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Molecular Chaperones: Opening and closing the Anfinsen cage
R. JOHN ELLIS
MOLECULAR CHAPERONES
Opening and closing the Anfinsen cage Dynamic interactions between chaperonins allow newly synthesized polypeptides to begin correct folding inside a transiently closed cage. Specialized chaperonins may be used to deal with recalcitrant proteins. The term 'Anfinsen cage' was introduced to describe an essential feature of the mechanism by which members of the chaperonin family of molecular chaperones increase the yield of correctly folded polypeptide chains [1]. Anfinsen's classical experiments on the refolding of pure denatured proteins demonstrated that polypeptide chains can self-assemble spontaneously in the test tube, in the absence of other macromolecules or energy expenditure [2]. Recent evidence shows, however, that protein folding in the cell requires interactions with pre-existing proteins that act as molecular chaperones [3], some of which hydrolyse ATP. These chaperones do not provide steric information for folding, but instead prevent, and possibly also reverse, the unproductive side-reactions that would otherwise result in nonfunctional conformations - hence the aptness of the term 'molecular chaperone'. The initial steps in protein folding, as the polypeptide chain emerges from the ribosome, are controlled by small chaperones of the hsp70/DnaJ/GrpE type, whereas at least some of the later folding steps occur inside 'cages' formed by the large oligomeric protein, chaperonin 60 [4]. These later steps are believed to resemble those occurring in the test tube in an Anfinsen refolding experiment - hence the term 'Anfinsen cage' is used to describe the structure responsible. It is not yet clear whether the final steps in folding to the native form occur inside or outside the cage: this may vary between proteins with different intrinsic folding rates (which can vary over a onehundred-fold range). It seems that chaperonins are required to assist protein folding in vivo because of two problems. Firstly, linear polypeptide chains collapse very rapidly into flexible compact structures that are intermediates in the folding process. These compact intermediates present hydrophobic surfaces that can aggregate together to form insoluble complexes which are incapable of progressing to the final native conformation. The extent of this problem varies with the polypeptide, but it is increased by both the high concentration of folding chains and the temperatures found inside cells. The second problem is that some folding intermediates do not aggregate, but appear to be kinetically trapped in a stable misfolded conformation: they cannot progress to the native state without binding to chaperonin 60 [5]. So how does chaperonin 60, and the related chaperonin 10, overcome these problems, considering that strong genetic evidence [6] supports the biochemical data from studies in vitro in showing that the chaperonins assist the folding of many unrelated polypeptides in living cells?
Recent experiments from Hartl's laboratory [7], combined with earlier studies, suggest a mechanism (see Fig. 1); the model should, at this stage, be regarded as a working hypothesis to stimulate further experimentation. Chaperonin 60 (GroEL in Escherichia col) assembles into two stacked rings of seven 60kD subunits, each of which has ATPase activity. Each ring encloses a central cage with dimensions of about 5x7nm. Chaperonin 10 (GroES in E. colt) assembles into a single ring of seven 10kD subunits [8]. Under most experimental conditions, one molecule of chaperonin 10 binds to one end of the chaperonin 60 oligomer, in the presence of Mg 2+-ADP, to form an asymmetric complex which is open at one end [1]. It is the cage - or cages, as there may be two separate cages per chaperonin 60 oligomer [1] - inside this complex that provides a sheltered space for the initiation of Anfinsen folding of single polypeptide chains. Thus, the problem of aggregation between folding molecules that occurs outside the cage is solved. Each chaperonin 60 oligomer acts as a cellular test tube, within which a polypeptide chain can start to fold in aqueous seclusion, an idea first published by Nilsson and Anderson [8]. In order for a mechanism of the type illustrated in Figure 1 to operate, binding of the compact folding intermediate to the chaperonin complex must be followed by its release into the cage, followed in turn by the initiation of correct folding and subsequent exit of the polypeptide at some point in the folding process. There is evidence from nuclear magnetic resonance (NMR) studies that, in some cases, the binding event may unfold the compact intermediate [9], so allowing a fresh attempt to be made at correct folding, as had been suggested by earlier kinetic studies [10]. In other cases, stable disulphide-bonded compact intermediates bind to chaperonin 60, suggesting that binding need not necessarily result in unfolding [11]. As chaperonin 60 can assist the folding of many different polypeptides, it is not surprising that the exact requirements for chaperonin 10 and nucleotides in vitro are found to vary from one polypeptide to another, but presumably one common mechanism operates within cells. This common mechanism involves an ATP-driven reaction cycle that continually switches the chaperonin machine through a set of different conformational states, until those regions of the polypeptide chain - as yet undefined that bind to the inside walls of the cage have been internalized in the tertiary structure of the folded protein [7]. The binding sites within the cage itself also have yet to be defined, but large-scale changes in the quaternary structure of chaperonin 60 that are induced by ATP have been
