereas actin cables extend from the
mother cell to the cortical patches (see Pruyne and Bretscher 2000b for a review).
Regarding the molecular motors involved in the mobilization of vesicles and microves-
icles that sustain polarity, it has been described that only a discrete number of the many
kinesins possessed by fungi are involved in the process: mainly kinesins 1, 3, and 7;
whereas the rest are involved in the mobilization of nuclei and in meiosis (reviewed by
Steinberg 2007b).
Once established, for the polarization end, it is characteristic that the polarized cells
acquire several special features: the presence of a cell wall with mechanical and struc-
tural characteristics that as already discussed is different from those of the mature wall;
the presence of a Spitzenkörper (already described, but see below); and, the presence of a
polarisome. The polarisome is a protein complex also involved in polarization by direct-
ing the localized assembly of actin filaments at the sites of polarization; in S. cerevisiae the
polarisome is made by proteins Bni1, Spa2, Pea2, and Bud6. Of these, Spa2 and Bud6 are
conserved in S. pombe and mycelial fungi (Pruyne and Bretscher 2000a; Virag and Harris
2006). A further characteristic is the presence of microdomains in the plasma membrane at
the apical region, known as lipid rafts. These are made by the accumulation of sterols and
sphingolipids. In A. nidulans, it was found that sterols were necessary for the correct posi-
tioning of Tea and TeaR, important cell-end markers, and probably of the whole cell-end
marker positioning system (Takeshita et al. 2008). The question of how sterols are localized
at the apex comes next. This dependence on localization of one component from the others
is a further example of the cause and effect dilemma. The observation of the existence of a
relationship between Cdc42 and formins with sterols has led Fischer, Zekert, and Takeshita
(2008) to suggest the existence of an interdependent correlation between Cdc42/formins,
cell-end marker, and sterol-rich domains. The role of these rafts is not clear enough, but it
is known that sphingolipids and ceramide, a simplified sphingolipid, play different roles
in cell signaling and the organization of the cytoskeleton (Futerman and Hannun 2004).
In S. cerevisiae, it was observed that mutants lacking their two ceramide synthases, Lag1
and Lac1, display severe alterations including some present in the cell wall (Schorling et al.
2001). In A. nidulans, by analysis of the effect of a compound extracted from the biocontrol
agent Lysobacter enzymogenes C3, it was concluded that lipid rafts made of ceramide, syn-
thesized by an enzyme specific to filamentous fungi, was essential for the establishment
and maintenance of cell polarity (Li et al. 2006). Another factor related to lipid metabolism
that was described as necessary to initiate and maintain cell polarity during the myce-
lial growth of A. nidulans was protein myristoylation, specifically of the ADP ribosylation
factor B (ArfB) (Lee and Shaw 2008). The authors suggested that the effect was related to
endocytosis, although this connection did not appear very strong. More convincing data
on the role of endocytosis in apical growth were provided by Abenza et al. (2009), who
observed the role of endocytic compartments in the movement and recycling of microtu-
bule plus ends close to the endocytic apical zone, although the role of this phenomenon in
cell wall expansion remained unexplained.
As already stated, apical growth in hyphae is related to the presence of an organ-
elle that occupies a central position at the apical zone. This organelle, the Spitzenkörper,
was originally described as a basophilic body by Brunswick (1924) in hyphae of Coprinus
starquilinus and Coprinus narcoticus. The structure of this organelle at the level of electron
microscopy was revealed years later by Girbardt (1955, 1957, 1969), who described that it
was made by the massive accumulation of vesicles, characteristically of two sizes: vesicles
and microvesicles, a result that was later confirmed by many authors (see below). In order
to understand the function of the Spitzenkörper, Girbardt prepared a classical time-lapse
film of the behavior of the Spitzenkörper in living hyphae of Polystictus, where the organ-
elle appeared as a dense body under phase contrast microscopy. It was observed that the
body anticipated any change in the direction of growth of the hyphae. Under strong illu-
mination, the Spitzenkörper disappeared bringing about the immediate suspension of
hyphal expansion and afterwards a new Spitzenkörper was slowly formed by the coales-
cence of what appeared to be vesicles, normally not at the apical site. The formation of the
new Spitzenkörper gave rise to reestablishment of hyphal growth in a direction corre-
sponding to the location of the new Spitzenkörper in the apical dome of the hyphae. New
Spitzenkörper formation also preceded the sites of branch or fibulae formation. All these
results constituted the evidence to consider that the Spitzenkörper had the role to direct
polarized (apical) growth of fungal hyphae.
As would be expected, the main components of the Spitzenkörper are vesicles and
microvesicles (we already mentioned the localization of chitosomes in the central part of
Spitzenkörper). They also contain ribosomes, microtubules, and microfilaments (Virag
and Harris 2006). As a general idea on the role of the Spitzenkörper, it is possible to sum-
marize it as follows: to serve as a collector of vesicles and microvesicles synthesized at
points farther from the hyphal tip and discharge them to the cell surface under a strict
program, which permits the correct synthesis of the plasma membrane and the cell wall
during cell expansion. According to this concept, function of the Spitzenkörper is also
related to the shape of fungal hyphae. Harris (2009) has recently suggested that a fur-
ther role of the Spitzenkörper may be related to developmental transitions in fungi. This
hypothesis was based on data published by Etxebeste et al. (2009), who described that the
transcription factor FlbB that regulates the expression of BrlA (the master transcriptional
regulator that controls sporulation in Aspergillus species) was located in the Spitzenkörper
present in the initial hyphae leading to conidiophore development. As an additional role
of FlbB in vegetative growth, the authors reported that flbB mutants had alterations in their
sensitivity to high osmotic pressure and in branching. Whether the presence of FlbB in the
apical region is incidental, or it really constitutes an additional functional component of
the Spitzenkörper, may be open to discussion.
How do vesicles accumulated in the Spitzenkörper move to the surface to discharge
their cargo in filamentous fungi is still a matter little understood. As discussed in the
previous chapter, migration and fusion of the vesicles and microvesicles to the plasma
membrane in fungi follow the same mechanism as in other cells, according to the SNARE
hypothesis described in Chapter 8. A multiproteic complex called the exocyst made by
eight proteins: Sec3, Sec5, Sec6, Sec8, Sec15, Exo70, and Exo84, has been described to be
involved in the docking of vesicles to the plasma membrane (Virag and Harris 2006).
9.3.3 Development of the hyphal shape

