Molecular chaperones.

ANNUAL REVIEWS

Further

Quick links to online content Annu. Rev. Biochem. 1991. 60:321-47 Copyright © 1991 by Annual Reviews Inc. All rights reserved

Annu. Rev. Biochem. 1991.60:321-347. Downloaded from www.annualreviews.org by Stanford University - Main Campus - Green Library on 07/20/12. For personal use only.

MOLECULAR CHAPERONES R . John Ellis Department of Biological Sciences, University of Warwick, Coventry, United Kingdom

Saskia M. van der Vies Molecular Biology Division, Central Research and Development Department, E. I. Du

Pont de Nemours and Company, Experimental Station, Wilmington, Delaware KEY WORDS:

assisted self-assembly, chaperonins, heat-shock proteins, protein folding,

tein assembly.

pro

CONTENTS PERSPECTIVES . . . . . . . . . . . . . . . . ...... .. . . . . . . . . . . . . . . . . . . . .. . . ... . . . . . . . . . . . . . . . . . . . . . . . ...... . . . . . . .

321

THE MOLECULAR CHAPERONE CONCEPT.............. . . . . . . . .... . . . . ................. . . The Principle of Protein Self-Assembly...... .. . . . . . . . . ... . .. .. . ............... . . . . .. . . . . . . . . . The Cellular Problem of Interactive Suifaces. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .. . . . . .

323 324 325 327 328

Assisted Self-Assembly: a Model for Chaperone Action................................... Origin of Molecular Chaperone . . ... . . . . .. . ................ . . . . . .. .............. ... . . ....... . .

EXAMPLES OF MOLECULAR CHAPERONES. . . ..... .. . . . . . . . . . . . . . . . .. . . . . . . . ... . . . . . . . . . The Nucleoplasmins ...... ............... ... . ... . . ..................... .... . ..... .. .

.

.

.

...

.

...

.

.

..

330

332

The Chaperonins..................................................................................

Heat-Shock Proteins 70 and 90...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . ...................

333 342

IMPLICATIONS AND SPECULATIONS . . . . . . . . . . .. . . . . . . . . . . . . ...............................

343

PERSPECTIVES

A continuing challenge for biochemists is to unravel those cellular processes that use the one-dimensional information in the genetic material to produce the three-dimensional structures that give proteins their biological properties. The detailed mechanism of polypeptide chain synthesis is well established, but it remains to "crack the second half of the genetic code," which ensures that these chains attain their functional conformations 0, l a). The current paradigm for protein biogenesis presented in textbooks of biochemistry (e.g. 321

0066-4154/91/0701-03 21$02.00


322

ELLIS

Ref. 2) derives from the classic work of Anfinsen (3) on the in vitro renatura tion of ribonuclease, and is based on the hypothesis of self-assembly. This hypothesis postulates that each polypeptide chain interacts with itself as it is synthesized to assume a folded conformation of lower free energy. This spontaneous process requires neither further input of energy nor any steric information extrinsic to the polypeptide itself. The conformation assumed by each polypeptide possesses the functional activity unique to that polypeptide. Such activity often includes the ability to bind to macromolecules, including other polypeptides, in a highly specific manner, so that complex oligomeric structures can also self-assemble from their component subunits. Such com plex structures often mediate their cellular functions in ways that require their subunits to transiently dissociate and reassociate. Many proteins are also unfolded to an approximately linear conformation in order to traverse mem branes en route to a wide array of subcellular compartments or to the extracellular medium; such proteins refold into their functional conformations once membrane transport is complete. The hypothesis of protein self-assembly postulates that in all these situa tions the interactions that occur within and between polypeptides are both necessary and sufficient to produce the functional conformation. According to this hypothesis it is thus sufficient to specify the sequences of nucleotides in the genes encoding the amino acid sequences of polypeptides in order to specify the information sufficient for the biogenesis of these polypeptides in their functional forms. Self-assembly is not presented as the universal mech anism for the genesis of biological structures, since exceptions, such as the requirement of scaffolding proteins for the assembly of some bacteriophages, are well-established. Nevertheless, most textbooks give the impression that self-assembly as described above is the predominant process for the biogenesis of protein-containing structures. The concept of molecular chaperones (4-10) qualifies this self-assembly hypothesis by suggesting that in many cases interactions within and between polypeptides and other molecules need to be controlled to reduce the probabil ity of formation of incorrect structures, i.e. structures that lack the functions required by their biological context. This control is exerted by pre-existing proteins acting as chaperones to inhibit incorrect molecular interactions. It is argued that in any given assembly process there is a certain probability that incorrect interactions will produce nonfunctional structures. Where this prob ability is small, self-assembly needs no assistance, but where it is high, molecular chaperones are essential to produce sufficient correct structures for cellular needs. Molecular chaperones are currently defined as a family of unrelated classes of protein that mediate the correct assembly of other polypeptides, but are not themselves components of the final functional structures (6). Known molecu lar chaperones do not convey steric information for either polypeptide folding


MOLECULAR CHAPERONES

323

or oligoIDI:!rization, so the principle of self-assembly is not violated in this sense. Rather the principle is qualified by the need in many cases for assistance by molecular chaperones; these function by binding to specific structural features that are exposed only in the early stages of assembly, and so inhibit unproductive assembly pathways that would otherwise act as kinetic dead-end traps and produce incorrect structures. The term chaperone is appropriate for this family of proteins because the role of the human chaperone is to prevent incorrect interactions between people, not to provide steric information for these interactions. The general concept of molecular chaperones developed over several years as a result of studies at Warwick University on the biogenesis of the chloro plast enzyme that fixes carbon dioxide in photosynthesis-ribulose bisphos phate carboxylase-oxygenase, or rubisco (8-10). It became clear by 1987 that this concept applies generally to a wide variety of processes involving pro teins within all types of cell-plant, animal, and bacterial. The growing number and variety of proteins that can be regarded as molecular chaperones suggest both that there is a widespread cellular need for chaperone function, and that se:lf-assembly in its strictest formulation may not be the predominant process for the biogenesis of macromolecular structures. The consequences of this conclusion for our understanding of the evolution of these structures have yet to be explored. The concept of molecular chaperones may be useful in medicine, since chaperone diseases may exist in which correct folding and/or oligomerization of particular proteins may fail due to changes in their chaperone.. It is also possible that some human viruses require host chaperones for their replication. Some, but not all, molecular chaperones are stress proteins, so it is possible that all stress proteins act as molecular chaperones. The molecular chaperone concept may also be useful in biotechnology, where problems often arise in using heterologous systems to produce valuable proteins in the required active form. In this article we first review the concept of molecular chaperones, and then discuss a few selected examples and speculate about directions for future research. The literature on molecular chaperones is growing rapidly as this novel concept is stimulating research in many laboratories, so that exhaustive coverage is no longer possible in one article. Several reviews have been published (6-9), as well as the first issue of a new journal, Seminars in Cell Biology, which is entirely devoted to all aspects of molecular chaperones (10). THE MOLECULAR CHAPERONE CONCEPT

Molecular chaperones are defined as a family of unrelated classes of protein that mediate the correct assembly of other polypeptides but that are not components of the functional assembled structures (6). The imprecise word


324

ELLIS

mediate is used deliberately in this definition because of our ignorance about the molecular details of chaperone action. The proposed function of chaperone proteins is to assist polypeptides to self-assemble by inhibiting alternative assembly pathways that produce nonfunctional structures. The term polypeptide chain-binding proteins has been proposed for essentially the same group of proteins in a stimulating review by Rothman (11). The history of the development of the molecular chaperone concept from work on the assembly of rubisco has been described (9), so only the key points are presented here.

The Principle of Protein Self-Assembly The term self-assembly is used in the literature with different meanings (12). In the context of molecular chaperones we use the term self-assembly to mean that the information that is both necessary and sufficient in principle to specify the three-dimensional structure, and hence the function, of each polypeptide, resides solely within the amino acid sequence of that polypeptide; thus a newly synthesized polypeptide chain should be able to attain its functional conformation in the intracellular environment with no assistance from other molecules. We are not referring to the more general idea that complex structures can reform after dissociation in vitro into their component parts with no further input of information; this clearly does not hold true for structures as complex as membranes, organelles, and some viruses, or even for proteins whose assembly involves irreversible processing steps. It is also important to appreciate that the term assembly in the chaperone context is used in a �ide sense. It embraces both the folding of polypeptide chains immediately after synthesis, and any oligomerization into larger structures with proteins or other macromolecules that may occur, as well as changes in the degree of folding or oligomerization that take place as part of the way in which these structures function subsequently. Such changes may occur when proteins are transported across membranes and are reactivated during recov ery from stresses such as heat shock. Anfinsen and his colleagues found that denatured purified ribonuclease refolds spontaneously in the absence of other proteins into an active enzyme (3). Many other protein-containing structures have been successfully rena tured in vitro to their functional conformations from their purified and sepa rated components (13-16), including structures as complex as the tobacco mosaic virus (17), and the subunits of bacterial ribosomes (18). These classic demonstrations are commonly cited in textbooks to support the notion that self-assembly is the dominant process for the biogenesis of functional protein containing structures. However, it has been pointed out by Creighton (19) that all the evidence for self-assembly comes from in vitro studies with isolated proteins so that the potential involvement of other proteins has been either