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Current Biology 1994, Vol 4 No 7 release of both ADP and chaperonin 10 (Fig. 1). The resulting binary complex of chaperonin 60 and polypeptide is stable in the absence of ATP; this is the form that was originally observed when it was discovered that newly synthesized chains of the large subunit of ribulose 1,5-bisphosphate carboxylase (rubisco) in chloroplast extracts are bound to an oligomeric protein made up of 60kD subunits [12]. In vivo, ATP binding weakens the interaction between the bound polypeptide and chaperonin 60, and induces the re-binding of chaperonin 10; the chaperonin 10 binds to either end of the chaperonin 60 oligomer with equal probability, to reform a ternary complex (binding to the opposite ring only is shown in Fig. 1). Chaperonin 60 oligomers with chaperonin 10 molecules bound to both ends have also been seen using electron microscopy, and these may be transient intermediates in cage assembly. Co-operative ATP hydrolysis is then postulated to induce the complete release of the polypeptide into the cage, so that all parts of the chain become available for Anfinsen folding more or less simultaneously. Polypeptide release tightens the binding of chaperonin 10 to chaperonin 60, so that if the polypeptide fails to fold to the stage at which its chaperonin 60-binding sites are internalized, it re-binds to the regenerated chaperonin 60/chaperonin 10/Mg 2+-ADP complex and the cycle repeats. Whether the polypeptide moves between the two rings of chaperonin 60 is unknown, but chaperonin 10 can be crosslinked to polypeptide that is bound to chaperonin 60. So, at some stage of the cycle at least, the polypeptide is contained in the ring of chaperonin 60 to which the chaperonin 10 is bound [13]. The extent to which larger polypeptides are buried within the cage is also unclear; rubisco large subunit bound to the chloroplast chaperonin 60 is digested by proteinase K bound to 100-micron diameter beads that are too large to enter the cage (my own unpublished data).
Fig.l. Proposed reaction cycle for chaperonin 60 and chaperonin 10 in the folding of nascent polypeptide chains. Adapted from [7,211. observed by negative-stain electron microscopy [1]. The defining principle of the mechanism outlined in Figure 1 is that ATP-induced conformational changes in chaperonin 60 drive alternative transient releases of either chaperonin 10 or the polypeptide chain [7]. The binding of a single molecule of a partially folded, compact intermediate to the complex containing chaperonin 60, chaperonin 10 and Mg 2 +-ADP triggers the
What is the role of the chaperonin 10 component? Firstly, chaperonin 10 stabilizes the adjacent ring in chaperonin 60 in a high-affinity state for binding ADP (not shown in Fig. 1, as ADP also binds to the distal ring, although more weakly). The chaperonin 60/chaperonin 10/Mg2+-ADP complex appears to be stable in the presence of ATP and the absence of unfolded polypeptide, but experiments show that there is in fact a dynamic equilibrium: ATP exchange with bound ADP induces the release of chaperonin 10 from chaperonin 60, followed by re-binding after the ATP is hydrolysed [7]. Secondly, when chaperonin 10 re-binds to chaperonin 60 upon exchange of ADP for ATP, it increases the co-operativity of subsequent ATP hydrolysis [14]; this co-operativity may be important to ensure simultaneous, rather than stepwise or random, release of all parts of the polypeptide chain. Thirdly, binding of chaperonin 10 to one or other end of the chaperonin 60 oligomer may prevent the folding polypeptide from exiting before internalization of its chaperonin 60-binding sites is complete. For example, in the absence of chaperonin 10, bound rhodanese is released from chaperonin 60 by the addition of ATP, but this released protein then aggregates rather than folding into active enzyme
DISPATCH [15]. We do not know the extent to which a polypeptide folds inside the cage before leaving, and the extent is likely to vary between different polypeptides. Thus chaperonin 10 acts rather like a jailer in controlling release from the cage, and it may achieve this by interacting directly with the polypeptide. A direct interaction between chaperonin 10 and a polypeptide that has been released but is still contained might also explain the variability in the requirement of chaperonin 60 for chaperonin 10 to assist the folding of different polypeptides [13].