Hyphae, as everybody knows have a cylindrical shape with a hemispheroidal shape at the
tip. The problem is to explain how hyphae from all fungi have the same shape irrespec-
tive of species, diameter, velocity of growth, or any other variable factor with the excep-
tion of those that selectively affect the morphogenetic pattern of the organism. Some early
mycologists tried to solve the problem by simple geometry simplifying the shape of the
apical zone (the dome) in the form of a hemisphere, and considering fungal hyphae to have
an apical type of growth. Accordingly, it was considered that growth followed a decaying
kinetics being maximal at the very tip and zero at the base of the dome. When data were
represented in a two-dimensional figure, using as a relative measurement of growth rate,
the cosine of the angle formed between a line (axis) located at the center of the hyphae
and lines traced from the center of the base of the dome to any point on the cell surface,
the shape obtained was a hemicircle. The model was a good approach to the problem but
many questions remained: (i) it was beyond the sensitivity of the methods to accurately
measure cell wall growth within the apical zone; (ii) the apical dome of hyphae is not
hemispherical but hemispheroidal, if they were, the ratio of length of the dome to the
base would be 1.0; and careful measurements using different fungi gave values between
2.48 and 4.46 (Trinci and Saunders 1977); and finally (iii) the parameters required by the
model have no cytological equivalents: what is the base of the dome, the center of the
hypha, apical pole, and angular coordinates? This question remained somewhat forgotten,
maybe because the pressure of the times was more in favor of molecular biology analysis,
and because the great gap in our (still) poor knowledge of the mechanical properties of
the cell wall. We should thank Bartnicki-García, Hergert, and Gierz (1989a, 1989b) who
opened their minds to new approaches to understand the mechanics of cell wall growth
in fungi. They originally conceived a very simple model by computer simulation where a
cell (originally of circular shape) grew by the addition of growth units (small squares on a
grid) provided by a vesicle supply center (VSC). These tutipotential growth units were ran-
domly distributed to the surface of the cell represented on a grid. Every time a growth unit
reached the cell surface, it changed from empty to full. The data obtained by this simplistic
model were extremely interesting. When the VSC remained at the center of the cell, this
increased in surface in a symmetrical way, as occurs during the period of spore swelling.
However, if the VSC moved away from the centre, growth started to occur asymmetrically,
the closer to the surface of the original cell it was maintained, the more the growing struc-
ture approached a tubular shape following this equation in Cartesian coordinates:
v = xcot V · x/N
where V is the rate of VSC linear displacement and N is the number of vesicles per time
unit (see Figure 9.3). In fact, all shapes shown by normal fungal cells could be matched
by equating the movements of the VSC. Interestingly, in the case of actual photographs
of mature hyphae, the position of the Spitzenkörper was found to correspond to that of
the VSC when the parameters of the photograph permitted adjusting them to those of the
model (see Figure 9.4).
VSC: vesicle supply center

