REVIEW

Protein folding in the cell: competing models of

chaperonin function
R. JOHN ELLIS’ AND F.-ULRICH HARTL
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom; and Cellular
Biochemistry and Biophysics Program, Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer
Center, New York, New York 10021, USA

ABSTRACF The long-standing view that polypeptide proteins fold inside living cells, where the conditions are
chains newly synthesized inside cells fold spontane- very different from those found in the test tubes of the
ouslyto their functional conformations in an energy- protein chemist?
independent fashion derives from the observation The rate of polypeptide chain production by some cells
that many pure denatured proteins will refold spon- is prodigious. Each cell of Escherichia coli growing at
taneously in vitro when the denaturant is removed. 37#{176}Cwith a doubling time of 40 mm makes about 1000
This view is being challenged by the alternative polypeptide chains of average mass 40 kDa every second
proposal that in vivo many chains need to be helped (2). These chains are contained in a cytoplasmic volume
to fold correctly by preexisting proteins acting as of only about 0.6 J.Lm3and are synthesized by about 15 x
molecular chaperones, some of which hydrolyse 1O ribosomes, which are almost touching one another
ATP. The need for molecular chaperones arises when arranged in polysomes. In addition, the effective
because the high concentrations of transiently inter- concentration of nascent chains is increased greatly be-
acting protein surfaces inside cells permit the forma- cause of the effect of macromolecular crowding. The lat-
tion of incorrect nonfunctional structures. The ter term describes the fact that the concentration of
best-studied family of molecular chaperones are macromolecules inside living cells is so high that a sig-
called the chaperonins, the archetypal examples be- nificant proportion of the cellular volume is physically
ing the GroEL and GroES proteins of Escherichia occupied and therefore unavailable to other macromole-
coli. The chaperornns increase the yield of correctly cules. For example, the total concentration of protein and
refolded polypeptide chains, both by decreasing their RNA inside E. coli is about 340 g/l (3). One effect of
propensity to aggregate with one another and by crowding is to increase the association of interacting
allowing polypeptides kinetically trapped in incor- macromolecules; macromolecular association constants
rect conformations to make fresh attempts to refold for proteins in E. coli are predicted to exceed those in di-
into the functional conformations. The mechanisms lute solution by several orders of magnitude (3), so this
by which the chaperonins achieve these remarkable effect will increase greatly the thermodynamic activity of
results are currently under debate. This review sur- nascent chains above that predicted from their concentra-
veys competing models for chaperonin action, and tions. Why is this high thermodynamic activity of nascent
emphasizes the importance when evaluating these chains important?
models of considering the intracellular environment It is known from studies of the refolding of pure dena-
in which the chaperonins have evolved to func- tured proteins that many polypeptides fold into 3-dimen-
tion.-Ellis, H. J., Hard, F.-U. Protein folding in the sional structures via compact partially folded
cell: competing models of chaperonin function. intermediates that display hydrophobic regions transiently
FASEBJ. 10, 20-26 (1996) on their surfaces. These regions can interact with one an-
other and so generate aggregates that may become insol-
Key words: molecular chaperones protein folding . GroEL uble. It is for this reason that the successful refolding of
GroES many pure denatured proteins in vitro is favored by using
low concentrations of protein and low temperatures, both
of which disfavor hydrophobic interactions (4). But these
PROTEIN FOLDING IS ARGUABLY THE SINGLE most impor-
devices are not available to cells, so how do they over-
tant process in biology because it converts linear come these problems?
polypeptide chains into 3-dimensional structures that en-
The concept of molecular chaperones proposes that the
dow proteins with all their vital activities. It is well estab-
high concentrations of interacting protein surfaces inside
lished that the steric information for this folding process
resides within the aminoacyl sequence of each chain, be-
cause pure denatured proteins will often refold spontane-
ously into the correct conformations when the denaturant 1To whom proofs and correspondence should be addressed.
2
is removed (1). But is this a complete description of how jxDorevmatlon: ut-mr ix, otnyorololate reauclase.