nitrogenase more efficiently than can the constitutive chaperoning. It is ironic to note that, although the discovery of the chaperonins stemmed from work on the synthesis of the enzyme rubisco in higher plant chloroplasts [18], this enzyme has still not been refolded successfully in an Anfinsen experiment. Perhaps the unusual occurrence of two related types of subunit in chaperonins 60 and 10 from chloroplasts [19,20], reflects some specialization of chaperonin function to deal with this very abundant but refractory protein.
Chaperonin 10 may also have other, more specific, roles. A recent paper [16] extends an earlier genetic observation that the correct assembly of the capsid protein, Gp23, of bacteriophage T4, unlike that of the proteins of bacteriophages lambda and T5, does not require chaperonin 10, even if chaperonin 10 is over-expressed. Instead, Gp23 assembly requires a bacteriophage-encoded protein, Gp31 [16]. Gp31 has no obvious sequence similarity with chaperonin 10, but it can substitute for chaperonin 10 in the assembly of bacteriophages lambda and T5 in E. coli in vivo, and in the chaperonin 60-assisted folding of bacterial rubisco both in vitro and in vivo. Gp31 forms complexes with chaperonin 60 from E. coli that resemble complexes of chaperonin 10 with chaperonin 60, as seen using electron microscopy (S.M. van der Vies and H.R. Saibil, personal communication).
Acknowledgements: I would like to thank Ulrich Hartl, Helen Saibil and Saskia van der Vies for their help and encouragement.
Gp31 is the first example of a co-chaperonin - a protein that is not obviously related to the chaperonins but is required for them to act in some cases (confusingly, chaperonin 10 is sometimes referred to as a co-chaperonin, but this is not strictly correct: chaperonin 10 was defined as a chaperonin both when the latter term was first introduced and when its sequence relationship to chaperonin 60 was noted). It is unclear why Gp31 is required instead of chaperonin 10 in the assembly of the bacteriophage T4 capsid protein. It may function exactly like chaperonin 10 but with improved efficiency, or it may have an additional unknown role. It is also possible that Gp31 and chaperonin 10 bind transiently to polypeptide exiting from a chaperonin 60 oligomer, and so play a role in the association of monomers into oligomers.
References
1. Saibil HR, Zheng D, Roseman AM, Hunter AS, Watson GMF, Chen S, auf der Mauer A, O'Hara BP, Wood SP,Mann NH, et a/: ATP induces large quaternary rearrangements in a cage-like chaperonin structure. Curr Biol 1993, 3:265-273. 2. Anfinsen CB: Principles that govern the folding of polypeptide chains. Science 1973,181:223-230. 3. Ellis RJ:Roles of molecular chaperones in protein folding. Curr Opin Struct Biol 1994, 4:117-122. 4.
5. 6. 7. B. 9. 10.
11. 12.
13. 14. 15.
