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328 Biochimica et Biophysica Acta, 946 (1988) 328-336


BBA 74243

Chitin synthetase activity ig bound to ~hRosomes and to the plasma membrane

in protoplasts of Saccharomyces cerevisiae

Alberto Flores Martinez * and Jaime Schwencke

Laboratoire d'Enzymologie du C.N.R.S., Gif-sur-Yvette (France)

(Received 12 July 1988)

Key words: Chitin synthetase; Chitosome; Protoplast; Plasma membrane; (S. cerevasme)

The sub-cellular distribution of chitin sTnthetase was studied in homogenates of Sacchuromyces cerevisiae
protoplasts. Use of a mild disruption method minimized rupture of vacuoles and ensuing contamination of
subceilular fractions by vacuolar proteinases. After fractiunation of whole or partially purified homogenates
through an isopycnic sucrose gradient chitin synthetase activity was found to be distributed between two
distinct particulate fractions with different buoyant density and particle diameter. When whole homogenates
were used, about 52% of the chitin synthetase loaded was localized in a microvesicular population identified
as chitosomes (diameter 40-110 nm; buoyant density ( d ) - 1.146 g/era3). Another vesicular population
containing 26% of the activity was identified as plasma membrane vesicles because of its large mean
diameter (260 nm), its high buoyant density ( d = 1.203 g / c m 3) and by the presence of the vanadate-sensi-
tire ATPase activity. Moreover, after surface labeling of protoplasts with aH.coneanavalin A, the label
cosedimented with the presumed plasma membrane vesicles. There was a negligible cross-contamination of
the chitosome fraction by yeast plasma membrane markers. In both the plasma membrane and the chitosome
fractious, the chitin synthetase was stable and essentially zymogenie. Activation of the chitosome fraction
produces microfibriis 100-250 nm in length. Our results st,p0oR the idea that chitosomes do not originate by
plasma membrane vesiculation but are defined sub-cellular organelles containing most of the chitin
synthetase in protoplasts of Saccharom¥ces cereoisiae.

Introduction have a buoyant density of ( d ) - 1.14-1.16 g/cm 3

in a sucrose isopycnic gradient [6,7]. Contrary to
The localization of chitin synthetase in actively this, the b~qk of the chitin synthetase has been
growing cells of yeast has been the matter of claimed to b~ attached to the plasma membrane
conflicting reports. A number of studies [1-=5] [8,9]. Although these differences may be in fact
describe a preferential location in microvesicles due to differences in strain and growth conditions,
(40-80 nm in diameter) called chitosomes which unequivocal assignment of chitin synthetase to
organelles is difficult for a number of reasons: (a)
harsh methods of cell disruption may produce
small artefactual membrane vesicles difficult to
* Present address: |nstituto de lnvestigacion en Biologia Ex- differentiate from pre-existing, physiologically im-
perimental, Facultad de Quimica, Universidad de Guana-
juato, Guanajuato, 36000 Mexico. portant vesicles, (b) pure preparations of cell
orga~elles are difficult to obtain without cross-
Correspondence: J. Schwencke, Laboratoire d'Enzymologie, contamination, (c) hydrolytic and proteolytic en-
C.N.ILS, 91198 Gif-sur-Yvette, France. zymes contained in the vacuolar compartment are
0005-2736/88/$03.50 © 1988 Elsevier Science Pubfishers B.V. (Biomedical Division)