X
Y Y
HYPHOID EQUATION y = x cot (xV/N) X
Figure 9.3 Schematic representation of the location and behavior of the vesicle supply center (VSC)
in the apical region of a hypha, and the hyphoid equation as a result of its activity. (Courtesy of S.
Bartnicki-García.)
VSC
SPK
Figure 9.4 Median section of the apical region of a hypha from N. crassa showing the spatial correla-
tion of the vesicle supply center and the Spitzenkörper. (Courtesy of S. Bartnicki-García.)
Very important conclusions from these preliminary experiments were that (i) it was
not necessary to invoke inexistent elements of the hypha to explain the mechanics of cell
wall growth, (ii) there was a possible correlation between VSC and Spitzenkörper, and (iii)
important perhaps not for the specific aspects of cell wall morphogenesis, but for geom-
etry in general, the existence of new family of curves, which because of their similarity to
fungal cells was named hyphoid.
To obtain the necessary experimental evidence for the model was not an easy task, and
it took several years of dedication by Bartnicki-García et al. before this was possible. The
tool employed was computer-enhanced videomicroscopy, with which the growth and tra-
jectory of the hyphae could be measured with great precision, and data were compared with
the theoretical expected results. The coincidence of the results provided strong experimen-
tal evidence (for example, Bartnicki-García, Bartnicki, and Gierz 1995a; Bartnicki-García,
Iso
ISOMETRIC
Ort
Rot
ORTHOGONAL
0 3
ROTATIONAL
6 12
Figure 9.5 Schematic representation of the three possible mechanisms of hyphal growh, and the
theoretical position of surface markers in each growth model. (Courtesy of S. Bartnicki-García.) (See
color insert.)
Bartnicki, and Gierz et al. 1995b) at least to the two-dimensional model. However, the
problem was that it failed to explain how the final shape at the three-dimensional level
was attained (Bartnicki-García et al. 2000). Although the logical conclusion is to consider
that the 2-D data are the projection of a 3-D object, the authors considered it necessary to
obtain a final answer to this uncertainty, and proceeded to determine experimentally how
the expansion of the cell wall involves one of the three possible mechanisms: (i) isometric
mode, where new material displaces the older one equally in three dimensions; (ii) orthog-
onal model, where displaced material moves perpendicularly to the existing surface, and
iii) rotational, where the walls maintains the shape of the 2-D rotated along the long axis
(see Figure 9.5). To perform these experiments, the authors used as external markers car-
bon particles placed in the medium that attached to the hyphae passing by. As internal
markers, they used small imperfections of the cell wall of the hyphae under observation.
Their results demonstrated that in all of the 18 hyphal tips analysed, the experimental data
only coincided with an orthogonal expansion of the cell wall, providing evidence that this
is the mechanism of expansion of the cell wall of hyphae (see Figure 9.6).
However, in this work the authors already warned that the shape of the hyphal tip
was not the only element necessary to define hyphal morphogenesis, and indicated that
it was necessary to rigorously define the spatial pattern of wall displacement (Gierz and
Bartnicki-García; 2001). Additionally, the authors discussed that the only physical force
that can generate an orthogonal expansion of the wall is turgor pressure, a demonstration
that put an end to a long dispute on the subject, and finally constituted evidence to dem-
onstrate that hyphae do not rotate during growth. It was well known that during growth,
sporangiophores from P. blakesleeanus rotate (for example, see Roelofsen 1950), probably by
reorientation of chitin microfibrils. The probability that this was a phenomenon common
to fungal hyphae had been previously entertained (for a discussion of the problem see
Ruiz-Herrera 1992).
R O I R O I
9 3 4
7.5 0.5 0
1
2
R O I R O I
Figure 9.6 Scheme showing the typical results of the movement of an external marker on the sur-
face of a fungal hypha. The solid lines and the corresponding letters represent the theoretical move-
ment of the growing cell surface (R: rotational; O: orthogonal; I: isometric). The circles indicate
the observed experimental results, showing that the fungal wall grows in the apical region by an
orthogonal mechanism. (Courtesy of S. Bartnicki-García.)
References
Abenza, J. A., Pantazopoulouose, A., Rodrıíguez, J. M. Galindo, A., and Peñalva, M. A. 2009. Long-
distance movement of Aspergillus nidulans early endosomes on microtubule tracks. Traffic
10:57–76.
Ahlquist, C. N. and Gamow, R. I. 1973. Phycomyces: Mechanical behavior of stage-II and stage-IV
sporangiophores. Plant Physiol. 51:586–587.
Akai, S., Fukutomi, M., Kunoh, H., and Shiraishi, M. 1976. “The Structure of the Spore Wall and Germ
Tube Change during Germination.” In The Fungal Spore. Form and Function, 2nd ed. Edited by D.
J. Weber and W. M. Hess, 355–410. New York: John Wiley and Sons.
Alcántara-Sánchez, F., Reynaga-Peña, C. G., Salcedo-Hernández, R., and Ruiz-Herrera, J. 2004.
Possible role of ionic gradients in the apical growth of Neurospora crassa. Ant. v Leeuwenhoek
86:301–311.
Baluska, F., Wojtaszek, P., Dieter Volkmann, D., and Barlow, P. 2003. The architecture of polarized cell
growth: The unique status of elongating plant cells. BioEssays 25:569–576.
Barelle C. J., Bohula, E. A., Kron, S. J., Wessels, D., Soll, D. R., Schäfer, A., Brown, A. J. P., and Gow,
N. A. R. 2003. Asynchronous cell cycle and asymmetric vacuolar inheritance in true hyphae of
Candida albicans. Eukaryot. Cell 2:398–410.