325

ignored or discounted. It is instructive to quote his words (19) on this point: "These generalizations about protein folding in vivo are extrapolations from in vitro studies and assume that folding is strictly a self-assembly process. There is no need to invoke special factors to explain folding in vivo, since intact prot,;!ins can fold by themselves in vitro, but their existence in the biosynthetic apparatus cannot by ruled out." The fact that the denaturation of many proteins is not completely reversible in vitro, especially at high protein concentrations where incorrect interactions may predominate, has not until recently raised serious concern about the general validity of the self-assembly hypothesis to describe the in vivo situation. One consequence of the assump tion that self-assembly operates widely in vivo is that much effort is currently being expended to determine the rules by which the primary structure of a polypeptide determines its functional conformation, an effort that has yet to bear fruit 0; l a). The molecular chaperone concept postulates that self-assembly as defined above is not the predominant process of protein assembly in vivo because increasingly, examples are being discovered where proteins will not assemble correctly unless assisted by other proteins. It is this latter group of proteins that we telm molecular chaperones, a suggestion made first at a meeting in Copenhag�m in 1987 (20) and subsequently published in Nature (4). The chaperone function is required because many cellular processes that involve protein assembly carry an inherent risk of malfunction owing to the number, variety, and flexibility of the weak interactions that hold proteins in their functional conformations. The cell thus continually faces the problem that incorrect interactions will generate nonfunctional structures. It is the role of molecular chaperones to combat this problem. The Cellular Problem of Interactive Surfaces

A number of fundamental cellular processes involve the transient exposure of interactive protein surfaces to the intracellular environment. The term in teractive surfaces refers to any regions of intramolecular or intermolecular contact between parts of the protein-containing structure that are significant in maintaining that structure. All these processes may have to combat the problem of incorrect interactions. Examples of such processes are as follows: 1. Protein synthesis: the aminoterminal region of each polypeptide is made before the carboxyterminal region. If the normal fate of the aminoterminal region is to interact with the carboxyterminal region of the same chain, what happens to the aminoterminal region before the carboxyterminal region is made-might it undergo incorrect interactions with itself or other cellular components? The chance of incorrect interactions occurring will be greater if the rate of synthesis is much slower than the rate of folding, so chaperones may be required during synthesis only for proteins that fold rapidly. The rate


326

ELLIS

of in vitro renaturation often exceeds that of in vivo assembly by several orders of magnitude, so it is not surprising that incorrect interactions are commonly observed in vitro ( 1). Thus the time required to refold denatured proteins in vitro varies from milliseconds to hours ( 13, 2 1), but the average protein in yeast is synthesized in about two minutes (22). Chaperones belong ing to the chaperonin group (Table 1) bind to nascent polypeptides syn thesized by cell-free extracts of Escherichia coli (23), while chaperones of the heat shock protein 70 (hsp 70) class bind transiently to many newly syn thesized polypeptides during their synthesis in vivo (24). It has been sug gested that the efficiency of folding of some proteins is increased by con trolled rates of translation in vivo ( 1, 25). 2. Protein transport: proteins that enter organelles such as the endoplasmic reticulum, mitochondrion, plastid, and the bacterial periplasm traverse the membrane in an unfolded or partially unfolded state (26). Such proteins are often synthesized by cytosolic ribosomes in the form of precursors. Such precursors have to be prevented from folding into a translocation-incompetent conformation before transport can proceed (27), and once inside the organelle the polypeptide refolds into its final functional conformation. Thus mech anisms to mediate folding and refolding are required on both sides of such membranes, and much current work on molecular chaperones concerns these processes in both bacteria (23, 26, 28, 29) and eukaryotic cells (30-32). 3. Protein function: in several examples, the normal functioning of oligomeric complexes involves changes in subunit-subunit interactions, so that regions previously involved in subunit contacts are transiently exposed to the intracellular environment. Such processes include DNA replication (33), the recycling of clathrin cages (34), and the assembly of microtubules. 4. Organelle biogenesis: many of the protein complexes found inside organelles such as plastids and mitochondria consist of subunits synthesized inside the organelle bound to other subunits synthesized by cytosolic ribo somes; the enzyme rubisco is a good example of such a complex. Thus in some cases modulation of the binding propensities of the subunits may be required before they can all be located together in the same organelle. It was the discovery of a chloroplast protein that binds noncovalently to newly synthesized subunits of rubisco, and thereby prevents incorrect interactions, that eventually led to the molecular chaperone concept (reviewed in 8, 9). 5. Stress responses: environmental stresses such as excessive heat often cause the denaturation of proteins and the formation of aggregates. To protect against such stresses, cells accumulate proteins that prevent the production of such aggregates by inhibiting the incorrect interactions that cause them and/or unscrambling the aggregates to allow correct reassembly (31 , 35). Many molecular chaperones are stress proteins that are abundant even in the absence of stress; thus the stress response can be viewed as an amplification of the basic chaperone function (24, 35, 36).



327

The self-assembly hypothesis in its strictest formulation implies that all the interactions between protein surfaces exposed in such processes as those listed above are totally correct, i.e. that they are both necessary and sufficient to produce the functional conformation. Since incorrect interactions have been observed both in vivo and in vitro (8, 14, 16), it seems more plausible to suggest that during a given assembly process there is a certain probability that incorrect interactions will lead to the formation of nonfunctional structures. Where this probability is low, molecular chaperones may not be required, but where it is high, they may be essential to produce enough functional structures to meet cellular needs. There is evidence that molecular chaperones are involved in all these processes as indicated above, and it was the accumula tion of this evidence that spawned the general concept of molecular chaperones (4, 6, 7, 9). How do chaperones mediate their essential functions? Assisted Self-Assembly: a Model for Chaperone Action

The molecular chaperone concept does not necessarily imply that chaperones convey steric information specifying assembly. Present knowledge on the formation of nucleosomes (37) and rubisco (38) shows that the chaperones involved in their assembly do not convey steric information, because correct structures can be formed in the absence of chaperones under certain, albeit unphysiological, conditions. Instead chaperones function by recognizing structural feature(s) of the interactive surface(s), which are accessible only during stages in the assembly process, or which appear as a result of stress of a mutation in an already assembled structure; chaperones have not been observed to bind to assembled functional proteins or (0 all unfolded proteins during in vitro renaturation (28). The chaperone binds to this feature nonco valently to form a stable complex in which incorrect assembly pathways are -inhibited. This binding is reversed under, as yet, undefined circumstances, which pemlit correct interactions to predominate. In some cases, but not all, this reversal requires the hydrolysis of ATP, probably to release the ligand, and the pre:sence of another protein to mediate the release step. The best studied systems supporting this model are the renaturation of bacterial rubisco (8, 38, 39) and the assembly of lambda phage heads (40). This model proposed for the action of molecular chaperones suggests that their action is rather subtle and is best described as assisting self-assembly. Thus the principle of protein self-assembly is not violated by the molecular chaperone concept, rather it is qualified. On this basis we distinguish two types of self-assembly: 1. Strict self-assembly: no factors other than the primary structure are re quired for the polypeptide to have a high probability of assembling correct ly within the intracellular environment.

328

ELLIS


2. Assisted self-assembly: the appropriate molecular chaperone is required in addition to the primary structure to allow correct assembly to predominate over incorrect assembly; such chaperones convey no steric information over and above that in the primary structure of the ligand. It is conceivable that some chaperones do convey steric information for other polypeptides to assemble correctly, i.e. that there are cases of directed assembly. No examples of such chaperones have yet been reported, and it is rather hard to imagine how evidence for them could be obtained until our knowledge of the structural aspects of chaperone action is much better developed. It is possible to represent this model for chaperone action graphically by plotting the free energy for protein assembly within the cell against two possible outcomes-either the protein assembles correctly to give a functional structure or it misassembles to give a nonfunctional structure. For some proteins the formation of the correct structure may be favored both kinetically and thermodynamically, and in such cases chaperones are not required (Fig ure la). In other cases misassembly may be kinetically favored, so that chaperones are required either to inhibit the misassembly pathway by present ing an energy barrier or to lower the activation energy of the correct pathway (Figure Ib). The molecular details of chaperone action are totally obscure at the present time, but studies have commenced in several crystallographic laboratories to determine the three-dimensional structures of chaperone-ligand complexes, and reports have already appeared describing the three dimensional structures of two chaperones (a fragment of hsp70 and the PapD protein) in the absence of ligand (41, 42). In vitro assays for chaperone function in the renaturation of rubisco (38, 39) and beta-Iactamase (43) have been developed using purified components, and their further exploitation should provide much valuable information. The basic postulate of the chaperone hypothesis is that chaperones function by preventing incorrect interactions, and thereby allow correct interactions encoded within the primary structure to predominate. Gething & Sambrook (44) have made the intriguing speculation that some chaperones might also maintain parts of a polypeptide chain in conformations that can eventually be converted into a wider variety of shapes than would otherwise be possible. Such hypothetical chaperones would thus expand the repertoire of interactions within and between polypeptides to produce structures that would not be possible in their absence.