The identification of Gp3 as a co-chaperonin raises the possibility that specialized chaperonins may exist to deal with particular polypeptides, the folding of which presents especial difficulties. So far, there are no clear examples, although some findings can be interpreted as providing evidence for their existence. For example, the soybean nodule bacterium Bradyrhizobium japonicum contains five very similar but distinct chaperonin 60-chaperonin 10 gene pairs, each under different transcriptional control, including one that is regulated by the nitrogen fixation regulatory protein, nifA [17]. The control by nifA may simply reflect a need to increase the amount of the chaperonin proteins available when nodules form - for example, if nodule formation requires unusually folded proteins or abnormally high rates of synthesis. Alternatively, and more interestingly, it may reflect a need for a specialized chaperonin system that can assemble
16. 17.
18. 19. 20.
21.
Hartl F-U, Hlodan R,Langer T: Molecular chaperones in protein folding: the art of avoiding sticky situations. Trends Biochem Sci 1994, 19:20-25. Peralta D, Hartman DJ, Hoogenraad NJ, Hoj PB: Generation of a stable folding intermediate which can be rescued by the chaperonins GroEL and GroES. FEBS Letts 1994, 339:45-49. Horwich AL, Low KB, Fenton WA, Hirshfield IN, Furtak K: Folding in vivo of bacterial cytoplasmic proteins: role of GroEL. Cell 1993, 74:909-917. Martin , Mayhew M, Langer T, Hartl F-U: The reaction cycle of GroEL and GroES in chaperonin-assisted protein folding. Nature 1993, 366:228-233. Nilsson B, Anderson S: Proper and improper folding of proteins in the cellular environment. Annu Rev Microbiol 1991, 45:607-635. Zahn R, Spitzfaden C, Ottiger M, Wuthrich R, Pluckthun A: Destabilization of the complete protein secondary structure on binding to the chaperone GroEL. Nature 1994, 368:261-265. Jackson GS, Staniforth RA, Halsall Dl, Atkinson T, Holbrook J, Clarke AR, Burston SG: Binding and hydrolysis of nucleotides in the chaperonin catalytic cycle; implications for the mechanism of assisted protein folding. Biochemistry 1993, 32:2554-2563. Hayer-Hartl MJ, Ewbank JJ, Creighton TE, Hartl F-U: Conformational specificity of the chaperonin GroEL for the compact folding intermediates of a-lactalbumin. EMBO J 1994, 13:3192-3202. Barraclough R,Ellis RJ:Protein synthesis in chloroplasts: IX Assembly of newly synthesised large subunits of ribulose bisphosphate carboxylase in isolated intact pea chloroplasts. Biochim Biophys Acta 1980, 608:19-31. Bochkareva ES,Girshovich AS: A newly synthesized protein interacts with GroES on the surface of chaperonin GroEL. Biol Chem 1992, 267:25672-25675. Gray TE, Fersht AR: Co-operativity in ATP hydrolysis by GroEL is increased by GroES. FEBS Lett 1991, 292:254-258. Martin J, Langer T, Boteva R, Schramel A, Horwich AL, Hartl F-U: Chaperonin-mediated protein folding at the surface of groEl through a 'molten globule' intermediate. Nature 1991, 352:36-42. van der Vies SM, Gatenby AA, Georgopoulos C: Bacteriophage T4 encodes a co-chaperonin that can substitute for Escherichia col GroES in protein folding. Nature 1994, 368:654-656. Fischer HM, Babst M, Kaspar T, Acuna G, Arigoni F, Hennecke H: One member of a GroESL-like chaperonin multigene family in Bradyrhizobium japonicum is co-regulated with symbiotic nitrogen fixation genes. EMBOJ 1993, 12:2901-2912. Ellis RJ:Molecular chaperones: the plant connection. Science 1990, 250:954-959. Hemmingsen SM, Ellis RJ:Purification and properties of ribulose bisphosphate carboxylase large subunit binding protein. Plant Physiol 1986, 80:269-276. Bertsch U, Soil , Seetheram R, Viitanen PV: Identification, characterisation and DNA sequence of a functional 'double' GroES-like chaperonin from chloroplasts of higher plants. Proc Natl Acad Sci USA 1992, 89:8696-8700. Ezzell C: How chaperonins monitor their protein charges. NIH Research 1994, 6:31-34.