liberated when cells are broken by t~e usual meth- was carefully resuspended in 1 M sorbito|, 25 mM
ods, and may interfere either with the zymogenic- Triethylamine (TEA)(HCI)(pH 8.0), 1 mM EDTA
ity or the stability of the chitin synthetase bound and kept 10 rniJ~ on ice. The protoplasts suspen-
to the sub-cellular fractions. sion was diluted 1" 4 with pre-warmed (30~C)
Most of these difficulties can now be overcome disrupting buffer: 0.36 M sorbitol, 25 mM TEA
using mild disruption conditions as those de- (HC1) (pH 8.0), 50 mM glucose, 2.5 mM K2CO3
scribed for the isolation of the fragile yeast and incubated 10 nfin at 30 ° C. Rupture of proto-
vacuoles [10,11]. Indeed, a mild disruption proce- plasts and vacuole liberation was assessed by
dure has been found to provide both a cyto- phase-contrast microscopy. Contanfination by
plasmic and a well preserved mitochondrial frac- soluble vacuolar proteinases was determined in a
tion with minimal contamination by vacuolar pro- 100000 × g supernatant of the lysate by measur-
teinases [12]. Here we describe the subcellular ing carboxypeptidase ¥ activity as in Ref. 12.
distribution of the chitin synthetase in Sac- Values of 10 to 15% were usually found with
charomyces cereoisiae homogenates after disrupt- reference to the total activity in the whole homo-
ing protoplasts in conditions minimizing organelle genate. Higher values (about 27% were found in a
rupture and hence, cross-contamination. 500 × g × 15 rain supernatant (Table I) indicating
that low density vacuolar vesicles are still present
Materials and Methods in the low speed supernatant.
Isopycnic sucrose gradient distribution of chitin
Chemicals. Uddine diphospho-N-acetyl-D-[U- synthetase. The protoplast lysate was centrifuged
14C]glucosamine ammonium salt (247 mCi/mmol) at 500 x g for 15 rnin (swinging bucket rotor) to
and [N-acetyl-3H] concanavalin A (47.6 Ci/mmol) remove vacuoles, intact protoplasts and debris.
were obtained from Amersham/Searle. [~,- The supernatant was applied to a Sepharo.~e 2B
32P]ATP ( > 5000 Ci/mmol) was purchased from column (15 × 5 cm) and eluted with 0.3 M sucrose,
New England Nuclear. Sodium deoxycholate was 20 mM Mops (Tr!s) (pH 7.0). ZymogenJc chitin
product No. 6504 from Merck. Crystallized a- synthetase eluted in the void volume. Void-volume
chymotrypsin (No. 17160) was obtained from fractions were pooled, sucrose was added to adjust
SERVA. Sodium orthovanadate, NADPH, sucrose the concentration to 20% (w/w) and used in con-
(gradient quality), N-benzoyl-L-tyrosine-p-nitro- junction with a .,.,,o ~ucrose solution to construct
anilide, p-nitrophenyl a-D-giucoside and buffer a 20 to 55% (w/w) linear sucrose gradient (36 ml).
substances were from Sigma Chemical Co. Sep- The gradients were centrifuged in a Beckman SW-
harose 2t:1 was from Pharmacia. All other chem- 28 rotor at 95 000 x g (rav) for 22 h. Fractions of
icals were of the highest purity available. 1 ml were collected from the bottom upwards by
Yeast strain and growth conditions. Sac- means of a peristaltic pump. In alternative experi-
charomyces~cerevisiae 1022 (ETH, Zurich; ATCC ments the whole lysate was adjusted to 20% sucrose
32167), kindly suppfied by Dr. A. Wiemken, was and used to prepare a 20 to 55% (w/w) linear
grown aerobically at 28"C in G-21 medium [13] sucrose gradient and the gradient centrifuged in
and harvested at the late exponential phase of similar conditions as described above.
growth. Enzymatic assays. Chitin synthetase was as-
Preparation, labeling and lysis of protoplast~. sayed according to Ruiz-Herrera and Bartniki-
Protoplasts were prepared and purified according Garcia [14] but using 60 # g / m l of a-chymotryp-
to the method of Sd,wencke et at. [12]. A suspen- sin to activate the zymogenic chitin synthetase.
sion of 1-10 9 prc::oplasts/ml in 0.6 M sorbitol, ATPase was assayed essentially as previously de-
10 mM MgCI 2, 2~ mM Mops ('Iris), pH 7.0 scribed [15], using 0.1 M ATP added to [~,-3~-P]ATP
(buffer A) was added of 0.25 #Ci 3HoconcanavalJn to a fina~ specific activity of 2.95 #Ci/rnOlo
A (dissolved in buffer A). After 10 rain at 30 ° C, Liberated [32p]P i WaS detern~ned as described in
protoplasts were collected by centrifugation Ref. 16 by scintillation counting after extraction
(swinging bucket rotor) at 3500 × g for 5 rain and into i s o b u t a n o l / c y c l o h e x a n e / a c e t o n e / w a t e r -
washed twice with ice-cold buffer A. The pellet saturated ammonium molybdate (750" 750" 15" 1,