Barelle, C. J., Richard, M. L., Gaillardin, C., Gow, N. A. R., and Brown, A. J. P. 2006. Candida albi-
cans VAC8 is required for vacuolar inheritance and normal hyphal branching. Eukaryot. Cell
5:359–367.
Barstow, W. E. and Pommerville, J. 1980. The ultrastructure of cell wall formation and of gamma
particles during encystment of Allomyces macrogynus zoospores. Arch. Microbiol. 128:179–189.
Bartnicki-García, S. and Lippman, E. 1972a. The bursting tendency of hyphal tips of fungi:
Presumptive evidence for a delicate balance between wall synthesis and wall lysis in apical
growth. J. Gen. Microbiol. 73:487–500.
Bartnicki-García, S. and Lippman, E. 1972b. Inhibition of Mucor rouxii by polyoxin D: Effects on chitin
synthetase and morphological development. J. Gen. Microbiol. 71:301–309.
Bartnicki-García, S. and Lippman, E. 1977. Polarization of cell wall synthesis during spore germina-
tion of Mucor rouxii. Exp. Mycol. 1:230–240.
Bartnicki-García, S., Bartnicki, D. D., and Gierz, G. 1995a. Determinants of fungal cell wall morphol-
ogy: The vesicle supply center. Can. J. Bot. 73:S372–S378.
Bartnicki-García, S., Bartnicki, D. D., Gierz, G., López-Franco, R., and Bracker, C. E. 1995b. Evidence
that Spitzenkörper behavior determines the shape of a fungal hyphae: A test of the hyphoid
model. Exp. Mycol. 19:153–159.
Bartnicki-García, S., Bracker, C. E., Gierz, R., Lopez-Franco, R., and Lu, H. 2000. Mapping the growth
of fungal hyphae: Orthogonal cell wall expansion during tip growth and the role of turgor. J.
Biophys. 79:2382–2390.
Bartnicki-García, S., Hergert, F., and Gierz, G. 1989a. Computer simulation of fungal morphogenesis
and the mathematical basis for hyphal (tip) growth. Protoplasma 153:46–57.
Bartnicki-García, S., Hergert, F., and Gierz, G. 1989b. “A Novel Computer Model for Generating
Cell Shape: Application to Fungal Morphogenesis.” In Biochemistry of Cell Walls and Membranes.
Edited by P. Kuhm et al., 43–60. Heidelberg: Springer-Verlag.
Bartnicki-García, S., Nelson, N., and Cota-Robles, E. 1968. Electron microscopy of spore germination
and cell wall formation in Mucor rouxii. Arch. Mikrobiol. 63:242–255.
Bartnicki-García, S. and Nickerson, W. J. 1962. Induction of yeast-like development in Mucor by car-
bon dioxide. J. Bacteriol. 84:829–840.
Bruswick, H. 1924. “Untersuchungen uber Geschlechts und Kernverhaltnisse bei der
Hymenomyzetengattung Coprinus.” In Botanische Abhandlungen, Vol. 5. Edited by K. Goebel,
1–152. Jena: Gustav Fisher.
Burnett, J. H. 1979. “Aspects of the Structure and Growth of Hyphal Walls.” In Fungal Walls and Hyphal
Growth. Edited by J. H. Burnett and A. P. J. Trinci, 1–25. Cambridge: Cambridge University
Press.
Mycol. 11:128–140.
Campbell, C. K. 1971. Fine structure and physiology of conidial germination in Aspergillus fumigatus.
Trans. Brit. Mycol. Soc. 57:393–402.
Cano, C. and Ruiz-Herrera, J. 1988. Developmental stages during the germination of Mucor sporan-
giospores. Exp. Mycol. 12:47–59.
Cantino, E. C. and Mills, G. L. 1983. “The Blastocladielan γ Particle: Once Viral Endosymbiont, Now
‘Chitosome’ Progenitor.” In Fungal Differentiation. Edited by J. E. Smith, 175–210. New York and
Basel: Marcel Dekker.
Castillo, L., Martinez, A. I., Garcera, A., Elorza, M. V., Valentin, E., and Sentandreu, R. 2003.
Functional analysis of the cysteine residues and the repetitive sequence of Saccharomyces cerevi-
siae Pir4/Cis3: The repetitive sequence is needed for binding to the cell wall β-1,3-glucan. Yeast
20:973–983.
Castillo, L., Martínez, A. I., Garcerá, A., García-Martínez, J., Ruiz-Herrera, J., Valentín, E., and
Sentandreu, R. 2006. Genomic response programs of Candida albicans following protoplasting
and regeneration. Fungal Genet. Biol. 43:124–134.
Castle, E. S. 1940. Discontinuous growth of single plant cells measured at short intervals and the
theory of intussusceptions. J. Cell. Comp. Physiol. 15:285–298.
Castle, E. S. 1958. The topography of tip growth in a plant cell. J. Gen. Physiol. 41:923–926.
Chang, F. and Martin, S. G. 2009. Shaping fission yeast and microtubules. Cold Spring Harb. Perspectives
Biol. 1:a001347.
Collinge, A. J. and A. P. Trinci. 1974. Hyphal tips of wild-type and spreading colonial mutants of
Neurospora crassa. Arch. Microbiol. 99:353–368.
Crombie, T., Gow, N. A. R., and Gooday, G. W. 1990. Influence of applied electrical fields on yeast and
hyphal growth of Candida albicans, J. Gen. Microbiol. 36:311–317.
d’Enfert, C. 1997. Fungal spore germination: Insights from the molecular genetics of Aspergillus nidu-
lans and Neurospora crassa. Fungal Genet. Biol. 21:163–172.
rouxii. J. Gen Microbiol. 130:2095–2102.
De Vries, S. C. and Wessels, J. G. H. 1982. Polarized outgrowth of hyphae by constant electrical field
during reversion of Schizophyllum commune protoplasts. Exp. Mycol. 6:95–98.
Drubin, D. G. and Nelson, W. J. 1986. Origins of cell polarity. Cell 84:335–344.
Elorza, M. V., Marcilla, A., Sanjuan, R., Mormeneo, S., and Sentandreu, R. 1994. Incorporation of
specifc wall proteins during yeast and mycelial protoplast regeneration in Candida albicans.
Arch. Microbiol. 161:145–151.
Elorza, M. V., Murgui, A., Rico, H., Miragall, F., and Sentandreu, R. 1987. Formation of a new cell wall
by protoplasts of Candida albicans: Effect of papulacandin B, tunicamycin and Nikkomycin. J.
Gen. Microbiol. 133:2315–2325.
Epstein, L., Bartnicki-García, S., Lippman, E., Bassein, S., and Amnuaykanjanasin, A. 2001.
Catastrophic wall rupture during conidial germination of a genetically tagged mutant of
Glomerella graminicola. Mycol. Res. 105(2):132–137.
Etxebeste, O., Herrero-García, E., Araujo-Bazán, L., Rodríguez-Urra, A. B., Garzía, A., and Espeso,
