Rapid Degradation of an Abnormal Protein in Escherichia coli Proceeds through Repeated Cycles of Association with GroEL
1999; Elsevier BV; Volume: 274; Issue: 53 Linguagem: Inglês
10.1074/jbc.274.53.37743
ISSN1083-351X
AutoresOlga Kandror, Michael Y. Sherman, Alfred L. Goldberg,
Tópico(s)Enzyme Structure and Function
ResumoMolecular chaperones are necessary for the breakdown of many abnormal proteins, but their functions in this process have remained obscure. The rapid degradation of the abnormal fusion protein CRAG in Escherichia coli requires the molecular chaperones GroEL, GroES, and trigger factor and proceeds through the formation of a CRAG-GroEL-trigger factor complex. Also associated with GroEL are smaller discrete fragments of CRAG. Pulse-chase experiments showed that these fragments were short-lived intermediates in CRAG degradation formed by C-terminal cleavages. Thus, CRAG degradation is not highly processive. In cells lacking the ClpP protease, the generation of these fragments and their subsequent degradation were much slower than in the wild type. Dissociation of CRAG from GroEL was necessary for its digestion by the ClpP protease, because in a groES temperature-sensitive mutant, CRAG was stable and accumulated on GroEL. Furthermore, the expression of a dominant GroEL mutant defective in substrate dissociation slowed degradation of both CRAG and the fragments. Therefore, we suggest that CRAG degradation proceeds through multiple rounds of substrate binding to GroEL, followed by their GroES-dependent dissociation, which allows further digestion by the protease. In this multistep process, GroEL and GroES function repeatedly, apparently to allow further degradation of CRAG and its fragments by the protease. Molecular chaperones are necessary for the breakdown of many abnormal proteins, but their functions in this process have remained obscure. The rapid degradation of the abnormal fusion protein CRAG in Escherichia coli requires the molecular chaperones GroEL, GroES, and trigger factor and proceeds through the formation of a CRAG-GroEL-trigger factor complex. Also associated with GroEL are smaller discrete fragments of CRAG. Pulse-chase experiments showed that these fragments were short-lived intermediates in CRAG degradation formed by C-terminal cleavages. Thus, CRAG degradation is not highly processive. In cells lacking the ClpP protease, the generation of these fragments and their subsequent degradation were much slower than in the wild type. Dissociation of CRAG from GroEL was necessary for its digestion by the ClpP protease, because in a groES temperature-sensitive mutant, CRAG was stable and accumulated on GroEL. Furthermore, the expression of a dominant GroEL mutant defective in substrate dissociation slowed degradation of both CRAG and the fragments. Therefore, we suggest that CRAG degradation proceeds through multiple rounds of substrate binding to GroEL, followed by their GroES-dependent dissociation, which allows further digestion by the protease. In this multistep process, GroEL and GroES function repeatedly, apparently to allow further degradation of CRAG and its fragments by the protease. trigger factor polyacrylamide gel electrophoresis isopropyl-1-thio-β-d- galactopyranoside In addition to their roles in protein folding and translocation (1Hartl F.U. Semin. Immun. 1991; 3: 5-16PubMed Google Scholar, 2Hartl F.U. Nature. 1996; 381: 571-580Crossref PubMed Scopus (3090) Google Scholar, 3Gething M.J. Sambrook J. Nature. 