Origin of Molecular Chaperone The history of the term molecular chaperone has been described repeatedly (4--10), but is rarely referred to by other authors. Some other authors also err


329


(a)

:r c

Incorrect

C orrect

c

Incorrect

Correct

Figure 1 Gra.phical representation of the energetic effects of chaperone action. The vertical axis (F) represents the free energy of protein assembly from the unfolded state (U) within the cellular environment, while the horizontal axis represents two possible results of assembly--either an inactive misassembled structure (I) or a functional correctly assembled structure (e). In example (a) the formation of the correct structure is favored both kinetically and thermodynamically; this is a case of strict self-assembly where chaperones are not required. In example (b) the formation of an incorrect structure is favored kinetically; the addition of a chaperone, indicated by the dotted lines, could either block the misassembly pathway, or lower the activation energy of the correct pathway. Example (b) is a case of assisted self-assembly. This method of representation was suggested by R. B. Freedman. Reprinted with permission from Ellis (10).

in restricting the term to proteins that mediate the folding of polypeptides but not other aspects of protein assembly and vice-versa, while others confuse the word chaperone with the word chaperonin, which describes just one class of the larger family of molecular chaperones. For these reasons we summarize the history of the term here, as well as justify its present biochemical usage. The term molecular chaperone was first used in the scientific literature by Laskey et al (45) as a succinct way of describing the function of nuc1eoplas min. Nucleoplasmin is an acidic soluble nuclear protein that mediates the in vitro assembly of nucleosomes from separated histones and DNA. When DNA is miJl:ed with histone monomers at physiological ionic strength, self assembly fails in a spectacular way-an instant precipitate forms. However, if the histones are first mixed with a molar excess of nuc1eoplasmin before addition of DNA, soluble nucleosome cores form; this assembly process does


330

ELLIS

not require ATP (37, 46). Nucleoplasmin binds to histones and thereby reduces their strong positive charge, but it does not bind to DNA or to assembled nucleosomes. Nucleoplasmin is required only for assembly, and is not a component of the functional nucleosome. Separate experiments show that the steric information for nucleosome assembly resides in the histones and not in nucleoplasmin, because nucleosomes can be formed in the absence of nucleoplasmin under appropriate but unphysiological conditions. The role of the nucleoplasmin is thus not to provide steric information for nucleosome assembly but to reduce the positive charges of the histone monomers, so that correct interactions with DNA are not swamped by incorrect interactions. In the words of Laskey et al (45), "We suggest that the role of the protein we have purified is that of a molecular chaperone which prevents incorrect ionic interactions between histones and DNA." Nucleoplasmin is thus the archetyp al molecular chaperone; our contribution has been to point out that the term can be usefully extended to describe a much larger range of unrelated proteins that function to prevent incorrect interactions in a variety of cellular pro cesses. The word chaperone is normally used to describe a particular and largely outdated form of social behavior by human beings. The Oxford English Dictionary (2nd edition) defines chaperone, or more correctly chaperon, as a "person, usually a married or elderly woman who, for the sake of propriety, accompanies a young unmarried lady in public as guide and protector." Thus the traditional role of the human chaperone, if described in biochemical terms, is to prevent improper interactions between potentially complementary surfaces. Moreover, human chaperones do not possess the steric information by which people interact nor are they present during married life. Molecular chaperone is thus a very appropriate term to describe proteins which prevent incorrect interactions between parts of other molecules, but which do not impart steric information or form part of the final functional structures. EXAMPLES OF MOLECULAR CHAPERONES

Table 1 lists some of the proteins that meet the definition of molecular chaperones; this list is not exhaustive since potential candidates are appearing rapidly, especially with respect to protein transport. It is also a matter for debate where to draw the line between molecules whose sale function is to act as chaperones and those with additional functions (see below). The family of molecular chaperones is defined functionally, whereas the classes within this family are defined on the basis that membcrs within a class are sequence related to one another but not to members of any other class. Three recent examples are of especial interest because these particular chaperones are

Table 1

The molecular chaperone family


Class

I.

Nucleoplasmins

Members

Alternative names

NucJeoplasrnin

37,45, 52

Protein XLNO-38

51,53

Protein Ch-N038

55

Nucieoplasmin S 2.

Chaperonins

Refs.

Chaperon in 60

56 Bacterial: groEL (E. coli)

5, 8,23,26, 28, 29,39,

65-kDa common antigen (mycobacteria)

62-64

40, 43,57-66,72-82 Plastid: rubisco subunit-binding protein

5,8, 9, 65,67, 83-90

Mitochondrial: hsp 60

32, 68, 92-98

mitonin HuCha60

102

Eukaryotic cytosol: TCP- l Chaperon in 10

groES Mitochondrial 3.

Heat shock proteins 70

9, 59

Bacterial:

Hsp68,72, 73; DnaK; clathrin

40,71 70 35, 42, 44, 97, 105-109

uncoating ATPase; BiP; grp75,78,80;hsc70,KAR2; SSA l -4; SSBl; SSCl; SSD l 4.

Heat shock proteins 90

5.

Signal recognition particle

6.

Subtilisin prosequence

47a

7.

Alpha-lytic factor prosequence

47b

8.

Ubiquitinated ribosomal proteins

48

9.

Trigger factor

28

Hsp83,87; HtpG

35,110-114 SRP

50

10. Sec B protein

28,29

II. Pap D protein

41

�

0 t"'" tTl (l c: t"'" >� (l ::c >"0 tTl � 0 Z tTl en

w w -


332

ELLIS

attached covalently to the molecules whose assembly they mediate, i.e. the pro-sequence of pro-subtilisin (47a) and alpha-lytic protease (47b), and the ubiquitin sequence present at the aminoterminus of two ribosomal precursor proteins in yeast and other eukaryotes (48). In all three cases the extra sequences improve the correct assembly of the functional structures, but are then removed. The advantage of these cotranslational chaperones is presum ably that they can bind their ligands without having to seek them out amid the crowd of other molecules. It can be argued that the presequences present on many proteins that are transported across membranes should also be regarded as cotranslational chaperones, since there is evidence that they function to prevent the folding of the remainder of the polypeptide into a transport-incompetent conformation (49). However, in several cases it has been found that the chaperone function of these presequences is not completely effective without the assistance of separate chaperones, e.g. the signal recognition particle in the case of cotrans lational protein transport (50), and the trigger factor, the SecB protein, and the bacterial chaperonin 60 protein (groEL) in the case of posttranslational protein transport (23, 28, 29). These observations raise the possibility that the correct assembly of some proteins reqUires the assistance of more than one chaperone, some of which may be transient sequences within the protein itself, and that in some cases these chaperones interact with one another to achieve the ultimate goal of promoting efficient correct assembly. Evidence for the involvement of more than one chaperone in the transport and assembly of imported mitochondrial proteins is beginning to emerge (see later). Two enzymes involved in protein assembly, i.e. protein disulfide isomerase and peptidyl-prolyl-cis-trans-isomerasc, arc not regarded as molecular chaperones, since the evidence suggests that, for example, disul fide bridges stabilize the native state but do not determine the spatial arrange ment of the polypeptide backbone ( la, 13). We predict that the list of molecular chaperones will grow steadily longer, not only because the recent recognition of the chaperone function is stimulat ing much research, but also because this function is apparently required by many cellular processes. Owing to space limitations, only the first four classes in Table I are now discussed. The Nucleoplasmins

Processes such as the replication of DNA, the transcription and processing of RNA, and the assembly of nucleosomes and ribosomes involve strong ionic interactions between positively charged proteins and negatively charged nucleic acids. In eukaryotic organisms, additional processes transport these proteins into the nucleus, and transport ribonucleoprotein particles and ribo somes out of the nucleus. Recent evidence has suggested that a group of



333

nucleop1asrnin-1ike proteins exist that may perform a variety of chaperone functions during these processes (51); this evidence, however, rests largely on guilt-by-association, since direct demonstrations of the functions of any of these nucleoplasmin-like proteins are lacking. The role of nucleoplasmin in the assembly of chromatin in vitro and in vivo has been reviewed recently (37), and is not discussed here. Immunogold localization studies show that nucleoplasmin is present not only throughout the nucleoplasm, but also along the whole length of lampbrush chromosome loops and with the nucleoli. Double-labeling experiments with an anti nucleoplasmin antibody and an anti-ribonucleoprotein antiserum show that both antigens are present on the same ribonucleoprotein particles on these loops, suggesting that nucleoplasmin may be involved in the assembly of these partides (52). Since th(! studies that led to the description of nUcleoplasmin as the first molecular chaperone (45), further work has revealed the existence of several other nuclear proteins that may fulfill a number of different but related chaperoning functions (51). One such protein (XLNO-38) shows striking sequence similarity to nucleoplasmin, but is located in the granular com ponent of the nucleolus (53). The first 124 amino acids of this protein show 6 1 % identity with the aminoterminal region of nucleoplasmin (53). This protein is not a component of mature ribosomes, but is associated with both large and small preribosomal subunit particles. Thus protein XLNO-38 may carry out chaperoning functions specific to the nucleolus, such as the assem bly of basic: proteins on ribosomal RNA precursors. It will be recalled that the self-assembly of ribosomes in vitro from purified RNA and ribosomal pro teins proceeds with difficulty (54), so it would be worth repeating these studies in the presence of purified protein XLNO-38. The homologous nu cleolar protein from chicken (termed protein Ch-N038) has been shown to shuttle between the nucleus and cytoplasm (55), suggesting that this protein may also escort newly synthesized ribosomal proteins into the nucleus or be required to maintain preribosoma1 particles in a conformation compatible with export from nucleus to cytoplasm. A protein closely related to nucleoplasmin has been purified from a Xeno pus kidney cell line (56). This protein is called nucleoplasmin S (S for somatic), and cross-reacts with monoclonal antibodies used to identify protein XLNO-38. However, sequence information will be required to determine the relationship of nucleop1asmin S to nucleoplasmin and protein XLNO-38. The Chaperonins