20 0892-6638/96/001 0-0020/$01 .50 © FASEB

 PthVItW

cells generate a need for mechanisms to prevent incorrect folding; the structure, evolution, properties, and functions
interactions between these surfaces; one such mechanism of both the GroE and TCP-1 groups of chaperonins are
is the existence of proteins that recognize and bind tran- discussed extensively in a book to be published shortly
siently to these surfaces and thus prevent their premature (12).
or inappropriate interaction. Such proteins are called mo-
lecular chaperones, and there is growing evidence that
many unrelated families of protein can act in this way DIFFERENT MODELS FOR THE FUNCTIONS
(5-7). With respect to protein folding, two families of mo- OF GroEL AND GroES
lecular chaperone have been recognized: the
DnaKJDnaJ/GrpE (or hsp7O) family, which bind to grow- The majority of research on the GroE chaperonins con-
ing polypeptide chains while they are being synthesized cerns their ability to increase the yield of correctly re-
by ribosomes and prevent premature folding; and the folded protein when a pure denatured protein is diluted
chaperonin family, which assist correct folding at a later from denaturant into a buffer containing GroEL, GroES,
stage, when complete chains have either just left the ribo- and adenine nucleotides (8). Analyses of this process by
some or have been transported into mitochondria and a variety of techniques in several laboratories have pro-
chioroplasts from the cytosol (8). Both families of duced a growing and broadly consistent body of data, but
chaperone increase the yield of correctly folded protein the interpretation of these data by different researchers
when used in a variety of in vitro or in organello experi- has produced different types of model.
ments (5-8). In this review we consider only the In the next section we discuss the relative merits and
chaperonins, and discuss the merits and problems of the problems of these models in the context of three conclu-
different models for how they function. sions about GroEIJES function we consider to be well
supported by in vitro data. Our aim is to define those as-
STRUCTURE AND PROPERTIES OF THE pects of GroE action observed in vitro that are mechanis-
CHAPERONINS tically essential to improve the yield of correctly refolded
protein when extrapolated as far as possible to in vivo
The term “chaperonin” was coined to describe one family conditions. It is not our aim to accommodate in these
of sequence-related molecular chaperone found in chioro- conclusions all the interpretations reported in the litera-
plasts, mitochondria, and eubacteria such as E. coli (9). ture, because we believe that, although some of these
This group of proteins are now called the GroE chaperon- may well describe accurately what happens under a par-
ins to distinguish them from a more distantly related ticular set of in vitro conditions, they have not been
group, called the TCP-1 chaperonins, found in archae- shown to be either mechanistically essential or relevant
bacteria and the eukaryotic cytosol (8, 10). The to what happens under in vivo conditions. It should also
chaperonins all have in common a large oligomeric struc- be remembered that detailed studies have been carried
ture consisting of two stacked rings of subunits, each out of only a few refolding proteins, especially rhodanese,
about 60 kDa in size and possessing ATPase activity. dihydrofolate reductase, malate dehydrogenase, and bac-
Each ring surrounds a central cavity that plays a key role terial rubisco, and how generally applicable these conclu-
in the functioning of this molecule. The GroE chaperon- sions are to other proteins remains to be seen. Proteins
ins have seven subunits per ring, and the TCP-1 differ greatly in the tendency of their compact intermedi-
chaperonins have eight or nine. The GroE group contains ate forms to aggregate and also vary over at least a 100-
in addition another smaller oligomer consisting of a sin- fold range in their rates of spontaneous refolding, so it is
gle ring of seven subunits, each about 10 kDa in size. unlikely that there can be one precise description of
The larger oligomer from E. coli is called GroEL and its chaperonin function that will apply to all proteins.
14 subunits are identical, whereas the smaller oligomer of Space restrictions do not permit us to cite all the many
7 identical subunits is called GroES. One oligomer of relevant references containing the evidence for these con-
GroEL binds to one oligomer of GroES in the presence of clusions, so here we cite the first publications favoring
adenine nucleotides to produce an asymmetric binary each conclusion; additional references are cited in recent
complex. In this complex the GroES sits over the cavity reviews (5-8) and a book (12). Figure 1 illustrates the
of one ring of GroEL, leaving the cavity in the other ring conclusions in diagrammatic form with respect to the re-
open to the medium. Electron microscopic studies sug- folding of dihydrofolate reductase.
gest that the cavities are not continuous between the
rings. The chaperonins from E. coli are by far the best
studied with respect to protein folding. The crystal struc- THREE CONCLUSIONS ABOUT GroEIJGroES
ture of GroEL is known to 2.8 A and reveals the presence FUNCTION IN VITRO
of apical, intermediate, and equatorial domains in each
subunit, although some parts of the apical and equatorial
Conclusion 1
domains are not clearly resolved (11).
The remainder of this review concerns the mechanism One chain of protein emerging from denaturant binds initially
of action of GroEL and GroES with respect to protein by noncovalent hydrophobic interactions as a compact foLd-