E. A. 2009. The bzip-type transcription factor FlbB regulates distinct morphogenetic stages of
colony formation in Aspergillus nidulans. Mol. Microbiol. 73:775–789.
Fischer, R., Zekert, N., and Takeshita, N. 2008. Polarized growth in fungi—Interplay between the
cytoskeleton, positional markers and membrane domains. Mol. Microbiol. 68:813–826.
Fuchs, B. B. and Mylonakis, E. 2009. Our paths might cross: The role of the fungal cell wall integrity
pathway in stress response and cross talk with other stress response pathways. Eukaryot. Cell
8:1616–1625.
Futerman, A. and Hannun, Y. A. 2004. The complex life of simple sphingolipids. EMBO J. 5:777–782.
Galun, E. 1972. Morphogenesis of Trichoderma: Autoradiography of intact colonies by (3H)
N-acetylglucosamine as a marker of new cell wall biosynthesis. Arch. Mikrobiol. 86:305–314.
Galun, M., Malki, D., and Galun, E. 1981. Visualization of chitin-wall formation in hyphal tips and
anastomoses of Diplodia natalensis by fluorescein-conjugated wheat germ agglutinin and [3]
N-acetyl-D-glucosamine. Arch. Microbiol. 130:105–110.
Gierz, G. and Bartnicki-García, S. 2001. Three-dimensional model of fungal morphogenesis based on
the vesicle supply center concept. J. Theor. Biol. 208:151–164.
Girbardt, M. 1955. Lebendbeobachtungen on Polystictus versicolor (L.). Flora 142:540–563.
Girbardt, M. 1957. Der Spitzenkörper von Polystictus versicolor (L.). Planta 50:47–59.
Girbardt, M. 1969. Die Ultrastruktur der Apikal region von Pilzhyphen. Protoplasma 67:413–441.
Gooday, G. W. 1971. An autoradiograph study of hyphal growth of some fungi. J. Gen. Microbiol.
67:125–133.
Gooday, G. W., Wei-Yun Zhu, W. Y., and O’Donnell, R. W. 1992. What are the roles of chitinases in the
growing fungus. FEMS Microbiol. Lett. 100:387–391.
Govindan, B., Bowser, R., and Novick, P. 1995. The role of Myo2, A yeast class V myosin, in vesicular
transport. J. Cell Biol. 128:1055–1068.
Gow, N. A. R. 1984. Transhyphal electrical currents in fungi. J. Gen. Microbiol. 130:3313–3318.
Harris, S. D. 2008. Branching of fungal hyphae: Regulation, mechanisms and comparison with other
branching systems. Mycologia 100:823–832.
Harris, S. D. 2009. The Spitzenkörper: A signaling hub for the control of fungal development? Mol.
Harris, S. D. and Momany, M. 2004. Polarity in filamentous fungi: Moving beyond the yeast para-
digm. Fungal Genet. Biol. 41:391–400.
Hasek, J. and Bartnicki-García, S. 1994. The arrangement of F-actin and microtubules during germi-
nation of Mucor rouxii sporangiospores. Arch. Microbiol. 161:363–369.
Hawker, L. E. and Abbott, P. McV. 1963. An electron microscope study of maturation and germina-
tion of sporangiospores of two species of Rhizopus. J. Gen. Microbiol. 32:295–298.
Herman, P. K. and Rine, J. 1997. Yeast spore germination: A requirement for Ras protein activity dur-
ing re-entry into the cell cycle. J. EMBO 16:6171–6181.
for roles of chitin biosynthesis and breakdown. Exp Mycol. 7:362–369.
Horio, T. and Oakley, B. R. 2005. The role of microtubules in rapid hyphal tip growth of Aspergillus
nidulans. Mol. Biol. Cell 16:918–926.
Howard, R. J. and Aist, J. R. 1977. Effects of MBC on hyphal tip organization, growth and mitosis of
Fusarium acuminatum, and their antagonism by D2O. Protoplasma 92:195–210.
Howard, R. J. and Aist, J. R. 1980. Cytoplasmic microtubules and fungal morphogenesis:
Ultrastructural effects of methyl benzimidazole-2-ylcarbamate determined by freeze-substitu-
tion of hyphal tip cells. J. Cell Biol. 87:55–64.
Hutzler, F., Gerstl, R., Lommel, M., and Strahl, S. 2008. Protein N-glycosylation determines functional-
ity of the Saccharomyces cerevisiae cell wall integrity sensor Mid2p. Mol. Microbiol. 68:1438–1449.
Koch, A. L. 1983. The surface stress theory of microbial morphogenesis. Adv. Microb. Physiol.
24:301–366.
Koch, A. L. 1985. How bacteria grow and divide despite of internal hydrostatic pressure. Can. J.
Koch, A. L. 1988. Biophysics of bacterial walls viewed as stress bearing fabric. Microbiol. Rev.
52:337–353.
Lee, S. C. and Shaw, B. D. 2008. ArfB links protein lipidation and endocytosis to polarized growth of
Aspergillus nidulans. Commun. Integr. Biol. 1:51–52.
Leighton, T. J. and Stock, J. J. 1970. Biochemical changes during fungal sporulation and spore germina-
tionI. Phenyl methyl sulfonyl fluoride inhibition of macroconidial germination in Microsporum
gypseum. J. Bacteriol. 101:931–940.
Lesage, G. and Bussey, H. 2006. Cell wall assembly in Saccharomyces cerevisiae. Microbiol. Mol. Biol.
Rev. 70:317–343.
Levin, D. E. 2005. Cell wall integrity signaling in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev.
69:262–291.
Li, S., Du, L., Gary Yuen, G., and Harris, S. D. 2006. Distinct ceramide synthases regulate polarized
growth in the filamentous fungus Aspergillus nidulans. Mol. Biol. Cell. 17:1218–1227.
López-Franco, R., Bartnicki-García, S., and Bracker, C. E. 1994. Pulsed growth of fungal hyphal tips.
Proc. Natl. Acad. Sci. USA 91:12228–12232.
Lopez-Romero, E., Ruiz-Herrera, J., and Bartnicki-García, S. 1982. The inhibitory protein of chitin
synthetase from Mucor rouxii is a chitinase. Biochim. Biophys. Acta 702:233–236.
Lovett, J. S. 1975. Growth and differentiation of the water mold Blastocladiella emersonii: Cytodifferentia
tion and the role of ribonucleic acid and protein synthesis. Bacteriol. Rev. 39:345–404.
Lowry, R. J. and Sussman, A. S. 1968. Ultrastructural changes during germination of ascospores of
Neurospora tetrasperma. J. Gen. Microbiol. 51:403–409.
Ma, H., Snook, L. A., Tian, C., Kaminskyj, S. G. W., and Dahms, T. E. S. 2006. Fungal surface remodel-
ling visualized by atomic force microscopy. Mycol. Res. 110:879–886.