R. John Ellis, Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK.
635
MOLECULAR CHAPERONES
Opening and closing the Anfinsen cage Dynamic interactions between chaperonins allow newly synthesized polypeptides to begin correct folding inside a transiently closed cage. Specialized chaperonins may be used to deal with recalcitrant proteins. The term 'Anfinsen cage' was introduced to describe an essential feature of the mechanism by which members of the chaperonin family of molecular chaperones increase the yield of correctly folded polypeptide chains [1]. Anfinsen's classical experiments on the refolding of pure denatured proteins demonstrated that polypeptide chains can self-assemble spontaneously in the test tube, in the absence of other macromolecules or energy expenditure [2]. Recent evidence shows, however, that protein folding in the cell requires interactions with pre-existing proteins that act as molecular chaperones [3], some of which hydrolyse ATP. These chaperones do not provide steric information for folding, but instead prevent, and possibly also reverse, the unproductive side-reactions that would otherwise result in nonfunctional conformations - hence the aptness of the term 'molecular chaperone'. The initial steps in protein folding, as the polypeptide chain emerges from the ribosome, are controlled by small chaperones of the hsp70/DnaJ/GrpE type, whereas at least some of the later folding steps occur inside 'cages' formed by the large oligomeric protein, chaperonin 60 [4]. These later steps are believed to resemble those occurring in the test tube in an Anfinsen refolding experiment - hence the term 'Anfinsen cage' is used to describe the structure responsible. It is not yet clear whether the final steps in folding to the native form occur inside or outside the cage: this may vary between proteins with different intrinsic folding rates (which can vary over a onehundred-fold range). It seems that chaperonins are required to assist protein folding in vivo because of two problems. Firstly, linear polypeptide chains collapse very rapidly into flexible compact structures that are intermediates in the folding process. These compact intermediates present hydrophobic surfaces that can aggregate together to form insoluble complexes which are incapable of progressing to the final native conformation. The extent of this problem varies with the polypeptide, but it is increased by both the high concentration of folding chains and the temperatures found inside cells. The second problem is that some folding intermediates do not aggregate, but appear to be kinetically trapped in a stable misfolded conformation: they cannot progress to the native state without binding to chaperonin 60 [5]. So how does chaperonin 60, and the related chaperonin 10, overcome these problems, considering that strong genetic evidence [6] supports the biochemical data from studies in vitro in showing that the chaperonins assist the folding of many unrelated polypeptides in living cells?
Recent experiments from Hartl's laboratory [7], combined with earlier studies, suggest a mechanism (see Fig. 1); the model should, at this stage, be regarded as a working hypothesis to stimulate further experimentation. Chaperonin 60 (GroEL in Escherichia col) assembles into two stacked rings of seven 60kD subunits, each of which has ATPase activity. Each ring encloses a central cage with dimensions of about 5x7nm. Chaperonin 10 (GroES in E. colt) assembles into a single ring of seven 10kD subunits [8]. Under most experimental conditions, one molecule of chaperonin 10 binds to one end of the chaperonin 60 oligomer, in the presence of Mg 2+-ADP, to form an asymmetric complex which is open at one end [1]. It is the cage - or cages, as there may be two separate cages per chaperonin 60 oligomer [1] - inside this complex that provides a sheltered space for the initiation of Anfinsen folding of single polypeptide chains. Thus, the problem of aggregation between folding molecules that occurs outside the cage is solved. Each chaperonin 60 oligomer acts as a cellular test tube, within which a polypeptide chain can start to fold in aqueous seclusion, an idea first published by Nilsson and Anderson [8]. In order for a mechanism of the type illustrated in Figure 1 to operate, binding of the compact folding intermediate to the chaperonin complex must be followed by its release into the cage, followed in turn by the initiation of correct folding and subsequent exit of the polypeptide at some point in the folding process. There is evidence from nuclear magnetic resonance (NMR) studies that, in some cases, the binding event may unfold the compact intermediate [9], so allowing a fresh attempt to be made at correct folding, as had been suggested by earlier kinetic studies [10]. In other cases, stable disulphide-bonded compact intermediates bind to chaperonin 60, suggesting that binding need not necessarily result in unfolding [11]. As chaperonin 60 can assist the folding of many different polypeptides, it is not surprising that the exact requirements for chaperonin 10 and nucleotides in vitro are found to vary from one polypeptide to another, but presumably one common mechanism operates within cells. This common mechanism involves an ATP-driven reaction cycle that continually switches the chaperonin machine through a set of different conformational states, until those regions of the polypeptide chain - as yet undefined that bind to the inside walls of the cage have been internalized in the tertiary structure of the folded protein [7]. The binding sites within the cage itself also have yet to be defined, but large-scale changes in the quaternary structure of chaperonin 60 that are induced by ATP have been