The chaperonins were originally defined (5) as a class of sequence-related molecular chaperones found in all bacteria examined (including the eu bacteria, archaebacteria, cyanobacteria, and rickettsiae), in all mitochondria


334

ELLIS

examined (including those from yeast, Tetrahymena, Drosophila, Xenopus, Zea mais leaves, and human cells), and in all plastids examined (including chloroplasts, chromoplasts, and etioplasts). The degree of sequence related ness within the chaperonin group is high; for example the yeast mitochondrial chaperonin 60 protein shares 45% identical residues with the wheat plastid chaperonin 60, and 54% identical residues with the E. coli chaperonin 60, so it is probable that these proteins are true evolutionary homologues. The proteins from bacteria and plastids now known as chaperonins have been studied over many years in different laboratories under a variety of names (Table 1 ). The distribution of the chaperonins, coupled with their high degree of sequence identity, is consistent with an ancient origin for this group, and with their appearance in mitochondria and plastids via endosymbiosis (57, 58). Recently, sequence comparisons have revealed a lesser similarity be tween the chaperonins and the protein TCP-l , encoded in the t-complex of mouse chromosome 1 7 (9, 59). This protein of previously unknown function occurs in the cytosolic compartment of animal cells (V. Lewis, K. Willison, unpublished data), and a monoclonal antibody against it detects a protein with a 60-kilodalton subunit in the cytosol of Pisum sativum leaves (G. M. F. Watson, N. H. Mann, R. J. Ellis, unpublished data). Presumably the TCP-l protein performs the same chaperone functions in the cytosolic compartment of eukaryotic cells that its relatives are known to perform inside bacteria, mitochondria, and plastids, and this possibility warrants investigation. The chaperonins are all abundant constitutive proteins that increase in amount after stresses such as heat shock ( 1 0), bacterial infection of mac rophages (60), and an increase in the cellular content of unfolded proteins (61 ). This stress response makes the chaperonins potent antigens in a variety of infections (62), and their presence in all organisms raises the potential danger of autoimmune disease (63, 64). The chaperonins are sometimes referred to as the heat shock 60 group of proteins, but we suggest that this is a poor name because it emphasizes just one specialized aspect of the broader roles of these proteins. There are two types of chaperonin that are sequence-related to each other. The larger type is called chaperonin 60 (cpn 60), since its subunit Mr is about 60,000, while the smaller type is called chaperonin 10 (cpn 1 0) with a subunit Mr of about 10,000. Comparison of sequences of chaperonin 10 from four bacterial species with those of chaperonin 60 from eight species (including plastid and mitochondrial representatives) reveals an amino acid match in 58 out of the total 1 03 residues of chaperonin 1 0; a match is scored whenever an amino acyl residue occurs in the same position in at least one chaperonin homologue and in at least one chaperonin 60 homologue (65). The region of the chaperonin 60 sequence that shows this similarity to the entire chaperonin 1 0 sequence extends from residue 1 0 1 of the chaperon in 60 sequence; the



335

remainder of the chaperonin 60 sequence shows no detectable similarity to the chaperonin 10 sequence so the evolutionary relationship of the two chapero nins remains unclear. Chaperonin 60 purified from bacteria (66), plastids (67), or mitochondria (68) is an oligomeric protein with a distinctive structure of 1 4 subunits, arranged in two rings of seven subunits each (the "double donut"). Chaperonin 60 from Escherichia coli (originally termed groEL) and mitochondria is a homo-oligomer, but the plastid chaperonin 60 contains equal amounts of two highly related polypeptides termed alpha and beta (65, 69). The chaperonin 10 type was until recently reported only from bacteria, where it occurs as an oligomer of seven identical subunits arranged in a single ring (40). However, a functional and structural equivalent to chaperonin 1 0 has been partially purified from the mitochondrial fraction o f beef and rat liver (70). Evidence is accumulating to support the view that these two chaperonins interact in a functional manner to mediate polypeptide assembly inside bacte ria and mitochondria. It is therefore likely that a chaperonin 10-like protein occurs inside plastids and perhaps also in the cytosol of eukaryotic cells. The bacterial and plastid chaperonin 60 oligomers respond to added MgATP by hydrolyzing it and dissociating reversibly to smaller forms; bacterial and mitochondrial chaperonin 10 bind to chaperonin 60 in the presence of MgATP and suppress the ATPase activity of the latter (38, 70, 7 1 ). These responses to ATP are involved in the mechanism of action of the chaperonins; information about this mechanism is becoming available for the bacterial and mitochondrial chaperonins. THE BACTERIAL CHAPERONINS The best-studied example is the chaperonin 60 of E. coli, whose function was first identified by its requirement for the assembly of bacteriophages lambda, T4, and T5 (40). The first step in the assembly of bacteriophage lambda is the formation of an oligomeric structure of 1 2 identical phage-encoded polypeptides. The correct formation of this preconnector, the basic structure on which head phage proteins assemble, involves the intermediate formation of a 1 : 1 noncovalent complex between a phage protein monomer and the chaperonin 60 oligomer (72, 73). In the absence of chaperonin 60 function, the head proteins of phage T4 form large insoluble aggregates that associate with the cell membrane (74). The function of chaperonin 10 is also required for correct phage head formation, since it is involved in the release of the phage protein from chaperonin 60, but neither chaperonin forms part of the assembled phage. The two chaperonins have been implicated in a number of processes in uninfected bacterial cells, and are required at all temperatures (40). For example, genetic evidence suggests a role in DNA replication (75, 76), cell division (77), and protein secretion (78, 79). Increased levels of the two chaperonins in E. coli will suppress a number of temperature-sensitive muta-


336

ELLIS

tions that cause defects in enzymes of intermediary metabolism and protein secretion (80). Such proteins include Sec Y, which is an integral membrane protein, suggesting that chaperonins may be involved in the assembly of membrane proteins as well as soluble proteins. High levels of the two chaperonins have also been shown to enhance the assembly of the foreign oligomeric protein rubisco into a functional enzyme when the latter is ex pressed in E. coli from a cloned cyanobacterial gene (81 ). It seems that by increasing the amount of the two chaperonins in the E. coli cell, either by heat shock, overexpression from a multicopy plasmid, or the presence of unfolded protein (61), the folding capacity can be enhanced, allowing the cell to cope with an increased demand for folding activity. This conclusion should be borne in mind by biotechnologists experiencing difficulty in persuading bacte rial cells to produce large amounts of foreign proteins in the required active form (82). In most cases it has been found that processes mediated by chaperon in 60 in vivo also require the function of chaperonin 1 0, an observation consistent with the ability of the two chaperonins to form a complex with each other (40). However, in vitro studies have shown that chaperonin 60 alone is capable of maintaining precursor polypeptides in conformations compatible with subsequent transport across membranes (28). The binding of the pre cursor molecule to the chaperonin 60 oligomer may either retard folding of the precursor or fix its folded state, thereby gaining time for the precursor to interact correctly with the transport machinery. In this model the precursor is released from chaperonin 60 by some component of the transport machinery rather than by chaperonin 1 0. Recently an in vitro system employing purified components has been developed that demonstrates directly the requirement of the two bacterial chaperonins for protein assembly (8, 38, 39). Rubisco purified from Rhodo spirillum rubrum is a dimeric protein whose active sites involve both subunits so that enzymic activity requires correctly assembled dimers. Treatment with high concentrations of guanidine HCI or urea denatures the dimer to inactive monomers containing little secondary structure. When the inactive protein solution is diluted 200-fold into buffer lacking denaturant at 25°C, less than 2% of the original rubisco activity appears after incubation for 2 h and less than 10% after 24 h; at high protein concentrations insoluble aggregates form in a few minutes. However, if the temperature of incubation is reduced below 25°e, the recovery of active enzyme increases, until all the activity is recovered at lOoe and below on incubation for 24 h. If the buffer at 25°e contains a molar excess of E. coli chaperon in 60 oligomer to rubisco mono mer, a stable 1 : 1 complex forms between the rubisco monomer and the oligomeric form of chaperonin 60. No other cellular factors are required for this binary complex to form, and it is stable for at least 1 6 h at 25°e. Addition