v/v). Carboxypeptidase Y activity was measured activity. Only 5 to 10% chitin synthetase activity
as described ht ReL 17 except for the addition of was found without proteolytic activation in the
0.5% deoxycholate, a-Glucosidase was assayed as 500 × g × 15 rain pellet while the activity in the
in ReL 18. 500 × g supernatant was 95-98% zymogenic.
Other methods. Protein was determined accord- Activation by a-chymotrypsin, in the optimal con-
ing to Bradford [19] using crystalline bovine serum ditions used here, gave more reproducible results
albumin as standard. The standard and all sam- than those obtained by trypsin activation. Addition
ples were previously digested with 1 M NaOH for of chymostatin after activation by a-chymotrypsin
30 rain and the NaOH neutralized. did not significantly enhanced the specific activity
of the chitin synthetase.
Distribution of chitin synthetase, vanadate-sensitioe
Cell disruption and chitin synthetase assay Mg 2 +-,4TPase and ~H-concanaoalin A label in sub-
The modification of the original method for cellular fractions obtained from ~H-concanavalin
mild protoplast disruption [12] was necessary be- labeled yeast protoplasts
cause the DEAE-Dextran was found to inhibit the As shown in Table I, a substantial amount of
activity of the plasma membrane-bound vanadate- chitin synthetase activity remains in the 500 × g x
sensitive Mg2+-ATPase. With the revised method 15 rain supernatant after protoplast lysis and coe-
(without DEAE-Dextran) about 60-70% of the lutes with the vanadate sensitive Mg2+-ATPase
protoplasts were disrupted while vacuolar integr- and the 3H-concanavalin A label in the void
ity was well preserved as indicated by the low volume after filtration through Sepharose 2B.
contamination of the 500x g × 15 rain super- Soluble vacuolar proteinases are largely entrapped
natant by the vacuolar carboxypeptidase Y (Table in vacuc,ies and hence sedimented in the 500 × g
I). Sub-cellular fractionation using DEAE-Dex- × 15 rain pellet as indicated by the carboxy-
tran as in the original disruption method [12], did peptidase Y activity (Table I). Soluble vacuolar
not alter the distribution of chitin synthetase proteoly~c activities remaining in the 500 x g × 15
described here. However, the activity of the rain supernatant were completely separated from
vanadate-sensitive Mg2+-ATPase activity bound the particulate fraction in the Sepharose 2B step.
to the plasma membrane fraction was much lower Also, a-glucosidase (a cytoplasmic marker) was
(not shown). Activation by partial proteolysis was undetectable in the particulate fraction elutiag in
required in order to measure chitin synthetase the void volume of the Sepharose 2B column.

Protoplasts were labeled with 3H.concanavalin A and washed bt:fore disruption (see Methods for details), n.d., not detected.

Total Total enzyme activity (/t mol/min) 3H-conca-

protein chitin vanadate~sensitive a-gluco- carboxy- navalin A
(roB) synthetase Mg 2+-ATPase sidase peptidase Y (tot~ clam)
Whole iysate 189.7 48.2 641 351 79.9 318546
$00 × g pellet a 57.6 27.0 595 121 54.8 161784
500× g sapernatant 117.7 21.9 417 196 22.0 74942
Sepharose 2B void
volume eluate 12.4 6.6 624 n.d. n.d. 38000

a T his pellet ( 5 0 0 × g × 1 5 rain) contains a few cells, intact protoplasts (about 35 to 40~ of the initial amount) as well as
agglomerates of permeabilized but otherwise non disrupted protoplasts.

B buoyant density [24] and by the fact that the

~.08 50 vanadate-sensitive Mg2+-ATPase [15,27] and the
3H-concanavalin A label [28,29] co-sediment with
-.06 o 4O them (Fig. 1, low panel). Both peaks of chitin
Io~o synthetase activity were highly zymogerfic i.e.
~.0~ without proteolytic activation less than 2% of the
total chitin synthetase activity could be measured
.5.0~ i in the chitosomal fraction and less than 5% in the
plasma membrane fraction. It is also interesting to