© Current Biology 1994, Vol 4 No 7
633
634
Current Biology 1994, Vol 4 No 7 release of both ADP and chaperonin 10 (Fig. 1). The resulting binary complex of chaperonin 60 and polypeptide is stable in the absence of ATP; this is the form that was originally observed when it was discovered that newly synthesized chains of the large subunit of ribulose 1,5-bisphosphate carboxylase (rubisco) in chloroplast extracts are bound to an oligomeric protein made up of 60kD subunits [12]. In vivo, ATP binding weakens the interaction between the bound polypeptide and chaperonin 60, and induces the re-binding of chaperonin 10; the chaperonin 10 binds to either end of the chaperonin 60 oligomer with equal probability, to reform a ternary complex (binding to the opposite ring only is shown in Fig. 1). Chaperonin 60 oligomers with chaperonin 10 molecules bound to both ends have also been seen using electron microscopy, and these may be transient intermediates in cage assembly. Co-operative ATP hydrolysis is then postulated to induce the complete release of the polypeptide into the cage, so that all parts of the chain become available for Anfinsen folding more or less simultaneously. Polypeptide release tightens the binding of chaperonin 10 to chaperonin 60, so that if the polypeptide fails to fold to the stage at which its chaperonin 60-binding sites are internalized, it re-binds to the regenerated chaperonin 60/chaperonin 10/Mg 2+-ADP complex and the cycle repeats. Whether the polypeptide moves between the two rings of chaperonin 60 is unknown, but chaperonin 10 can be crosslinked to polypeptide that is bound to chaperonin 60. So, at some stage of the cycle at least, the polypeptide is contained in the ring of chaperonin 60 to which the chaperonin 10 is bound [13]. The extent to which larger polypeptides are buried within the cage is also unclear; rubisco large subunit bound to the chloroplast chaperonin 60 is digested by proteinase K bound to 100-micron diameter beads that are too large to enter the cage (my own unpublished data).
Fig.l. Proposed reaction cycle for chaperonin 60 and chaperonin 10 in the folding of nascent polypeptide chains. Adapted from [7,211. observed by negative-stain electron microscopy [1]. The defining principle of the mechanism outlined in Figure 1 is that ATP-induced conformational changes in chaperonin 60 drive alternative transient releases of either chaperonin 10 or the polypeptide chain [7]. The binding of a single molecule of a partially folded, compact intermediate to the complex containing chaperonin 60, chaperonin 10 and Mg 2 +-ADP triggers the
What is the role of the chaperonin 10 component? Firstly, chaperonin 10 stabilizes the adjacent ring in chaperonin 60 in a high-affinity state for binding ADP (not shown in Fig. 1, as ADP also binds to the distal ring, although more weakly). The chaperonin 60/chaperonin 10/Mg2+-ADP complex appears to be stable in the presence of ATP and the absence of unfolded polypeptide, but experiments show that there is in fact a dynamic equilibrium: ATP exchange with bound ADP induces the release of chaperonin 10 from chaperonin 60, followed by re-binding after the ATP is hydrolysed [7]. Secondly, when chaperonin 10 re-binds to chaperonin 60 upon exchange of ADP for ATP, it increases the co-operativity of subsequent ATP hydrolysis [14]; this co-operativity may be important to ensure simultaneous, rather than stepwise or random, release of all parts of the polypeptide chain. Thirdly, binding of chaperonin 10 to one or other end of the chaperonin 60 oligomer may prevent the folding polypeptide from exiting before internalization of its chaperonin 60-binding sites is complete. For example, in the absence of chaperonin 10, bound rhodanese is released from chaperonin 60 by the addition of ATP, but this released protein then aggregates rather than folding into active enzyme
DISPATCH [15]. We do not know the extent to which a polypeptide folds inside the cage before leaving, and the extent is likely to vary between different polypeptides. Thus chaperonin 10 acts rather like a jailer in controlling release from the cage, and it may achieve this by interacting directly with the polypeptide. A direct interaction between chaperonin 10 and a polypeptide that has been released but is still contained might also explain the variability in the requirement of chaperonin 60 for chaperonin 10 to assist the folding of different polypeptides [13].