Martin, F. J., Nicolas, G., and Villanueva, J. R. 1973. Chemical changes in the cell wall of conidia of
Penicillium notatum during germination. Can. J. Microbiol. 19:789–796.
Mateeva, Z., Petrov, P., Venkov, P., and Hadjiolov, A. A. 1976. Electron microscopic study of the lysis
of an osmotic-sensitive yeast mutant. J. Microscopic. Biol. Cell 26:73–74
McGillivrary, A. M. and Gow, N. A. R. 1986. Applied electrical fields polarize the growth of mycelial
fungi. J. Gen. Microbiol. 132:2515–2525.
Merson-Davies, L. A. and Odds, F. C. 1989. A morphology index for characterization of cell shape in
Candida albicans. J. Microbiol. 135:3143–3152.
Momany, M. 2002. Polarity in filamentous fungi: Establishment, maintenance and new axes. Curr.
Opinion Microbiol. 5:580–585.
Money, N. P. 1990. Measurement of turgor pressure. Exp. Mycol. 14:416–425.
Money, N. P. and Harold, F. M. 1992. Extension of the water mold Achlia: Interplay of turgor and wall
strength. Proc. Natl. Acad. Sci. USA 89:4245–4249.
Money, N. P. and Howard, R. J. 1996. Confirmation of a link between fungal pigmentation, turgor
pressure, and pathogenicity using a new method of turgor measurement. Fungal Genet. Biol.
20:217–227.
Morales, M. and Ruiz-Herrera, J. 1990. Light-induced changes in the vesicular apparatus of
Phycomyces sporangiophores during the phototropic response. Photochem. Photobiol. 52:223–227
Murgui, A., Elorza, M. V., and Sentandreu, R. 1986. Tunicamycin and papulacandin B inhibit incor-
poration of specifc mannoproteins into the wall of Candida albicans regenerating protoplasts.
Necas, O. 1961. Physical conditions as important factors for the regeneration of naked yeasts proto-
plasts. Nature 192:1664–1665.
Necas, O. and Svoboda, A. 1981. “Morphogenesis in Protoplasts.” In Yeast Cell Envelopes. Biochemistry,
Biophysics, and Ultrastructure. Edited by W. N. Arnold, 105–127. Boca Raton, FL: CRC Press.
Obregon, A., Monzalvo, S., Calvo-Mendez, C., and Ruiz-Herrera, J. 1990. Ultrastructural and chem-
ical alterations in germinating spores of Mucor rouxii (Zygomycetes), induced by two com-
pounds which inhibit their developmental pattern. Crypt. Bot. 1:323–331.
Oort, A. P. J. 1931. The spiral-growth of Phycomyces. Kon. Ned. Akad. Wet. Proc. 34:564–575.
Ortega, J. K. E., Gamow, R. I., and Ahlquist, C. N. 1975. Phycomyces: A change in the mechanical prop-
erties after a light stimulus. Plant Physiol. 55:333–337.
Ortega, J. K. E., Manica, K. J., and Keanini, R. G. 1988. Phycomyces: Turgor pressure behavior during
the light and avoidance growth responses. Photochem. Photobiol. 48:697–703.
Ortega, J. K. E., Zehr, E. G., and Keanini, R. G. 1989. In vivo creep and stress relaxation experiments
to determine the wall extensibility and yield threshold for the sporangiophores of Phycomyces.
J. Biophys. 56:465–475.
Pruyne, D. and Bretscher, A. 2000a. Polarization of cell growth in yeast. I. Establishment and mainte-
nance of polarity states. J. Cell Sci. 113:365–375.
Pruyne, D. and Bretscher, A. 200b. Polarization of cell growth in yeast. II. The role of the cortical actin
cytoskeleton. J. Cell Sci. 113:571–585.
Regalado, C. M., Sleeman, B. D., and Ritz, K. 1995. Aggregation and collapse of fungal wall ves-
icles in hyphal tips: A model for the origin of the Spitzenkörper. Phil. Trans. R. Soc. Lond. B.
352:1963–1974.
Reinhardt, M. O. 1892. Das Waschsthum der Pilzhyphen. Jahrb. Wiss. Bot. 23:479–565.
Remsen, C. C., Hess, W. M., and Sassen, M. M. A. 1967. Fine structure of germinating Penicillium
megasporum conidia. Protoplasma 64:439–451.
Rico, H., Carrillo, C., Aguado, C., Mormeneo, S., and Sentandreu, R. 1997. Initial steps of wall proto-
plast regeneration in Candida albicans. Res. Microbiol. 148:593–603.
Riquelme, M., Gerhard, G., and Bartnicki-García, S. 2000. Dynein and dynactin deficiencies affect the
formation and function of the Spitzenkörper and distort hyphal morphogenesis of Neurospora
crassa. Microbiology 146:1743–1752.
Rodriguez, L., Laborda, F., and Sentandreu, R. 1979. Patterns of wall synthesis in Saccharomyces cere-
visiae. Curr. Microbiol. 2:293–297.
Roelofsen, P. A. 1950. The origin of spiral growth in Phycomyces sporangiophores. Rec. Travaux
Botaniques Neerlandais 42:72–110.
Rønne, M. and Bøye, H. A. 1977. Cell wall synthesis in Allomyces macrogynus. Cytobios. 19:159–170.
Ruiz-Herrera, J. 1994. Polyamines, DNA methylation, and fungal differentiation. Crit. Rev. Microbiol.
20:143–150.
Ruiz-Herrera, J., León-Ramírez, C., Alvarez, E., and Venkov, P. 1998. Increased activity of wall hydro-
lytic enzymes may explain the phenotype of Saccharomyces cerevisiae fragile mutants. Curr.
Ruiz-Herrera, J., Valenzuela, C., Martinez-Cadena, G., and Obregon, A. 1989. Alterations in the
vesicular pattern and wall growth of Phycomyces induced by the calcium ionophore A 23187.
Saunders, P. T. and Trinci, A. P. J. 1979. Determination of tip shape in fungal hyphae. J. Gen. Microbiol.
110:469–473.
Sawin, K. E. and Nurse, P. 1998. Regulation of cell polarity by microtubules in fission yeast. J. Cell
Biol. 142:457–471.
Schmid, J. and Harold, F. M. 1988. Dual roles for calcium ions in apical growth of Neurospora crassa.
J. Gen. Microbiol. 134:2623–2631.
Schorling, S., Vallee, B., Barz, W. P., Riezman, H., and Oesterhelt, D. 2001. Lag1p and Lac1p are essen-
tial for the Acyl-CoA-dependent ceramide synthase reaction in Saccharomyces cerevisiae. Mol.
Biol. Cell 12:3417–3427.
Seiler, S. and Plamann, M. 2003. The genetic basis of cellular morphogenesis in the filamentous fun-
gus Neurospora crassa. Mol. Biol. Cell 14:4352–4364.
Sellen, D. B. 1980. “The Mechanical Properties of Plant Cell Walls.” In The Mechanical Properties of
Biological Materials. Edited by V. F. V. Vincent and J. D. Currey, 135. Cambridge: Cambridge