337

of MgATP" potassium ions, and chaperonin 10 then results in the discharge of this complex, and the recovery of up to 80% of the original rubisco activity in the form of dimers. The number and order of steps in this latter process are not known, so it is not clear if the chaperonins are required in the dimerization step as we:11 as in the folding step. What is clear, however, is that the formation of a binary complex between chaperonin 60 and some folding intermediate of rubisco is an obligatory step in the chaperonin-dependent refolding pathway. This folding intermediate is also observed in the spontaneous folding of rubisco at 15°C and can be trapped by chaperonin 60 into a stable complex, thus inhibiting the spontaneous folding process. A similar observation has been made in the refolding of prebeta-lactamase; however, in this system retardation of refolding is reversed by ATP hydrolysis in the absence of chaperonin 10 (43). Recent work has extended the findings made with the rubisco refolding assay to citrate synthase from pig heart mitochondria, the reactivation of which after dilution from guanidine Hel also requires the presence of the two bacterial chaperonins and MgATP (103). Chloroplast and mitochondrial chaperonin 60 will partially replace bacterial chapcronin 60 in the rubisco refolding assay (39), while the mitochondrial chaperonin 10 will partially replace the bacterial chaperonin 10 (70). Addition of the chaperonins at temperatures below 25°C stimulates the spontaneous renaturation rate by tenfold. While it is likely that the chaperonins act catalytically in vivo, this has not been demonstrated so far in this in vitro system because the tendency of unfolded rubisco subunits to aggregate unless mopped up immediately by chaperonin 60 is so great. These in vitro observations confirm the proposal (6) that chaperones con vey no steric information for protein assembly, but rather act kinetically to assist self-assembly. The development of such in vitro reconstitution systems will pave the way for a more detailed undcrstanding of the steps in chaperonin action. It should be possible to use optical techniques to monitor changes in the environment of suitable chromophores present in the renaturing polypeptide to supplement information about the nature of the chaperonin ligand interaction that will eventually come from structural studies of crystals of these complexes. The general picture that is emerging for the function of the bacterial chaperonins has been summarized by Georgopoulos & Ang (40) as follows. Chaperonin 60 binds to unfolded forms of many polypeptides during pro cesses such as protein synthesis and protein transport. This binding maintains the polypeptides in a state that (a) prevents them from misfolding, (b) assists them in arriving at their correct intra- and inter-molecular folding pattern, (c) allows them to be more readily transported, and (d) permits proteases to degrade them. For some of these processes chaperonin 10 and MgATP are also required. Whether the chaperonins also bind to the interactive surfaces of

338

ELLIS

folded subunits that have transiently dissociated from a larger complex as part of the function of that complex remains to be established, but such binding might account for the suppression of mutations in proteins of the bacterial DNA replication complex by increases in the cellular content of the chapero nins (75, 76). The plastid chaperonin 60 was discovered in the author's (RJE) laboratory during studies on the biogenesis of the chloro plast enzyme rubisco, which catalyzes the first step in the photosynthetic pathway of carbon dioxide fixation (8, 9, 83). The chloroplast rubisco enzyme consists of eight large subunits that are synthesized inside the chloro plast and eight small subunits that are imported into the chloroplast after synthesis as higher-molecular-weight precursors in the cytosol. From the observation made by Roger Barraclough in 1980, that rubisco large subunits newly synthesized by isolated intact chloroplasts associate with a large oligomeric chloroplast protein prior to their assembly into the rubisco holoen zyme, it was hypothesized that the binding of the large subunits to this chloroplast protein may be an obligatory step in the assembly of the rubisco enzyme, thus suggesting that the latter is not a spontaneous process (83). The binding of newly synthesized rubisco large subunits to the oligomeric chloro plast protein resembles that of denatured Rhodospirillum ruhrum rubisco subunits to the bacterial chaperonin 60 in its stoichiometry and stability. It was later shown that imported rubisco small subunits also bind noncovalently to the same chloroplast protein (84, 85). Since then it has been found from cDNA sequence determinations that this chloroplast protein is closely related to both the groEL protein of E. coli and the heat shock protein 60 of mitochondria, a finding that led to the new name of the chaperonins for this group (5). Compelling evidence that the plastid chaperonin 60 is obligatorily involved in the assembly of plant rubisco has proved elusive; the best evidence in favor of this view is the inhibition by plastid chaperon in 60 antibodies of the appearance of in vitro-synthesized rubisco large subunits in an oligomeric form comigrating with pre-existing rubisco holoenzyme in chloroplast ex tracts (86, 87). The plastid chaperonin 60 contains equal amounts of two polypeptides called alpha and beta, which show about 50% sequence identity (65, 88). The arrangement and distribution of these two polypeptides within the chaperonin 60 oligomer is not known. Each polypeptide is encoded by a small number of nuclear genes, whose transcription is slightly enhanced by heat shock. Etio plasts also contain plastid chaperonin 60, and the amount of this protein increases when etiolated shoots of Pisum sativum are exposed to light (89). It is likely that plastid chaperonin 60 fulfills a multifunctional role within


THE PLASTID CHAPERONINS



339

this organelle, analogous to bacterial chaperonin 60. A wide range of polypeptides are found associated with chaperonin 60 after transport as precursors into isolated intact chloroplasts; these include foreign polypeptides such as chloramphenicol acetyltransferase, as well as glutamine synthetase and the beta subunit of the thylakoid ATP synthase. However, not all imported polypeptides associate with the chaperonin 60 in a detectable fash ion, e.g. f�:rredoxin and superoxide dismutase, indicating some specificity of binding (90). It is possible that the presequence of these imported polypeptides is either required for or enhances the binding of the polypeptide to chaperon in 60; mature rubisco small subunit, for example, does not bind to chaperonin 60. It has been reported that both the presequence and regions within the mature E. coli outer membrane protein A (omp A) participate in the recognition of the E. coli chaperonin 60 in vitro (28). Release of the imported chloroplast polypeptides by the plastid chaperonin 60 has been shown to require the hydrolysis of ATP (90). It is highly probable that plastids contain a form of chaperonin 1 0 (70), but no purification has yet been reported. The range of plastid processes requiring chaperonin 60 has yet to be explored, but by analogy with the bacterial situation may include plastid DNA replication, plastid division, and the assembly of oligomeric complexes such as the photosystems of the thylakoid membranes. The fact that some photo system polypeptides are transported into the thylakoid lumen at the non appressed regions, where they are transported for some distance before combining with other polypeptides anchored in appressed regions, suggests another potential intracellular site for chaperone action (91); by analogy with the endopla.smic reticulum however, any chaperones functioning in the thyla koid lumen may be members of the hsp 70 class rather than the chaperonin class. Another obvious goal for future research is the development of an in vitro system for the renaturation of the chloroplast rubisco enzyme from denatured subunits, based on the success that has been achieved with the simpler bacterial mbisco (8, 38, 39). Crop plant mbisco is a potential target for improvement by genetic engineering in view of its major influence on crop productivity, but all attempts to produce active mutant forms of such rubiscos in E. coli have so far becn unsuccessful, apparently because of the failure of the rubisco subunits to be released from the bacterial chaperonin 60 (8). The development of an in vitro reconstitution system for crop plant rubisco using the plastid chaperonins would overcome this block. MITOCHONDRIAL CHAPERONINS The most recently identified chaperonin 60 is the one located in the matrix of mitochondria from plant, animal, and protistan cells (68, 92, 93). The gene encoding the mitochondrial chaperonin 60 is part of the nuclear genome in yeast, and as in E. coli, the

THE


340

ELLIS

protein is essential for cell viability at all temperatures. The concentration of this protein increases twofold when Tetrahymena cells are exposed to high temperatures (94). The proposed requirement for chaperones in the refolding and oligomeriza tion of polypeptides that are transported across membranes (5, 7, 68) was elegantly confirmed with the aid of a yeast strain carrying a mutation in the mitochondrial chaperonin 60 gene. In this temperature-sensitive mutant (mif4), human ornithine transcarbamylase fails to form an enzymically active trimer in the mitochondrial matrix, but transport and processing of the pre cursor polypeptide is unaffected (95). Similarly, there are defects in the assembly of the mitochondrial ATPase complex in this mutant; the imported beta subunit fails to assemble into the ATPase complex , while two other subunits normally destined for the intermembrane space stay in the matrix because of a failure of the mechanism that re-exports them from the matrix (95). Analogous to the observations made with the chloroplast chaperonin 60, the mitochondrial chaperonin 60 is capable of binding both to polypeptides newly synthesized inside mitochondria (93) and to a number of unrelated proteins that are imported into isolated mitochondria (96). Whereas imported polypeptides bound to partially purified chaperonin 60 are sensitive to pro tease and hence regarded as unfolded, the addition of ATP reduces their protease sensitivity without leading to release from the chaperonin; this observation suggests that folding takes place on the surface of the chaperonin 60 oligomer (96). Subsequent release of this folded polypeptide presumably requires another factor, the most likely candidate being chaperonin 10. The report that a protein partially purified from liver mitochondrial fractions can substitute for the E. coli chaperonin 10 function in the in vitro renaturation of bacterial rubisco strongly suggests the existence of a chaperonin 10 homo logue in mitochondria (70). These initial findings have sparked more detailed studies on the involve ment of chaperonin 60 and other chaperones in protein transport and assembly in mitochondria, some of which are leading to a reassessment of earlier conclusions. Thus it used to be thought that ATP is required to unfold precursor proteins prior to their transport across the mitochondrial mem branes, as well as to provide the energy for transport across contact sites. Recent findings suggest instead that ATP is required only to prevent the formation of incorrectly folded structures in the cytosol in conjunction with the cytosolic heat-shock 70 proteins (hsp 70), and to release imported polypeptides from binding to chaperonin 60 and the mitochondrial hsp 70 chaperone after import (32). The new model postulates that it is the binding to the matrix hsp 70 chaperone of the polypeptide that is traversing the contact site that provides the driving force for transport. This binding also facilitates the unfolding of the precursor polypeptide on the cytosolic side of the contact