note that a-glucosidase (a cytoplasmic marker),
carbo~vpeptidase Y, proteinase B (soluble vacuo-
lar markers [12]), and a-mannosidase (a marker
..... ",, ,. 50
for vacuolar membranes [20]) were not detected in
the sucrose gradient (data not shown), indicating
"¢" "" ~. 40
that there was no cross-contamination by either
soluble vacuolar proteinases, entraped soluble cyo
toplasnfic enzymes or vacuolar membranes°
~. 5
,~ 2 .,20 Distribution and qzzantification of chitin synthetase
after sucrose gradient centrifugation of a whole pro-
0 10 20 30 t
toplasts lysate
Selective loses of chitoso~a! and/or the plasma
Fig. 1. Isopycnic sucrose gradient centrifugation of a partially
membrane fraction may occur when using the
purified vesicular fraction. The void volume eluate of the semi-purified Sepharose 2B void volume as a start-
Sepharose 2B column obtained as detailed under Materials and ing material. Therefore we decided to use whole
Methods was used to construct a 20 to 55% (w/w) linear protoplasts lysates to construct the sucrose gradi-
sucrose gradient. Tubes were centrifuged at 95 000 × g (ray) for ent. In order to facilitate the idenfif;¢duon of
24 h. Top panel: e, 3H-concanavalin A; [I, chitin synthetase
activity. Bottom panel: o, 3H.concanavalin A; A,
vesicles originating from the yeast plasma mem-
Mg2+-ATPase activity; ~, Mg:'+-ATPase activity assayed in brane, protoplasts were labeled with 3H-con-
the presence of 50 #M sodium vanadate. canavalin A before disruption. The resulting whole

Distribution of chitin synthetase after sucrose gradi-

ent centrifugation of a partially purified fraction 50 ' "~..1 2 "....... ",, ",,, A S~

In four separate experiments when the void

" .10 - , g
volume of the Sepharose 2B column was subjected
to isopycnic sucrose gradient centrifugation • ~..08 4m
(20-557o), two peaks of chitin synthetase activity o30 . ~ .o6 .7
were reproducibly obtained (Fig. 1). The main
peak (peak 13) contained microvesicles having the
u~2o. u)
same size distribution (40-110 nm; median diame- ,| ~.O2
,~ ~
ter: 75 nm), electron microscopic appearance (Fig.
3B)and buoyant density (1.1463 g/cm 3) described o lo 20 30
for chitosomes [1,2,7]. The second peak (peak A)
Fig. 2. ~sopycnic sedimentation profile of chitin synthetase (~)
consisted of larger vesicles (190-350 rim; median and 3H-concanavalin A (®) fi'om a whole protoplast lysa~e.
diameter 260 rim), smooth appearance (Figs. 3A 1 Protoplasts were lysed as described in Materials and Methods
and 3A z) and higher buoyant density (1.203 and the whole homogenate used to build a linear sucrose
g/cm3). Vesicles in peak A appear to correspond gradient (20 to 55~; w/w) a~.d centrifuged at 95000x g (ray)
to plasma-membrane particles as indicated by their for 24 h.


&k: P r

,,~ i!~ "~:i


Fig, 3. Electronmicrographsof particles from peaks A and B of the sucrose gradient(Fig. 1, top panel). Negative stain. Bar = 200
nm. (At and A 2) plasma-membranefraction.(B) Chitosomefraction. Proctoid(Pr) and cycloid(cy) forms.

lysate was made 20% with solid sucrose and used total chitin synthetase activity loaded while 52%
to obtain a 20-55% (w/w) sucrose gradient. After of the activity was found in the chitosomes peak.
a single isopycnic centrifugation chitin synthetase Directly measurable chitin synthetase activity in
distribution was similar to that obtained above for these fractions (i.e. without proteolytic activation)
the mixed vesicle population present in the void- varied between 0.5 and 1% of the total activity in
volume fraction after Sepharose 2B chromatograo the chitosomal fraction and between 3 and 5% in
phy. Two main peaks of chitin synthetase were the plasma membrane ghost vesicles.
detected (Fig. 2), one (peak A) with a specific
gravity of 1.199 g / c m 3 (plasma membrane) and Electron microscopy of vesicles separated by iso-
the other (peak B) with specific gravity of 1.146 pycnic sucrose gradient
g / c m 3 (chitosomes). In these conditions, the Further evidence that chitin synthetase activity
plasma membrane peak accounted for 26% of the is in fact bound to two different types of sub-