COMPETING MODELS OF CHAPERONIN FUNCTION 21

REVIEW

compact forms (or molten globules) contain secondary

structures and often display some surface hydrophobic
residues that cause them to aggregate with one another
even at low concentrations, i.e., below 1 j.tM. In the pres-
ence of GroEL the compact forms do not aggregate with

‘go’ one another, but instead bind to GroEL (13), one chain
usually binding to one GroELJGroES complex. This bind-

‘.7 7ADP.
ing represents
originally
chloroplasts
the essential molecular
postulated for the chaperonin
(14, 15). The bound forms maintain
chaperone function
found inside
their
compact nature, as shown by tryptophan fluorescence and
dye-binding characteristics (16), and by protection of am-
ide hydrogens against exchange (17).
2 Negative-stain electron microscopy has located the
7 AlP
7 ATP, bound compact protein in the central cavity of GroEL
(18). The protein is bound between the apical domains of
each subunit, as demonstrated indirectly by electron mi-
croscopic visualization of gold particles coated with dena-
tured protein (19), and directly by cryoelectron
microscopy of GroEL/GroES complexes containing bound
3 cc’ malate dehydrogenase (20). This conclusion is supported
by mutational studies that link the loss of ability to bind
116
compact intermediates with the location of hydrophobic
woES. residues on the inner surface of the apical domains as re-
71’I O vealed by the crystal structure (21). Location of these hy-
drophobic protein binding sites on the inner face may
7 ADP
serve to prevent the aggregation of GroEL oligomers with
7 ATP
one another. This binding face is poorly defined in the
4 #{163}111 crystal structure, which may reflect the flexibility
quired both for GroEL to bind a wide range of different
re-

proteins and the conformational changes required for the

reversal of binding resulting from the action of GroES
and adenine nucleotides (22).
Figure 1. Model for the folding of dihydrofolate reductase (DHFR) by The asymmetric binding of one GroES oligomer to one
GroEL and GroES. ADP, the seven subunits in a GroEL ring that are end of the GroEL oligomer has been demonstrated by
bound to CroES in a high-affinity ADP state; ATP, the seven subunits in both negative staining (18, 23) and cryoelectron micros-
a GroEL ring in the ATP-bound state; U, unfolded polypeptide substrate
copy (20). This binding requires either ATP or ADP, and
as compact folding intermediate (open sphere). 1, Polypeptide binding
facilitates dissociation of tightly bound ADP and GroES. 2, Polypeptide
it has been shown that the preferential binding of these
transiently bound in nucleotide-free ring. 3, AlP binds and GroES nucleotides to one ring introduces asymmetry between the
reassociates with the polypeptide-containing ring of GroEL. DHFR pro- two rings, even in the absence of GroES (24). The gen-
tein is released into the GroEL cavity where it folds to the native state eration of this asymmetry by negative cooperativity is one
(hatched sphere) (GroES reassociation with the free GroEL ring is not
example of the multiple allosteric interactions displayed
shown). The seven subunits of GroEL that interact with GroES hydrolyze
by the GroEL oligomer, which is reflected in the confor-
AlP. This step may be required for the folding of certain polypeptide
substrates but is not necessary for the folding of DHFR. 4, GroES bound mational changes detected by cryoelectron microscopy
stably to GroEL subunits in the ADP state, enclosing the folded DHFR (20). It is possible to distinguish between the two rings of
in the GroEL cavity. 5, ATP binding and hydrolysis in the opposite GroEL GroEL in the GroEIJGroES complex, because the bind-
ring leads to GroES dissociation and folded DHFR emerges into the bulk ing of GroES to one ring protects the subunits of that ring
solution. 6, Incompletely folded protein is recaptured before it leaves
against removal of their 16 carboxy-terminal residues by
GroEL and may undergo unfolding to allow structural rearrangement in
preparation for another folding cycle in the GroEL cavity. Blue ring -
proteinase K whereas the subunits in the ring not bound
GroES; green rings - GroEL to GroES have these residues removed (18, 22). That
GroES protects the subunits of the GroEL ring to which it
is bound was first concluded from experiments in which
ing intermediate to the central cavity of the ring open to the GroES was cross-linked to the subunits of GroEL that are
medium of one asymmetric binary GroELIGroES complex. protected against proteinase K (22). This conclusion has
The random coil forms of polypeptide chain found in been recently confirmed by an independent approach us-
denaturants such as 8M urea or 6M guanidinium chloride ing an immobilized form of GroEL (25).
collapse in milliseconds into a mixture of more compact The validity of the cross-linking data has been ques-
forms when the denaturant is diluted sufficiently. These tioned on the grounds that the results are artefacts pro-