nitrogenase more efficiently than can the constitutive chaperoning. It is ironic to note that, although the discovery of the chaperonins stemmed from work on the synthesis of the enzyme rubisco in higher plant chloroplasts [18], this enzyme has still not been refolded successfully in an Anfinsen experiment. Perhaps the unusual occurrence of two related types of subunit in chaperonins 60 and 10 from chloroplasts [19,20], reflects some specialization of chaperonin function to deal with this very abundant but refractory protein.
Chaperonin 10 may also have other, more specific, roles. A recent paper [16] extends an earlier genetic observation that the correct assembly of the capsid protein, Gp23, of bacteriophage T4, unlike that of the proteins of bacteriophages lambda and T5, does not require chaperonin 10, even if chaperonin 10 is over-expressed. Instead, Gp23 assembly requires a bacteriophage-encoded protein, Gp31 [16]. Gp31 has no obvious sequence similarity with chaperonin 10, but it can substitute for chaperonin 10 in the assembly of bacteriophages lambda and T5 in E. coli in vivo, and in the chaperonin 60-assisted folding of bacterial rubisco both in vitro and in vivo. Gp31 forms complexes with chaperonin 60 from E. coli that resemble complexes of chaperonin 10 with chaperonin 60, as seen using electron microscopy (S.M. van der Vies and H.R. Saibil, personal communication).
Acknowledgements: I would like to thank Ulrich Hartl, Helen Saibil and Saskia van der Vies for their help and encouragement.
Gp31 is the first example of a co-chaperonin - a protein that is not obviously related to the chaperonins but is required for them to act in some cases (confusingly, chaperonin 10 is sometimes referred to as a co-chaperonin, but this is not strictly correct: chaperonin 10 was defined as a chaperonin both when the latter term was first introduced and when its sequence relationship to chaperonin 60 was noted). It is unclear why Gp31 is required instead of chaperonin 10 in the assembly of the bacteriophage T4 capsid protein. It may function exactly like chaperonin 10 but with improved efficiency, or it may have an additional unknown role. It is also possible that Gp31 and chaperonin 10 bind transiently to polypeptide exiting from a chaperonin 60 oligomer, and so play a role in the association of monomers into oligomers.
References
1. Saibil HR, Zheng D, Roseman AM, Hunter AS, Watson GMF, Chen S, auf der Mauer A, O'Hara BP, Wood SP,Mann NH, et a/: ATP induces large quaternary rearrangements in a cage-like chaperonin structure. Curr Biol 1993, 3:265-273. 2. Anfinsen CB: Principles that govern the folding of polypeptide chains. Science 1973,181:223-230. 3. Ellis RJ:Roles of molecular chaperones in protein folding. Curr Opin Struct Biol 1994, 4:117-122. 4.
5. 6. 7. B. 9. 10.
11. 12.
13. 14. 15.