University Press.
Semighini, C. P. and Harris, S. D. 2008. Regulation of apical dominance in Aspergillus nidulans hyphae
by reactive oxygen species. Genetics 179:1919–1932.
Sietsma, J. H. and Wessels, J. G. H. 1979. Evidence for covalent linkages between chitin and β-glucan
in a fungal wall. J. Gen. Microbiol. 114:99–108.
Sietsma, J. H. and Wessels, J. G. H. 1981. Solubility of (1→3)-β-D/(1→6)-β-D-glucan in fungal walls:
Importance of presumed linkage between glucan and chitin. J. Gen. Microbiol. 125:209–212.
Slayman, C. L. and Slayman, C. W. 1962. Measurement of membrane potentials in Neurospora. Science
136:876–877.
Staebell, M. and Soll, D. R. 1985. Temporal and spatial differences in cell wall expansion during bud
and mycelium formation in Candida albicans. J. Gen. Microbiol. 131:1467–1480.
Stålhammar-Carlemalm, M. 1976. An electron microscope study of spore germination in Fusarium
sambucinum var. Coeruleum. Trans. Brit. Mycol. Soc. 67:83–90.
Steinberg, G. 2007a. Hyphal growth: A tale of motors, lipids, and the Spitzenkörper. Eukaryot. Cell
6:351–360.
Steinberg, G. 2007b. Preparing the way: Fungal motors in microtubule organization. Trends Microbiol.
15:14–21.
Sudbery, P. E. Regulation of polarised growth in fungi. 2008. Fungal Biol. Rev. 22:44–55.
Takeshita, N., Higashitsuji, Y., Konzack, S., and Fischer, R. 2008. Apical sterol-rich membranes are
essential for localizing cell end markers that determine growth directionality in the filamentous
fungus Aspergillus nidulans. Mol. Biol. Cell 19:339–351.
Takeuchi, Y., Schmid, J., Caldwell, J. H., and Harold, F. M. 1988. Transcellular ion currents and exten-
sion of Neurospora crassa hyphae. J. Membr. Biol. 101:33–41.
That, T. C., Rossier, C., Barja, F., Turian, G., and Roos, V. P. 1988. Induction of multiple germ tubes in
Neurospora crassa by antitubulin agents. Eur. J. Cell Biol. 46:68–79.
Trinci, A. P. J. and Collinge, A. J. 1975. Hyphal wall growth in Neurospora crassa and Geotrichum can-
didum. J. Gen. Microbiol. 91:355–361.
Trinci, A. P. J. and Saunders, P. T. 1977. The growth of fungal hyphae. J. Gen. Microbiol. 103:243–248.
Tsukahara, T. 1968. Electron microscopy of swelling and germinating conidiospores of Aspergillus
niger. Medical Mycol. 6:185–191.
Van Schaftingen, E. and Van Laere, A. J. 1985. Glycerol formation alter the breaking of dormancy
of Phycomyces blakesleeanus spores. Role of an interconvertible glycerol-3-phosphatase. Eur. J.
Biochem. 148:399–404.
Venkov, P. V., Hadjiolov, A. A., Bataner, E., and Schlezinger, D. 1974. Saccharomyces cerevisiae sorbitol
dependent fragile mutants. Biochem. Biophys. Res. Commun. 56:599–604.
Virag, A. and Harris, S. D. 2006. The Spitzenkörper: A molecular perspective. Mycol. Res. 110:4–13.
Wendland, J. 2001. Comparison of morphogenetic networks of filamentous fungi and yeast. Fungal
Genet. Biol. 34:63–82.
Wendland, J. and Philippsen, P. 2001. Cell polarity and hyphal morphogenesis are controlled by mul-
tiple Rho-protein modules in the filamentous ascomycete Ashbya gossypii. Genetics 157:601–610.
B C
A
GPI PROTEIN-S-S-PROTEIN
PIR PROTEIN-S-S-PROTEIN
O
P
O O
β-1,6 GLUCANS β-1,3 GLUCANS
CHITIN
Figure 3.1 Schematic representation of the fungal cell wall structure. (A) Scheme representing the
association of bound proteins and polysaccharides. Noncovalently bound proteins are not repre-
sented in the scheme. (B) Representation of the organization of the fungal cell wall. Thin waving
lines, chitin; straight red lines, β-glucans; circles, proteins. (C) Electron microscopy section of a
germinating spore of Mucor rouxii showing the structure of the cell walls, both original and from
the germ tube.
Figure 4.4 Laser scanning confocal microscopy (LSCM) of a hyphal apex of Neurospora crassa
expressing chitin synthase 1 tagged with green fluorescent protein (Chs 1-GFP). (A) Phase-contrast
showing mitochondria, the Spitzenkörper (Spk) at the apical dome and other high-density intracel-
lular organelles. (B) Fluorescent channel showing strong localization of Chs1-GFP at the Spk. (C)
Merged images of A and B. Scale bar: 5 μm. (Courtesy of Meritxel Riquelme.)
Figure 8.3 Stratification of the Spitzenkörper in a hyphal apex of N. crassa by laser scanning confo-
cal microscopy. Left panel: GS-1-GFP localized at the outer layer of the Spitzenkörper (Spitzenring).
Center panel: Chs1-mChFP localized at the core of the Spitzenkörper. Right panel: merged image.
Scale bar: 10 µm. (Courtesy of E. Sánchez-León, J. Verdín, and M. Riquelme.)
D P
A B
D
3
C
VESICLES (per mµ2)
2.5 PROXIMAL
DISTAL
2
1.5
1
0.5
0
2.5 5 7.5 10 12.5
DISTANCE (FROM THE APEX) (mµ)
Figure 9.2 The role of vesicles and microvesicles in wall synthesis during the phototropic response
of Phycomyces blakesleeanus stage I sporangiophores. (A) After asymmetric illumination, the spo-
rangiophore, which behaves as a convergent lens, bends and grows towards the light source. (B)
Section of the apical (growing) zone of the sporangiophore used to determine the number of micro
and macrovesicles in the proximal (P) and distal (D) zones. (C) Data reveal a decrease in the number
of vesicles in the distal side, indicating its faster rate of mobilization to increase the growth rate
of this side in comparison to the proximal one. (Modified from Morales, M. and Ruiz-Herrera, J.,
1990, Light-Induced Changes in the Vesicular Apparatus of Phycomyces Sporangiophores during the
Phototropic Response, Photochem. Photobiol. 52:223–227.)
Iso
ISOMETRIC
Ort
Rot
ORTHOGONAL
0 3
ROTATIONAL
6 12
Figure 9.5 Schematic representation of the three possible mechanisms of hyphal growh, and the
theoretical position of surface markers in each growth model. (Courtesy of S. Bartnicki-García.)