22 Vol. 10 January 1996 The FASEB Journal ELLIS AND HARTL

 HLVILW

duced by the use of trichloroacetic acid as protein pre- the evidence that the compact intermediate binds to the
cipitant (26), but the same results are obtained in the ab- central cavity of GroEL (18-21) or with the evidence that
sence of trichloroacetic acid (M. Mayhew and F. U. Harti, GroES binds, unbinds, and rebinds continually to GroEL
unpublished results). The conclusion that GroES protects in the presence of ATP (22). The observations leading to
the subunits in the GroEL ring to which it is bound is this suggestion are more readily interpreted in terms of
consistent with the structural data, as the carboxy-termi- the reported failure of the cross-linking agent used to in-
nal residues removed by proteinase K are located deep in stantly prevent the ATP-dependent interaction of the
the cavity (11) and GroES binding can be readily envis- GroEL and GroES so that the apparent stability of ternary
aged to prevent access of proteinase K to the cavity of the complexes observed is an artefactual consequence of the
ring to which GroES is bound. Proteinase K treatment of cross-linking assay (see footnote 21 in ref. 33).
GroEL/GroES complexes labeled with either cx-[32P]az-
ido-ATP or -ADP shows that it is the seven subunits of Conclusion 3
GroEL in the ring bound to GroES that bind and hydro-
lyse ATP preferentially (22). Hydrolysis of this ATP to Binding of GroES to one ring of GroEL creates a totally
ADP produces a GroEL/GroES/ADP complex; this com- enclosed cavity by triggering large conformational
plex is more stable than other complexes, and thus may changes in the ring to which it is bound; in this cavity
be the form that binds newly synthesised polypeptides in the compact intermediate is released and can start to fold
vivo (27). in a protected environment. Multiple rounds of release
and rebinding within the cavity may be required for some
Conclusion 2 proteins until their hydrophobic surfaces are internalized.
The more folded protein diffuses away when the GroES
The binding of compact intermediate to the open ring of unbinds on hydrolysis of ATP.
the GroEL/GroES/ADP complex triggers the release of The binding of GroES to one ring of GroEL creates an
GroES and ADP; binding of ATP results in rebinding of enclosed dome-shaped cavity underneath the GroES that
the GroES with roughly equal probability to either, but is larger (width 80 A and height 65 A) than the cavity
not both, rings of GroEL. found in one ring of GroEL in the absence of GroES (20).
Because the cavity in one GroEL ring is blocked by It is tempting to speculate that the compact intermediate
GroES, the compact intermediate must bind initially to may be released into this cavity where it can start to fold
the cavity in the opposite ring, as the electron micro- in isolation from other compact intermediates. One reason
scopic data indicate (20). However the binding of com- for this capping by GroES may be that the rate of diffu-
pact intermediate causes the GroES to dissociate, and in sion of compact intermediates in free solution is much
the presence of ATP it can then rebind to either, but not faster than the rate of folding of many proteins, even al-
both, rings (22); this is a consequence of the negative co- lowing for the reduction in diffusion rates produced by
operativity of ATP binding, so that ATP binds to one ring macromolecular crowding (3); thus, an uncapped ring
only (24), and is necessary for GroES to bind to that ring. could release a compact intermediate into the intracellu-
The data in support of some aspects of this conclusion lar environment with some of its surface hydrophobic
have been questioned. The observation that binding of a residues still exposed and hence run the risk of causing
compact intermediate favors release of GroES from aggregation with similar molecules. This view is equiva-
GroEL has been attributed to the effects of residual con- lent to regarding each GroEL/GroES complex as a mini-
centrations of guanidinium chloride in the refolding buff- test tube in which a single compact intermediate can start
er rather than to a direct effect of the intermediate (28). to fold in isolation, and has been termed the “Anfinsen
Clearly it is important to check for possible effects of cage” model in honor of the pioneer of in vitro protein re-
such residual denaturant in all protein refolding experi- folding (34).
ments, but a reexamination of this possibility has con- The first observation consistent with this idea was the
firmed the original conclusion for the conditions used, report that casein does not reduce the ATP-induced re-
which are different from those in which effects of the de- folding of denatured rhodanese bound to GroEL in the
naturant are observed (25). It is possible to make ternary presence of GroES (16). Casein has some affinity for
complexes of GroEL and GroES in which both ends of GroEL, even when undenatured, and thus competes with
each oligomer of GroEL are bound to GroES, and it has denatured rhodanese for binding to GroEL. Measurements
been proposed that such ternary complexes may be obli- of the amount of ATP required for the refoLding of rho-
gate intermediates in the functioning of the GroE danese by the GroEL/GtoES complex give a value of
chaperonins (29, 30). However, recent work from two about 130 mol ATP/mol rhodanese (16). The hydrolysis
laboratories provides no support for the functional impor- of ATP by the GroEIIGroES complex is cooperative (35),
tance of such ternary complexes in protein refolding (25, so that several rounds of hydrolysis of about 14 ATP
31, 32). The additional suggestion that such ternary com- molecules each are required to refold 1 mol of rhodanese.
plexes are stable and functional in protein refolding, and In the absence of both casein and GroES, ATP hydrolysis
that the compact intermediate must therefore bind to the causes the release of the bound rhodanese into the me-
outside surface of the complex (33), is not consistent with dium, where it aggregates slowly, but when casein is pre-