Fig. 4. Electron micrograph of chitosomes after a-chymotrypsin activation. Negatively stain. Bar = 200 nm. (A) General picture
showing most!y cycloid (Cy) forms. (B) l~crofibrils (F).

cellular fractions was obtained after negative chitosomes either alone, agglomerated or bound to
staining and electron microscopy examination of the large vesicles was found in this fraction. Con-
the two peaks separated by isopycnic sucrose versely, the chitosomal fraction, was found to be
gradient centrifugation. Thus, the microvesicle virtually free of plasma membranes.
population wk,~ch equilibrates at a density of 1.146
g/cm 3 (Fig. 3) shows the same morphological Discmsion
characteristics of those already described for the
yeast chitosomes [1,7,21], namely spheroidal par- Chitosomes were originally proposed to convey
ticles 40-110 nm in diameter (mean diameter: 75 zymogenic chitin synthetase activity to the sites of
nm) (Fig. 3B). A characteristic central depression chitin synthesis iA the plasma membrane [1,2,6].
(cycloid form) was found in about 25~ of the In spite of the strong collective evidence indicat-
population while the rest of the particles did not ing their physiological reality (reviewed in Ref.
show it (Proctoid form). After chymotrypsin 21), their existence as true subcellular structures
activation no essential changes were observed in has been questioned [22]; it was argued that they
the chytosomal population, both cycloid and proc- may be artefactual vesicles derived from the
toid forms were seen (Fig. 4A). Microfibrils plasmalemma during cell breakage since yeast
(100-280 nm long), presumably of chitin, were plasma-membrane has been claimed to contain
also observed (Fig. 4B). The vesicular population the bulk of the chitin synthetase activity [8]. How-
equilibrating at a buoyant density of d = 1.203 ever, since c~tin synthesis most ~robably occurs
g/cm 3 was found to be quite different from at the Flasma membrane, chitosomes should be
chitosomes both in diameter (mean diameter: 260 intrinsically able tc reco~.~zc and bi~d ~o plasma
rim) and appearance since they are seen as col- mer ibrane 'in vivo' and probably also 'in vitro'
lapsed, smooth vesicles resembling plasma mem- d~,ing or after disruption of yeast cells. Thus we
brane ghosts (Figs. 3A 1 and 3A 2)- No evidence of believe that the opposite a~tefa~ must also be

0 I~
considered i.e. most of the chitin synthetase activ- g
ity originally in chitosomes may become bound to ¢:

E .08 1( "U" 1.8 "1

plasma membrane fragments during cell breakage -e

by the usual harsh mechanic~3 methods. This

would explain claims indicating that most if not ¢ .06 1; ~ " .6
all of the chitin synthetase activity is bound to UJ

plasma membrane vesicles [8,9]. In an effort to U) m g

help to resolve the controversy, we isolated chito-
somes and plasma membrane fragments after dis-
ruption of yeast protoplasts by a modification of a

' i ~"

,4, m


very mild method described by Schwencke et al.