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Fungal Cell Wall

Structure, Synthesis, and Assembly

1. Viruses and Plasmids in Fungi, edited by Paul A. Lemke

No claim to original U.S. Government works

International Standard Book Number-13: 978-1-4398-4838-8 (eBook - PDF)

Chapter 2 Cell wall composition.............................................................................................. 7

Chapter 3 Structure of the fungal cell wall..........................................................................29

4.4.5 Regulation of chitin synthases............................................................................ 53

Chapter 6 Fungal glucans........................................................................................................73

Chapter 8 Cytological aspects of cell wall synthesis.......................................................123

Chapter 9 Cell wall growth and expansion........................................................................ 149

Table 1.1 Main Functions of the Cell Walls

Table 1.2 Structural Components of the Cell Wall from

Cell wall composition

Table 2.1 Classification of Chemotypes of Fungal Cell Walls

2.2 Cell wall composition

Table 2.2 Components of the Fungal Cell Walls

Table 2.3 Main Sugars Identified in Hydrolyzates of Fungal Cell Walls

N-acetylglucosamine); Mann = mannose; GalN = galactosamine (a product of polysaccharides containing

of Ascomycota, and in a more restricted number of Basidiomycota species, some of them

glucose > mannose > glucosamine > galactose

Important heteropolysaccharides from fungal cell walls are galactomannans, described

2.2.2 Cell wall proteins

2.2.3 Lipids from the cell walls

2.2.5 Inorganic salts in the walls

2.3 Differentiation of the cell wall chemical composition

applying chitin determination as an indicator of the biomass of wood-decay fungi during

(Buecher and Phaff 1968) and mannans in Trigonopsis variabilis (Sentheshanmuganathan

Structure of the fungal cell wall

3.2 Methods for the study of cell wall structure

β-1,6 GLUCANS β-1,3 GLUCANS

3.3 Organization of the cell wall

3.4 The cell wall surface

3.5 Microfibrilar structure of the cell wall

values of 109–250 μm were obtained for C. albicans, Paracoccidioides brasiliensis, H. capsula-

3.6 Associations of the components of the cell wall

3.7 Mechanical characteristics of the fungal cell wall

4.2 Structure of chitin

4.3 Chitin distribution in nature

4.4 Chitin biosynthesis

4.4.2 CHS gene multiplicity in fungi

4.4.3 Classification and possible evolution of fungal chitin synthases

4.4.4 Structure of chitin synthases

4.4.5 Regulation of chitin synthases

4.4.6 The mechanism of chitin biosynthesis

UDP-GlcNAc + (GlcNAc)n → (GlcNAc)n+1 + UDP

4.5. Inhibitors of chitin synthases

4.6 Cytology of chitin biosynthesis

5.2 The functions of chitosan

5.3 Chitosan biosynthesis and genetic control and

Table 5.1 In Vitro Synthesized Chitosan Is Made by a Mixture of Glucosamine and

number origin (cm) of precursor glucosaminea acetateb

6.2 Structure of glucans

6.3 Role of fungal glucans

to humans. P. brasiliensis is a dimorphic human pathogen that grows in a mycelial form in

6.4 Synthesis of glucans

6.4.2 Introduction to the synthesis of β-1,3-glucans

6.4.3 Stimulators and inhibitors of β-1,3-glucan synthases

structure is somewhat different to papulacandins. Interestingly, it was a common obser-

6.4.4 Purification of β-1,3-glucan synthases

6.4.5 Genes encoding β-1,3-glucan synthases

6.4.6 Structure and phylogenetic characteristics of β-1,3-glucan synthases

To analyze the phylogenetic relationships among β-1,3-glucan synthases from fungi

5.3 Chitosan biosynthesis and genetic control and

6.5 Other glycosyl transferases involved

7.4.4 Further glycosylation of the inner core to form

Fungal cell wall: Structure, synthesis, and assembly

Fungal cell wall: Structure, synthesis, and assembly

9.2.2 The physical stages of nascent and mature wall and their