COMPETING MODELS OF CHAPERONIN FUNCTION 23

U#{149} V U TV

sent this aggregation is strongly enhanced (16). Thus, a mutant form of GroEL that can hind compact intermedi-
GroES is required for rhodanese to be released in a form ates but is unable to release them. The capping of bound
that is either competent for refolding correctly or at least intermediate by GroES confirms previous observations
has a much reduced propensity to aggregate in the pres- that binding and rebinding of GroES can occur to both
ence of casein. This conclusion implies that structural re- rings of GroEL (22) and that GroES can be cross-linked
arrangements that produce this form take place when the to a compact intermediate, provided the latter is bound to
rhodanese is associated with the GroELJGroES complex. GroEL (39).
Similar results were obtained subsequently for the The observation that GroEL can release bound compact
GroEL/GroES mediated refolding of malate dehydro- intermediates in nonnative form into the solution has led
genase and citrate synthase (36). to the proposal that the main function of the chaperonin
When these observations were made, there was no evi- is to bind intermediates in such a way as to cause partial
dence about the location of the bound compact intermedi- unfolding, i.e., breakage of some noncovalent bonds, and
ate, but with hindsight they can be interpreted in terms of then to release the more unfolded intermediates into the
the role of GroES in displacing the intermediate into the solution in each round of ATP hydrolysis for spontaneous
enclosed cavity, where it can rebind and unbind from refolding to occur in free solution (29, 38, 40). The no-
GroEL several times until its binding sites are internal- tion that binding to GroEL may cause partial unfolding is
ized. The fact that there is overlap between intermediate- not inconsistent with the view that binding and rebinding
binding sites and GroES-binding sites on GroEL (21) is occurs within the GroEL cavity, despite a claim to the
consistent with this interpretation. The more folded inter- contrary (40). Compact intermediates of mitochondrial
mediate can then diffuse out from the cavity when the malate dehydrogenase become trapped in a kinetically
GroES unbinds and, in the case of slower folding pro- stable form when incubated at 36#{176}C; these intermediates,
teins, complete its folding to the correct conformation in however, do not aggregate and can be caused to refold to
free solution once the danger of aggregation has passed. the native conformation by adding GroEL, GroES, and
Recent work with dihydrofolate reductase (DHFR)2 sup- ATP (41). This example supports the view that some pro-
ports this interpretation. teins require the chaperonins to fold correctly not to
DHFR is a monomeric protein that refolds spontane- avoid a potential aggregation problem, but to reverse sta-
ously with high efficiency in vitro. Nevertheless this pro- ble misfolded states. There are, however, three problems
tein binds to the GroEL homologue of isolated Neurospora with the proposal that refolding occurs spontaneously in