1992; 355: 33-45Crossref PubMed Scopus (3575) Google Scholar, 4Welch W.J. Curr. Opin. Cell Biol. 1991; 3: 1033-1038Crossref PubMed Scopus (121) Google Scholar), molecular chaperones are also necessary for the selective degradation of certain proteins with highly abnormal conformations. This role for the molecular chaperones is best documented inEscherichia coli but has also been demonstrated in yeast, animal cells, and mitochondria (5Sherman M. Goldberg A.L. Feige U. Morimoto R.I. Yahara I. Bolla B. Stress-inducible Cellular Responses. Birkhäuser Verlag, Basel, Switzerland1996: 57-78Crossref Google Scholar, 6Hayes S.A. Dice J.F. J. Cell Biol. 1996; 132: 255-258Crossref PubMed Scopus (163) Google Scholar). Moreover, the breakdown of different abnormal polypeptides appears to require different chaperones and distinct ATP-dependent proteases (7Sherman M. Goldberg A.L. EMBO J. 1992; 11: 71-77Crossref PubMed Scopus (173) Google Scholar, 8Kandror O. Busconi L. Sherman M. Goldberg A.L. J. Biol. Chem. 1994; 269: 23575-23582Abstract Full Text PDF PubMed Google Scholar). For example, if alkaline phosphatase fails to be secreted into the periplasm, it does not fold correctly and is rapidly degraded by protease La (lon) in a process requiring DnaK (the Hsp70 homolog in bacteria), and its cofactors, DnaJ and GrpE (7Sherman M. Goldberg A.L. EMBO J. 1992; 11: 71-77Crossref PubMed Scopus (173) Google Scholar). By contrast, the rapid breakdown of the recombinant fusion protein, CRAG, requires the ClpP protease as well as GroEL, GroES, and trigger factor (TF)1 (8Kandror O. Busconi L. Sherman M. Goldberg A.L. J. Biol. Chem. 1994; 269: 23575-23582Abstract Full Text PDF PubMed Google Scholar, 9Kandror O. Sherman M. Rhode M. Goldberg A.L. EMBO J. 1995; 14: 6021-6027Crossref PubMed Scopus (90) Google Scholar).The precise roles of chaperones in these degradative processes are still uncertain. One critical property of the molecular chaperones is that they selectively bind to unfolded proteins, and it has been suggested that association of an unfolded polypeptide with the chaperone may serve to promote substrate recognition by the cell's ATP-dependent proteases (5Sherman M. Goldberg A.L. Feige U. Morimoto R.I. Yahara I. Bolla B. Stress-inducible Cellular Responses. Birkhäuser Verlag, Basel, Switzerland1996: 57-78Crossref Google Scholar, 6Hayes S.A. Dice J.F. J. Cell Biol. 1996; 132: 255-258Crossref PubMed Scopus (163) Google Scholar). Alternatively, the chaperones may function together with proteases during the degradative process, preventing aggregation of the unfolded substrates, promoting their unfolding, or helping to maintain them in a conformation that can be readily digested by cellular proteases.The major goal of the present study was to clarify the role of the molecular chaperones, GroEL and GroES, in protein breakdown. The unique structure of the fusion protein CRAG provides many experimental advantages for such studies. This rapidly degraded protein contains at its N terminus an unfolded 12-amino acid domain of the Cro repressor, followed by an IgG-binding domain of protein A, and 14 amino acids derived from β-galactosidase at its C terminus (10Hellebust H. Uhlen M. Enfors S.O. J. Bacteriol. 1990; 172: 5030-5034Crossref PubMed Google Scholar). Because of the protein A domain, CRAG can be easily isolated from cell extracts together with associated proteins by affinity chromatography on an IgG-Sepharose column. By this approach, GroEL, TF, and CRAG were shown to form in vivo ternary complexes, containing one TF molecule, one GroEL dodecamer, and one CRAG molecule (or CRAG fragment) (11Kandror O. Sherman M. Moerschell R. Goldberg A.L. J. Biol. Chem. 1997; 272: 1730-1734Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The formation of these complexes appears to be an initial, rate-limiting step in CRAG degradation, and increased expression of GroEL/GroES promotes complex formation and CRAG degradation (8Kandror O. Busconi L. Sherman M. Goldberg A.L. J. Biol. Chem. 1994; 269: 23575-23582Abstract Full Text PDF PubMed Google Scholar, 9Kandror O. Sherman M. Rhode M. Goldberg A.L. EMBO J. 1995; 14: 6021-6027Crossref PubMed Scopus (90) Google Scholar). Even in cells not expressing CRAG, some TF forms complexes with GroEL (11Kandror O. Sherman M. Moerschell R. Goldberg A.L. J. Biol. Chem. 1997; 272: 1730-1734Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), and these GroEL-TF complexes show a