[12]. This method preserves vacuolar integrity and
z .02
' [ N-.2
a TM

minimizes contamination of homogenates by 0 10 20 30


vacuolar proteinases preventing the ~ton-physio- FRACTION NUMBER

logical 'activation' of zymogenic chitin synthetase Fig. 5. Linear-log sucrose gradient centrifugation of partially
purified vesicular fraction. The void volume eluate of a Sep-
by protease B [23-25]. Using this mild disruption harose 2B column (see Materials and Methods) was con-
method, chitin synthetase activity is found in two centrated against Aquacid IIA. 1 ml of concentrate was charged
different particulate fractions after isopycnic over a gradient prepared accordi: gly to Brakke and Van Pelt
sucrose gradient centrifugation. One fraction con- [30] and centrifuged in a SW.28 r~,tor at 90000× g (ray) for 2.5
tain collapsed smooth surface vesicles (mean di- h. 1.1 ml fractions wece collet;~.ed and assayed for chitin
synthetase (B), acid phosphatase (C,) and /~-l,3-exoglucanase
ameter: 260 nm) when examined by electron nil. (A).
croscopy negative staining and equi~brate at a
buoyant density of d - 1.203 g/cm 3. This fraction
shows a variety of characteristics that correspond against the idea that chitosomes may originate by
to plasma membrane ghost vesicles. Indeed yeast fragmentation of the yeast plasma membrane.
plasma membrane vesicles has been found to equi- When protoplast homogenates are subjected to
librate between d = 1.18-1.23 g/cm 3 [26] and at a single isopycnic centrifugation, some other
1.22 g/cm 3 [27] in a sucrose gradient. Also, the membrane-bound enzymatic activities, i.e. acid
characteristic vanadate-sensitive Mg2+-ATPase of phosphatase and fl-l,3-exoglucanase can be found
the yeast plasma membrane [15,27] co-sediments in the chitosomal fraction [4]. However, after a
with this heavier peak. Moreover, in protoplasts linear-log sucrose gradient centrifugation [30] these
labeled with 3H-concanavalin A which binds activities no longer contaminated the chitosomal
strongly to the external surface of the yeast fraction (Fig. 5). Similar results have been found
plasmalemma [28,29] the peak of radioactivity co- during the purification of Candida albicans chito-
migrated with the heavier peak of chitin syn- somes [5].
thetase activity, further indicating its plasma- Both chitin synthetase fractions were found to
lemmal origin. The main peak of chitin synthetase be 95 to 99~ zymogenic indicating that whatever
activity can be identified as chitosomes because of the mechanism for chitin synthetase activation it
its microvesicular size (mean diameter: 75 nm), its does not occur during purification. The absence of
morphological characteristics, the production of contamination by vacuolar proteinases, was an
microfibrUs after partial proteolytic activation and absolute requirement for maintaining zymogenic-
its low buoyant density ( d - 1.146) [1,2,7,21]. Sig- ity. This is in accordance with the reported in
nificantly, this fraction was found to be essentially vitro artefactual 'activation' of the zymogenic
free of contamination by plasma membrane chitin synthetase by vacuolar proteinase B [23-25].
vesicles as indic:~ted by the presence of only back- Recent evidence indicates the existence of two
ground levels of the 3H-concanavalin A label, by c,hitin synthetases (Chsl, Chs2) encoded by the
the lack of vanadate sensitive Mg 2+-ATPase (Fig. genes: CHS1 and CHS2 [31,32]. Chitin synthetase
1 low panel) and by the absence of/~-l,3-glucan 1 (Chsl) has been proposed to correspond to the
synthetase (not shown). These results clearly argues proteolytically activatable type, while Chs2 has

been proposed either not to be proteolytically and Mme J. Nappr, for expert handl/ng of the
activatable [32] or zymogen/c [33]. Therefore a manuscript. A Flores has been supported by
clear cut differentiation of the type of ch/tin syn- CONACYT, CINVESTAV-IPN and the Urfiver-
thetase present in ch/tosomes cannot be estab- sity of Guanajuato, Mexico.
fished on the basis of zymogerficity. Moreover, the
lack of requirement for the proteolytic activation References
reported for Chs2 [32] is also doubtful because of
the probable contamination with vacuolar pro- 1 Bracket, C.E, Ru/z-Hen.era, J. and Bartn/cki-Garcia, S.
teinases of the 'mixed-membrane' preparation (1976) Prec. Natl. Acad. Sci. USA 73, 4570-4574.
used. On the other hand in EDTA-treated 'mixed 2 Bartnicki°Garcia, S., Bracker, C.E., Reyes, E. and
membrane' preparations CC ~ appears to be Ruiz-Herrera, .L (1978) Exp. Mycoi. 2, 173-192.
3 Hanseler, E., Nyhlen, L.E. and Rast, D.M. (1983) Exp.
strongly inhibitory to Chsl but not to Chs2 [32]. Myco|. 7, 17-30.
Recent work within purified sub.cellular fractions 4 Schwenke, J., FIores, A. and Vofisek, J. (1984) in Microbial
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(1987) Exp. Mycol. 11,331-338.
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Acad. Sci. USA 72, 3952-3955.
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Acknowledgements 25 7ubenko, G.$., Mitchell, A.P. and 3ones, E.W. (1979) Prec.
Natl. Acad. Sci. USA 76, 2395-2399.
We are grateful to Jean Laporte for the electron 26 Schibeci, A., Rattray, .I.B.M. ~nd Kidby, D.K. (1973) Bio-
micrographs. We wish to thank Mme J. Mauger chim. Biophys. Acta 311, 15-25.

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