mitochondria after import into the matrix space and folds free solution when the intermediates are released from
to a native-like protease-resistant state on addition of GroEL.
ATP (37). This finding was the first to indicate that the First, no experiment has been reported that definitively
fact that a protein refolds spontaneously in vitro with no shows that folding occurs while the compact intermediate
difficulty does not necessarily mean that it folds sponta- is in free solution containing GroEL. Kinetic studies of
neously in vivo. Recent studies have shown that the com- the refolding of the small protein barnase are consistent
pact intermediate of DHFR can refold all the way to the with the view that this protein refolds while bound to
native state while the protein remains within the GroEL GroEL and not in free solution (42). Barnase refolds
cavity (M. Mayhew, A. da Silva, and F. U. Hartl, unpub- spontaneously very rapidly, but the rate of refolding is re-
lished results). This conclusion depends on the fact that duced by addition to GroEL because the compact inter-
it is possible to measure the final refolded state of DHFR mediate binds to GroEL; it is not, however, reduced to
while it remains in the cavity by assaying for the ability zero by addition of a 50-fold excess of GroEL, as ex-
of the GroEL/GroES/DHFR complex to bind the substrate pected if refolding occurs only in free solution. Second,
analog methotrexate. The holes in the sides of GroEL are such a mechanism would not offer protection against ag-
large enough to allow small molecules to enter the cavity gregation when the intermediates are jumping between
capped by GroES (11). In one type of experiment, a GroEL oligomers, but genetic experiments indicate that
modified form of DHFR was cross-linked to GroEL in the aggregation is a real problem in vivo, because at least
central cavity after formation of the binary GroEL/DHFR 30% of the soluble proteins of E. coli aggregate or mis-
complex. Addition of both GroES and ATP to this com- fold when GroEL function is disrupted (43). There is a
plex then generates the ability of the complex to bind suggestion that GroEL can bind to small aggregates and
methotrxate. Similar experiments with noncross-linked reverse their formation, but so far this interpretation rests
DHFR confirm that refolding occurs when GroES binds solely on indirect kinetic evidence (40). Third, it can be
to the ring to which the compact intermediate is bound. calculated that a compact folding intermediate 50 kDa in
DHFR that is bound to the opposite ring is either re- size takes only about 0.5 ms to diffuse 100 nm, the aver-
tained in an unfolded state or is released in a not yet age distance between GroEL oligomers in a typical pro-
completely folded conformation that is still capable of re- tein refolding experiment (see Fig. 2). This is a much
binding to another GroEL oligomer in the solution. This shorter time than it takes the average protein to refold;
rebinding was established in experiments similar to those even allowing for the probability that binding is not diffu-
first reported by Weissman et al. (38) using trap-GroEL, sion-limited and that several transfers may take place,