The identification of Gp3 as a co-chaperonin raises the possibility that specialized chaperonins may exist to deal with particular polypeptides, the folding of which presents especial difficulties. So far, there are no clear examples, although some findings can be interpreted as providing evidence for their existence. For example, the soybean nodule bacterium Bradyrhizobium japonicum contains five very similar but distinct chaperonin 60-chaperonin 10 gene pairs, each under different transcriptional control, including one that is regulated by the nitrogen fixation regulatory protein, nifA [17]. The control by nifA may simply reflect a need to increase the amount of the chaperonin proteins available when nodules form - for example, if nodule formation requires unusually folded proteins or abnormally high rates of synthesis. Alternatively, and more interestingly, it may reflect a need for a specialized chaperonin system that can assemble
16. 17.
18. 19. 20.
21.
Hartl F-U, Hlodan R,Langer T: Molecular chaperones in protein folding: the art of avoiding sticky situations. Trends Biochem Sci 1994, 19:20-25. Peralta D, Hartman DJ, Hoogenraad NJ, Hoj PB: Generation of a stable folding intermediate which can be rescued by the chaperonins GroEL and GroES. FEBS Letts 1994, 339:45-49. Horwich AL, Low KB, Fenton WA, Hirshfield IN, Furtak K: Folding in vivo of bacterial cytoplasmic proteins: role of GroEL. Cell 1993, 74:909-917. Martin , Mayhew M, Langer T, Hartl F-U: The reaction cycle of GroEL and GroES in chaperonin-assisted protein folding. Nature 1993, 366:228-233. Nilsson B, Anderson S: Proper and improper folding of proteins in the cellular environment. Annu Rev Microbiol 1991, 45:607-635. Zahn R, Spitzfaden C, Ottiger M, Wuthrich R, Pluckthun A: Destabilization of the complete protein secondary structure on binding to the chaperone GroEL. Nature 1994, 368:261-265. Jackson GS, Staniforth RA, Halsall Dl, Atkinson T, Holbrook J, Clarke AR, Burston SG: Binding and hydrolysis of nucleotides in the chaperonin catalytic cycle; implications for the mechanism of assisted protein folding. Biochemistry 1993, 32:2554-2563. Hayer-Hartl MJ, Ewbank JJ, Creighton TE, Hartl F-U: Conformational specificity of the chaperonin GroEL for the compact folding intermediates of a-lactalbumin. EMBO J 1994, 13:3192-3202. Barraclough R,Ellis RJ:Protein synthesis in chloroplasts: IX Assembly of newly synthesised large subunits of ribulose bisphosphate carboxylase in isolated intact pea chloroplasts. Biochim Biophys Acta 1980, 608:19-31. Bochkareva ES,Girshovich AS: A newly synthesized protein interacts with GroES on the surface of chaperonin GroEL. Biol Chem 1992, 267:25672-25675. Gray TE, Fersht AR: Co-operativity in ATP hydrolysis by GroEL is increased by GroES. FEBS Lett 1991, 292:254-258. Martin J, Langer T, Boteva R, Schramel A, Horwich AL, Hartl F-U: Chaperonin-mediated protein folding at the surface of groEl through a 'molten globule' intermediate. Nature 1991, 352:36-42. van der Vies SM, Gatenby AA, Georgopoulos C: Bacteriophage T4 encodes a co-chaperonin that can substitute for Escherichia col GroES in protein folding. Nature 1994, 368:654-656. Fischer HM, Babst M, Kaspar T, Acuna G, Arigoni F, Hennecke H: One member of a GroESL-like chaperonin multigene family in Bradyrhizobium japonicum is co-regulated with symbiotic nitrogen fixation genes. EMBOJ 1993, 12:2901-2912. Ellis RJ:Molecular chaperones: the plant connection. Science 1990, 250:954-959. Hemmingsen SM, Ellis RJ:Purification and properties of ribulose bisphosphate carboxylase large subunit binding protein. Plant Physiol 1986, 80:269-276. Bertsch U, Soil , Seetheram R, Viitanen PV: Identification, characterisation and DNA sequence of a functional 'double' GroES-like chaperonin from chloroplasts of higher plants. Proc Natl Acad Sci USA 1992, 89:8696-8700. Ezzell C: How chaperonins monitor their protein charges. NIH Research 1994, 6:31-34.
R. John Ellis, Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK.
635