341

site and provides both the energy and the vectorial aspects of the transport process-in other words this binding pulls the polypeptide into the mitochondrion. The hsp 70 chaperone then mediates the transfer of the imported polypeptide to the matrix chaperonin 60 for correct assembly to be assisted (97). There is some evidence to support such a model for animal mitochondria; many newly synthesised mitochondrial proteins made by HeLa cells are transiently attached to either hsp 70 or the chaperonin 60, as judged by immunoprecipitation of extracts of pulse-labeled cells; this binding is released by incubation with ATP. Some evidence was also obtained for the existence of an ATP-dissociable complex of chaperonin 60 and hsp 70 (L. A. Mizzen, A. N. Kabiling, W. 1. Welch, submitted for publication). If all imported mitochondrial proteins need the aid of the chaperonins in order to assemble, it is predicted that imported chaperonin polypeptides would also require the assistance of the already-present chaperonin 60 oligo mer. This appears to be the case; when synthesis of the wild-type chaperonin 60 is induced in the yeast mif4 mutant under conditions where the mitochond rial chaperonin function is absent, the imported chaperonin 60 polypeptides fail to form the characteristic double-donut structure (97a). In other words, functional preexisting chaperonin 60 is required inside the mitochondrion in order that new chaperonin 60 can be assembled from imported subunits. This requirement can be viewed as one aspect of the principle that mitochondria do not arise by self-assembly but only by the division and growth of existing mitochondria. Chaperonin 60 from E. coli. on the other hand, is capable of self-assembly after denaturation with urea in vitro (97b). This self-assembly of bacterial chaperonin 60 requires MgATP and is stimulated by chaperonin 1 0. These observations are interpreted to mean that the chaperonin 60 oligo mers that are formed initially then self-chaperone the assembly of the remain ing monomers. A more speculative function for mitochondrial chaperonin 60 has recently been proposed (98, 1 1 5). Mutant lines of Chinese hamster ovary cells were selected for their resistance to anti-mitotic drugs such as podophyllotoxin, and a number of these were found to contain electrophoretic variants of either the mitochondrial chaperonin 60 (called P I by Gupta) or the constitutive cytosolic hsp 70 protein (called P2). Podophyllotoxin is believed to act by inhibiting the formation of tubulin dimers and so preventing the assembly of microtubules. Gupta suggests that hsp 70 facilitates the transfer of newly synthesized tubulin polypeptides into the mitochondrial matrix where the chaperonin 60 assists their assembly into dimers. These dimers are then exported without unfolding into the cytosol for use in microtubule assembly. The processes of microtu bule assembly and disassembly are in principle excellent candidates for assistance by molecular chaperones, since they involve the transient exposure of interactive protein surfaces, while the in vitro assembly of tubulin dimers,


342

ELLIS

as distinct from that of microtubules, has never been reported. However, the presumed cytosolic chaperonin 60 homologue, protein TCP- I , would seem a more plausible candidate for such a chaperone than the homologue located inside the mitochondrion, and there is no evidence that tubulin polypeptides are imported into mitochondria. The model also possesses two features for which there are no precedents; these are the export of a mitochondrial protein from the matrix into the cytosol and the passage of a dimeric protein across the mitochondrial membrane without disassembly. However, this model has several testable features and these should be explored, since progress in science often comes from pursuing the unexpected. Heat-Shock Proteins 70 and 90

The molecular chaperone concept can accommodate the functions of stress proteins if it is assumed that the primary effect of stress is to cause the appearance of interactive surfaces that are recognized by chaperones. Thus heat shock causes proteins to denature and form incorrect aggregates, while heat-shock proteins inhibit these processes by binding to the interactive surfaces exposed by high temperature. The stress response can thus be viewed as an amplification of a preexisting function that all cells require for their operation under nonstress conditions. Recent studies of the hsp 70 and 90 classes of stress protein have been reviewed and support the notion that these proteins act as molecular chaperones (35 , 104). Members of the hsp 70 class occur in both prokaryotes (Dna K protein in E. coli) and in several compartments of eukaryotic cells, e.g. several members in the cytosol, including the clathrin-uncoating ATPase , the BiP protein in the endoplasmic reticulum, and a member in the mitochondrial matrix. There is evidence that these proteins can interact with polypeptides during a variety of assembly processes in such a way as to prevent the formation of nonfunctional structures. For example, the BiP protein stabilizes nascent unfolded polypeptides as they emerge into the lumen of the endoplasmic reticulum during cotranslational protein transport (44), while the cytosolic hsp 70 proteins encoded by the SSAI and SSA2 genes of yeast maintain precursor polypeptides destined to enter the endoplasmic reticulum or mitochondrion in a translocation-competent conformation ( 1 05-107). The hsp 70 member in the mitochondrial matrix is required for protein translocation and the correct folding of proteins imported into the matrix (97). Pelham suggested in a seminal review ( 108) that heat-shock proteins not only mediate assembly processes but also promote the disassembly of proteins that have been damaged as a result of stress. Cycles of binding of a heat-shock protein to interactive surfaces exposed by the stress were suggested to be followed by ATP-mediated release, which triggers conformational changes in



343

the denatured protein that favor correct reassembly. No report has yet appeared that any chaperone can mediate the reappearance of the original biological activity from insoluble protein aggregates in vitro, but a recent remarkable finding shows that the Dna K protein from E. coli causes the reappearance of RNA polymerase activity when incubated with heat denatured inactive polymerase and MgATP ( l 09). Evidence is emerging that suggests that members of the hsp 90 class act as molecular chaperones in the mechanism of signal transduction by steroid receptors. In the absence of steroid hormones, the steroid receptor is bound to hsp 90 and is unable to activate transcription of steroid-controlled genes. Addition of steroid hormone displaces the bound hsp 90 and produces a receptor capable of activating transcription ( 1 1 0- 1 1 3) . However, this effect is not just one of steric hindrance of the DNA-binding site by hsp 90 since genetic studies in yeast suggest that receptors can be activated by hormone only if they are bound to hsp 90 initially. In other words, hsp 90 facilitates the subsequent response of the receptor to the hormone by binding to the receptor and allowing it to maintain an activatable conformation ( 1 1 4). IMPLICATIONS AND SPECULATIONS

The growing evidence for the involvement of several distinct classes of molecular chaperone in a variety of fundamental cellular processes is forcing a re-examination of some basic concepts. Self-assembly in the strict sense may be much more limited than the current paradigm implies, and it may have to be replaced by assisted self-assembly as the predominant process for the biogenesis of protein-containing structures. It follows that the evolution of these structures must involve not only the amino acyl sequences of the structural components but also those of their chaperones. The involvement of chaperones in the assembly of many proteins must be taken into account by those endeavoring to "crack the second half of the genetic code." There are: also implications of the molecular chaperone concept for medi cine and biotechnology. Besides the fact that those molecular chaperones that are stress proteins are also dominant immunogens in human bacterial in fections and are involved in certain autoimmune diseases (62-64) , there is the possibility that chaperone diseases exist in which the correct assembly of a particular protein fails because of changes in its sequence that affect recogni tion by the (:haperone, or because of changes in the sequence of the chaperone itself; the mitochondrial myopathies are obvious candidates here. Diseases with such a qmse may in the future become treatable by overexpression of the appropriate chaperone from inserted genes, analogous to the suppression by this means of certain mutations affecting protein assembly in E. coli (80) . The fact that some bacteriophages require the chaperonins of E. coli in order to

344

ELLIS

replicate raises the possibility that some viruses of plant and animal cells also require the assistance of host chaperones for their replication. This possibility should be explored to see if it is possible to mutate these chaperones to the


disadvantage of viral replication without harming the host cell. There is much commercial interest in expressing genes for useful proteins in heterologous systems , which offer advantages such as cost, purity, and the readiness with which structural alterations in the proteins can be made. However , production of recombinant proteins in bacteria is limited in many cases by the failure of the protein to assemble correctly into an active

conformation; a common problem is that the protein forms insoluble inclusion bodies (99, 1 00). The molecular chaperone concept suggests that researchers encountering such problems should re-examine the biogenesis of the protein of interest by the cells in which it occurs naturally to determine whether chaperones are involved . Such re-examination should include short pulse

label experiments, since chaperones are typically involved early in biogenesis, and the use of nondenaturing analytical techniques that allow the detection of complexes linked by noncovalent bonds. If chaperones are found to be involved, consideration should be given to the desirability of co expressing the genes for this chaperone in the same heterologous cells that are making the protein of interest in the hope that correct assembly will be favored. It may also be possible to develop defined in vitro systems contain

ing chaperones that will produce active proteins from denatured inclusion bodies, analogous to the one used with such success for bacterial rubisco (8, 38, 39). There are two key questions to be addressed by future research on molecu lar chaperones. Firstly, what is the structural basis by which a given

chaperone recognizes and binds to some feature(s) present in a wide variety of unrelated proteins but which is accessible only in the early stages of assem bly? Studies on the binding of short synthetic peptides to chaperones are already shedding some light on this problem ( 1 0 1 ) , but the final answer will come from studies of the crystal structures of chaperones containing bound polypeptide ligands. Secondly, how do bound chaperones exert their func-' tion? It is our current view that chaperones work by inhibiting incorrect

assembly pathways, but the answer to this question will come"from the development of in vitro assembly systems using pure components whose conformations can be readily m easured .