24 Vol. 10 january 1996 The FASEB journal ELLIS AND HARTL

 HtvItw

these values suggest that compact intermediates will not

have time to refold appreciably in free solution while
jumping between GroEL oligomers.
Recent repetition of these jumping experiments with
the same refolding protein, rhodanese, indicates that only
25% of the bound rhodanese leaves GroEL in a single 8
round of ATP hydrolysis; of the remaining 75% bound
rhodanese, 5% refolds to completion during this single
round (J. Martin and F. U. Hartl, unpublished results).
These results are consistent with the view that only a lOOnm
fraction of the compact intermediates fold to a committed
state upon release into GroEL cavities capped by GroES. In v/tm rnvtvo
In other words, the molecules of partially folded protein Figure 2. The difference in macromolecular crowding between in vitro
do not all behave identically when released into GruEL systems in which GroE chaperonins are studied and the in vivo situation
cavities; such varied behavior is expected on the grounds in which they evolved to function. Each square represents the face of a
cube with an edge 100 nm in length. The left-hand square (in vitro)
that a large range of conformational possibilities exists,
illustrates the 1.5 p.M concentration of GroEL, similar to that used used
and therefore many potential refolding pathways. in many protein refotding experiments; there is one GroEL oligomer per
It remains to consider why the GroEL releases 25% of cube (colored yellow, and approximately to scale). The right-hand square
the bound rhodanese in a partially refolded state: is this (In vivo) illustrates the 3 p.M concentration of GroEL in the cytoplasm of
a mechanistically essential part of GroEL function or is it E. coil; there are about two GroEL oligomers (yellow) per cube, and the
an artefact produced by the use of conditions far removed whole cytoplasm consists of about 600 such cubes. The other structures
in the right-hand square represent the sizes and concentrations of the
from those found in vivo, where GroEL evolved to func-
macromolecular components in the cytoplasm that create crowding by
tion? We suggest that the latter is the case because a no- occupying so much space; thus, each cube contains in addition to two
table feature of all in vitro studies of chaperonin function GroEL molecules about 30 ribosomes (green), about 340 tRNA and
published so far is the absence of macromolecular crowd- mRNA molecules (purple), and about 500 other protein molecules (red).
ing, a prominent aspect of the intracellular environment. The right-hand square is reprinted with permission from Goodsell (1991),
Trend.s Biochem. Sci., vol. 16, pp. 203-206, with permission.
Figure 2 represents this dramatic difference in visual
form.
It is possible to create a highly crowded environment
in vitro by adding large amounts of certain high molecu-
lar weight polymers, such as Ficoll 70 and Dextran 70
(3). Use of such polymers with GroEL shows that crowd- on the cellular contents and in vitro activities of GroEL,
ing inhibits the jumping of rhodanese refolding interme- and from the rates of protein synthesis in vivo, the frac-
diates between GroEL oligomers observed in their tion of total cellular protein that GroEL could interact
absence (J. Martin and F. U. Hartl, unpublished results). with under defined growth conditions. For E. coli cells
This observation can be interpreted in terms of the large growing at 37#{176}C with a doubling time of 40 mm and con-
increase in macromolecular association constants pro- taining on average about 1000 GroEL oligomers per cell,
duced by the crowding effect (3). These studies demon- it can be calculated that GroEL could assist the folding of
strate, at least for rhodanese, that there is no mechanistic about 7.5% of all the polypeptides folding in the cyto-
requirement for the release of partially folded intermedi- plasm, i.e., excluding those exported into or across the
ates into free solution in nonnative conformations that are cellular membranes (the full calculation can be found in
not yet committed to reach the native state without fur- Chapter 1 of ref 12). This calculation assumes that each
ther interaction with the GroEIIGroES system. GroEL oligomer can fold one average polypeptide chain
in 20 s at 37#{176}C. This value is derived from current in vi-
tro estimates of GroEL activity, but it may be worth
HOW MANY PROTEINS USE GroEL TO FOLD searching for intracellular factors and/or conditions that
IN VIVO? increase this rate.
Finally, there are four known intracellular compart-
The genes encoding GroEL and GroES are essential for ments where proteins fold but that do not appear to con-
viability and the proteins are required for growth at all tain any type of chaperonin: the endoplasmic reticulum,
temperatures. A recent genetic study used a temperative- intermembrane mitochondrial space and intrathylakoid
sensitive mutation to shut off the production of GroEL in chloroplast lumen of eukaryotic cells, and the periplas-
vivo, and concluded that a minimum of about 30% of the mic space of bacteria. Thus, it is certainly not the case
soluble proteins of E. coli requires GroEL to fold cor- that the folding of all proteins in cells involves the
rectly (43). However, it is difficult to rule out that only a chaperonins, but the rapidly growing list of other types of
small number of proteins actually interact with GroEL, molecular chaperone cautions against concluding that the
the folding of the remainder being disturbed by indirect folding of any protein in nature occurs in a spontaneous
effects. An alternative approach is to calculate from data manner.

COMPETING MODELS OF CHAPERONIN FUNC11ON 25

rV UVT

22. Martin, J., Mayhew, M., Longer, 1., and Hard, F.-U. (1993) The reaction
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26 Vol. 10 January 1996 The FASEB Journal ELLIS AND HARTL