ACKNOWLEDGMENTS

We thank the following for generous access to unpublished data: A . A . Gatenby, R . S . Gupta, S . M . Hemmingsen, A . L . Horwich, R. Jaenicke, V.


345

Lewis, N. H. Mann, W. Neupert, N. Pfanner, G. M. F. Watson, and W. 1 . Welch; and the Science and Engineering Research Council for financial support.


Literature Cited 1 . Jaenicke . R. 1 988. Protein Structure and Protein Engineering, pp. 1 6-36. Mossbach Colloq. 39. Berlin/Heidel berg: Springer - Verlag 1a.

Jaenick,e, R. press

1 99 1 . Biochemistry. In

2. Watson,

J. D . , Hopkins, N. H . , Roberts, 1 . W. , Steitz, J . A. , Weiner, A. M. 1987. Molecular Biology of the Gene, 1 : 159. Menlo Park, Calif: Benja min/Cummings. 744 pp. 4th ed.

3. Anfinsen , C. B. 1 973. Science 1 8 1 :22330

4. Ellis, R. 1. 1987 . Nature 328:378-79 5. Hemmingsen, S. M . , Woolford, c . , van der Vies, S. M . , Tilly, K . , Denni s, D. T . , et al. 1988. Nature 333:330-34 6. Ellis, R. J . , Hemmingsen, S. M. 1989 .

Trends Biochem. Sci. 1 4 :339-42 7. Ellis, R. J . , van der Vies, S. M . , Hem mingsen S. M. 1989. Biochem. Sac. Symp. 55: 145-53 8. Gatenby . A. A . , Ellis, R. J. 1990. Annu. R,�v. Cell. Bioi. 6 : 1 25-49 9. Ellis, R. J. 1 990. Science 250:954-59 10. Ellis, R. J . , ed. 1990. Seminars in Cell Biology , Vol. 1 . Philadelphia: Saunders Scientific. 72 pp. 1 1 . Rothman, J. E. 1 989. Cell 59:59 1-601 1 2 . Miller, A. 1 984. Developmental Control in Animals and Plants, ed. C. F. Gra ham, P. F. Wareing, pp. 373-95 . Ox ford: Blackwell Scientific. 5 1 9 pp. 2nd ed. 1 3 . Fischer, G . , Sc hmidt , F. X. 1990. ..

Biochemistry 29:2205- 1 2

14. Jaenicke, R . , Rudolph, R . 1989. Protein Structure: A Practical Approach, ed. T. E. Creig hton, pp. 1 9 1-223. Ithaca, NY: 1LR Press 1 5 . Creighton,

T. E. 1 990.

Biochem.

1.

270: 1 - 1 6 1 6. Jaenicke, R . 1 987. Prog. Biophys. Mol.

BioI. 49: 1 1 7-237 1 7 . Klug, A. 1979. Harvey

Leet. 74: 14 1-72 1 8 . Nomura, M. 1 973. Science 1 79: 864-73 1 9. Creighton, T. E. 1 984. The Proteins, p. 3 1 2 . New York: Freeman. 5 1 5 pp. 20. Ellis, R. J . , van der Vies, S. M . , Hem mingsen, S. M. 1 987. Plant Molecular Biology , L ife Sci . , NATO-AS! Ser. A,

1 40:33-40, ed. D. von Wettstein, N-H. Chua. New York: Plenum. 697 pp. 2 1 . Jaenicke, R . , Rudolph, R, 1 986. Methods Enzymol. 1 3 1 :2 1 8-50

22. Petersen , N. S . , McLaughlin, C. S . 1 973. J . Mol. Bioi. 8 1 :33-45 23. Bochkareva, E. S . , Lissin, N. M . , Gir shovich, A. S. 1988 . Nature 336:25457 24. Beckman, R . P . , Mizzen, L. A . , Welch, W. J. 1 990. Science 248:850-54 25. Purvis , I. J . , Bettany, A. J. E . , San tiagu, T . C . , Cuggins, J. R . , Duncan, K . , et at . 1 987. J. Mal. BioI. 193:4 1 317 26. M eyer, D . I. 1988. Trends Biochem. Sci. 1 3 : 47 1 -74 27 . Rothman, J. E . , Kornberg , R. D. 1986.

Nature 322:209-1 0 28. Lecker, S . , Lill, R . , Ziegelhofer, T . , Georgopoulos, C . , B assf ord P. J . , e t al. ,

1 989. EMBO J. 8 :2703-9 29. Weiss, J . B . , Ray, P. H . , Bassford , P.

1. 1988. Proc. Natl. Acad. Sci. USA 85:8979-82 30. Hartl, F-U . , Neupert, W. 1990. Science 247:930-38

3 1 . Pelham, H. R. B. 1989. EMBO J. 8 : 3 1 7 1-76 32. Pfanner, N . , Rassow, J . , Guiard, B . , Soliner, T . , Hartl, F-U . , Neupert, W . 1990. 1. B ioi Chem. 265 : 1 6324-29 33. Alberts, B. M. 1 987. Philos. Trans. R . Soc. London, Ser. B 3 1 7:395-420 34. Rothman, J. E . , Schmid, S. L. 1986. .

Cell 46:5-9

35. Morimoto, R. T . , Tissieres, A . , Geor gopoulos , c . , eds. 1 990. Stress Proteins in Biology and Medicine, Monogr. 1 9 . 36.

New York: Cold Spring Harbor Lab. 450 pp. Pelham, H. R. B. 1 990. See Ref. 35,

pp. 287-99

37. Dilworth, S. M . , Dingwall, C. 1988. BioEssays 9:44-49 38. Viitanen, P. V . , Lubben, T. H. , Reed, J . , Goloubinoff , P . , O Keefe , D. P . , et al. 1990. Biochemistry 29:5665-7 1 39. Goloubinoff, P. , Christeller, J. T. , Gatenby, A. A . , Lorimer, G. H. 1 989. '

Nature 342:884-89


346

ELLIS

40. Georgopoulos, c . , Ang, D. 1990. See Ref. 1 0 , pp. 1 9-25 4 1 . Holmgren, A . , Branden, C-I. 1989. Na ture 342:248-5 1 42. Flaherty, K. M . , DeLuca-Flaherty, C . , McKay, D . B . 1990. Nature 346:62328 43. Laminet, A. A . , Ziegelhoffer, T. , Geor gopoulos, c . , Pluckthun, A . 1990. EMBO J. 9:23 1 5- 1 9 4 4 . Gething, M-J . , Sambrook, J . 1 990. See Ref. 1 0 , pp. 65-72 45 . Laskey , R. A . , Honda, B. M . , Mi lls , A. D . , Finch, J . T. 1978. Nature 275:41620 46. Blow, J. J . , Dilworth, S. M . , Dingwall, C . , Mills, A . D . , Laskey, R . A . 1987.

Philos. Trans. R. Soc. London, Ser. B 3 1 7:483-94 47a. Zhu , X . , Ohta, Y . , Jordan, F . , Inouye , M. 1989. Nature 339:483-84 47b. Silen, J. L . , Agard, D. A. 1989. Na ture 34 1 :462-64 48. Finley, D . , Bartel , B . , Varsharsky, A . 1 989. Nature 338:394-401 49. Verner, K . , Schatz, G. 1988. Science 24 1 :347-58 50. Walter, P. , Lingappa, V. R. 1986. Annu. Rev. Cell Bioi. 2:499-5 1 6 5 1 . Dingwall, C . , Laskey, R . A . 1990. See R ef. 1 0 , pp. 1 1- 1 7 5 2 . Moreau, N . , Angelier, N . , Bonnanfant Jais, M . L . , Gonnon, P . , Kubisz, P. 1 986. J. Cell Bioi. 103:683-90 5 3 . Schmidt-Zachmann, M . S . , Hugle-Darr, B . , Franke, W. W. 1987. EMBO J. 6 : 1 88 1-90 54. Nomura, M . , Held, W. A. 1974. Ribo somes, ed. M. Nomura, A. Tissieres, P. Lengyel . Cold Spring Harbor, NY: Cold Spring Harbor Lab. 5 5 . Borer, A . A . , Lehner, C. F . , Eppenber ger, H. M . , Nigg, E. A. 1989. Cell 56:379-90 56. Cotten, M . , Chalkey, P. 1 987. EMBO J. 6:3945-54 57. Picketts, D. J . , Mayanil . C. S. K . , Gup ta, R . S . 1 989. J. Bioi. Chem. 264: 1 2001-8 58. Gupta, R. S . , Picketts, D. J . , Ahmad, S . 1 989. Biochem. Biophys. Res. Commun. 1 63:780-87 59. Gupta, R. S. 1990. Biochem. Int. 20:833-41 60. Buchmeier, N. A . , Heffron, F. 1990. Science 248:730-32 6 1 . Parsell, D. A . , Sauer, R. T. 1989. Genes Dev. 3 : 1 226-32 62. Young, D. B . , Lathigra , R . , Hendrix, R. W . , Sweetser, D . , Young, R. A . 1 988. Proc. Natl. Acad. Sci. USA 85:4267-70

63. Young, D. B . 1990. See Ref. 1 0 , pp. 27-35 64. Young, D. B . , Mehlert, A . , Smith, D . F . 1 990. S ee Ref. 35, pp. 1 3 1-65 65. Martel, R . , Cloney, L. P . , Pelcher, L . E . , Hemmingsen, S . M . 1 990. Gene 94: 1 8 1-87 66. Hendrix, R. W. 1 979. J. Mol. Bioi. 1 29:375-92 67. Pushkin, A. V . , Tsuprun, V. L . , Solov jera, N. A . , Shubin, V. V . , Evstigneera, Z. G . , Kretovitch , W. L. 1 982. Biochim. Biophys. Acta 704:379-84 68 . McMullin, T. W . , Hallberg, R. L. 1988. Mol. Cell. Bioi. 8:37 1-80 69. Hemmingsen, S. M . , Ellis, R. J . 1 986. Plant Physiol. 80:269-76 70. Lubben, T. H . , Gatenby, A . A . , Donaldson, G . K . , Lorimer, G . H . , Viitanen, P . V . 1990. Proc. Natl. Acad. Sci. USA 87:7683-87 7 1 . Chandrasekhar, G. N . , Tilly, K . , Wool ford, C . , Hendrix, R. W . , Georgo poulos, C. 1986. J. Bioi. Chem. 26 1 : 1 24 14-19 72. Murialdo, H . 1979. Virology 96:34 1-67 73. Kochan, J . , Murialdo, H. 1 983. Virolo

gy 1 3 1 : 1 00-15

74. Takano, T., Kakefuda, T. 1 972. Nature New Bioi. 239:34-37 75. Fayet , 0 . , Louarn, I-M . , Georgopoulos, C. 1986. Mol. Gen . Genet. 202:43545 76. Jenkins, A. J . , March, J . B . , Oliver, I. R . , Masters, M . 1986. Mol. Gen. Genet. 202:446-54 77 . Miki, T. , Orita, T. , Furuno, M . , Horiuchi, T . 1988. J . Mol. Bioi. 20 1 :327-38 78. Phillips, G. J . , Silhavy, T. I . , 1 990. Nature 344:882-84 79. Kusukawa, N . , Yura, T . , Ueguchi, c . , Akiyama, Y . , Ito, K . 1989. EMBO J. 8:35 1 7-2 1 80. Van Dijk, T. K . , Gatenby , A. A . , LaRossa, R . A . 1 989. Nature 342:45 153 8 1 . Goloubinoff, P . , Gatenby, A . A . , Lorimer, G . H . 1989. Nature 337:44-47 82. G atenby, A. A . , Vi itane n , P. V . , Lorimer, G . H . 1990. Trends Biotech nolo 8:354-58 83. Barraclough, B . R . , Ellis, R. I. 1 980. Biochim. Biophys. Acta 608: 1 9-3 1 84. Ellis, R. J . , van der Vies, S. M. 1988. Photosynth. Res. 1 6: 1 0 1- 1 5 85. Gatenby, A . A . , Lubben, T. H . , Ahl qvist, P. , Keegstra, K. 1988. EMBO J . 7 : 1 307- 1 4 8 6 . Cannon, S . , Wang , P . , Roy, H . 1 986. J , Cell Bioi. 1 03 : 1 327-35 87. Roy, H. 1989. Plant Cell 1 : 1035-42


MOLECULAR CHAPERONES 88. Hemmingsen, S. M. 1990. See Ref. 10, pp. 47-54 89. Lennox, C. R . , Ell i s, R. J . 1 986. Biochem . Soc. Trans. 14:9-11 90. Lubben, T. H . , Donaldson, G. K . , V iitanen , P . V. , Gatenby, A . A . 1989. Plant Cell 1 : 1 223-30 91. Anderson, J . M . , Andersson, B. 1988. Trends Biochem. Sci. 13:351-55 92. Horwich, A. L. , Neupert, W . , Hartl, F-U. 1990. Trends Biotechnol. 8:12631 93. Hallberg, R. L. 1990. See Ref. 10, pp. 37-45 94. McM ulliin , T. W. , Hallberg , R . L. 1 987. Mol. Cell BioI. 7:4414-23 95. Cheng, :M. Y . , Hartl,F-U . , Martin, J . , Pollock, R. A . , Kalousek, F. , e t al. 1989. Nature 337:620-25 96. Ostermann , J., Horwich, A. L., Neupert . W . , Hartl, F-U. 1989. Nature 341:125·-30 97. Kang, P-J . , Ostermann, J . , Shilling, J . , Neupert,. W. , Craig, E. A. , e t a l . 1990. Nature 348:137-43 97a. Cheng, M . Y . , Hartl , F-U. , Horwich, A . L. 1990. Nature 348:455-58 97b. Lissin, N. M . , Venyaminov, S. Yu. , Girshovi ch , A. S. 1990. Nature 348: 339-42 98. Gupta, R. S. 1990. Trends Biochem. Sci. 15:415-18 99. M itraki , A., King, J. 1989. Bioi Techno/. 7:690-97 100. Schein, C H. 1989. Bio/Technol. 7:1141��9 101. Flynn, G. C , Chappell , T. G. , Roth-

102.

103 . 104. 105.

\06. 107. \08. \09. 1 1 0.

Ill.

1 1 2.

347

man, J. E. 1989. Science 245:38590 Jindal, S., Dudani, A. K. , Singh,B . , Harley, C B . , Gupta, R . S. 1989. Mol. Cell. B ioi. 9:2279-83 Buchner, J . , Schmidt, M . , Fuchs, M . , Jaenicke, R . , Rudolph, R . , e t al . 1991. Biochemistry. In press Ellis, R. J. 1990. Nature 346:710 Deshaies, R. J . , Koch, B. D. , Wemer Washburne , M . , Craig, E. A., Schek man, R. 1988. Nature 332: 800-5 Chirico, W. J . , Waters, M. G . , BlobeJ, G. 1988. Nature 332:805-9 Murakami, H . , Pain, D . , B lobel , G . 1988. J. Cell BioI. 1 07:2051-57 Pelham,H. R. B. 1986. Cell 46:959--61 Skowyra, D. , Georgopoulos, C, Zylicz, M . 1990. Cell 62: 939-44 Groyer, A. , Schweizer-Groyer, G . , Cadepond, F. , Mariller, M . , Bau1ieu, E-E. 1987. Nature 328:624-26 Sanchez, E. R . , Meshinchi, S . , Tienrun groj , W. , Schlesinger, M . J . , Toft, D . 0 . , Pratt, W . B. 1987 J. Bioi. Chem. 262:6986-9 1 Willmann, T . , Beato, M. 1986. Nature 324:688-91

1 1 3. Klein-Hitpass, L . , Tsai, S . Y . , Weigel, N. L., Allen, G. F . , Riley, D., et al. 1990. Cell 60:247-57 114. Picard, D . , Khurseed, B . , Garabedian, M. J . , Fortin , M. G., Lindqui st , S. , Yamamoto , K. R. 1990. Nature 348: 166-68 l i S. Gupta, R. S. 1 990. Biochem. Cell Bioi. 68:1352-63

Molecular chaperones and neuronal proteostasis.

Molecular chaperones. Unfolding protein folding.

Are prions misfolded molecular chaperones?

Do nucleic acids moonlight as molecular chaperones?

The nucleotide exchange factors of Hsp70 molecular chaperones.

ATP-dependent molecular chaperones in plastids--More complex than expected.

Adhesin presentation in bacteria requires molecular chaperones and ushers.

The Clp proteins: proteolysis regulators or molecular chaperones?

Protein Quality Control by Molecular Chaperones in Neurodegeneration.

Promoting Neuronal Tolerance of Diabetic Stress: Modulating Molecular Chaperones.

Cyanobacterial heat-shock response: role and regulation of molecular chaperones.

Development-dependent regulation of molecular chaperones after hypoxia-ischemia.

Non-equilibrium conformational dynamics in the function of molecular chaperones.

Regulation of GPCR Anterograde Trafficking by Molecular Chaperones and Motifs.

The role of heat-shock proteins as molecular chaperones.

Chaperones classified.

Chaperones in Neurodegeneration.

The Chemical Biology of Molecular Chaperones--Implications for Modulation of Proteostasis.

Mass spectrometry quantifies protein interactions--from molecular chaperones to membrane porins.

RNAs as chaperones.

ATPase subdomain IA is a mediator of interdomain allostery in Hsp70 molecular chaperones.

In vivo substrates of the lens molecular chaperones αA-crystallin and αB-crystallin.

Possible Function of Molecular Chaperones in Diseases Caused by Propagating Amyloid Aggregates.

A novel mechanism for small heat shock proteins to function as molecular chaperones.