higher affinity for CRAG and for various other unfolded proteins than does GroEL alone (11Kandror O. Sherman M. Moerschell R. Goldberg A.L. J. Biol. Chem. 1997; 272: 1730-1734Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). This capacity of TF to enhance that association of GroEL with CRAG can account for the finding that increased expression of TF also promotes CRAG degradation (9Kandror O. Sherman M. Rhode M. Goldberg A.L. EMBO J. 1995; 14: 6021-6027Crossref PubMed Scopus (90) Google Scholar).To elucidate the degradative pathway and the precise roles of GroEL and GroES in CRAG degradation, we have studied the fate of CRAG molecules after binding to GroEL. We show here that the digestion of CRAG occurs not by a single highly processive mechanism but through the formation of discrete short-lived polypeptide intermediates. Moreover, this process appears to involve multiple cycles of binding of these polypeptides to GroEL followed by GroES-mediated dissociation from GroEL, which seems to allow proteolytic digestion by ClpP. Degradation of CRAG, like that of most proteins, requires ATP, but the biochemical basis for this energy requirement is unclear (8Kandror O. Busconi L. Sherman M. Goldberg A.L. J. Biol. Chem. 1994; 269: 23575-23582Abstract Full Text PDF PubMed Google Scholar), because none of the ATPases that are known to associate with ClpP (ClpA, ClpB, or ClpX) are essential for this degradative process (8Kandror O. Busconi L. Sherman M. Goldberg A.L. J. Biol. Chem. 1994; 269: 23575-23582Abstract Full Text PDF PubMed Google Scholar). Data presented in this paper suggest that the energy required for GroEL/GroES function might account for the ATP utilized in the degradation of CRAG.DISCUSSIONSeveral mechanisms have been proposed to account for the requirement for molecular chaperones in protein degradation; for example, the chaperones have been proposed to maintain substrates in a soluble nonaggregated form (13Gragerov A. Nudler E. Komissarova N. Gaitanaris G.A. Gottesman M.E. Nikiforov V. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10341-10344Crossref PubMed Scopus (184) Google Scholar, 14Gottesman S. Wickner S. Maurizi M.R. Genes Dev. 1997; 11: 815-823Crossref PubMed Scopus (468) Google Scholar) or to facilitate their initial recognition in prokaryotes by cellular proteases (7Sherman M. Goldberg A.L. EMBO J. 1992; 11: 71-77Crossref PubMed Scopus (173) Google Scholar) or in eukaryotes by ubiquitination enzymes (15Lee D.H. Sherman M. Goldberg A.L. Mol. Cell. Biol. 1996; 16: 4773-4781Crossref PubMed Scopus (125) Google Scholar, 16Yaglom J.A. Goldberg A.L. Finley D. Sherman M.Y. Mol. Cell. Biol. 1996; 16: 3679-3684Crossref PubMed Scopus (60) Google Scholar). These models can not apply to CRAG and its fragments, which are all soluble, monomeric proteins. The results presented here demonstrate that GroEL and GroES play a quite different role in this nonprocessive pathway, in which the chaperones and the proteases carry out complementary reactions at multiple steps during the degradation of CRAG, as summarized in Fig. 7. The pathway proposed in Fig. 7 is strongly supported by several observations: 1) GroEL, especially when in complexes with TF, binds strongly to CRAG (9Kandror O. Sherman M. Rhode M. Goldberg A.L. EMBO J. 1995; 14: 6021-6027Crossref PubMed Scopus (90) Google Scholar); 2) GroEL is found in vivo in complexes with both full size CRAG and the smaller fragments, which were shown to be intermediates in CRAG degradation; and 3) both the formation and further breakdown of these intermediates requires binding to GroEL and GroES-dependent dissociation of CRAG from GroEL into the cytosol, indicating that the chaperonin must function repeatedly in the course of degradation.Because we were unable to demonstrate any GroES in the GroEL-CRAG complexes (8Kandror O. Busconi L. Sherman M. Goldberg A.L. J. Biol. Chem. 1994; 269: 23575-23582Abstract Full Text PDF PubMed Google Scholar, 11Kandror O. Sherman M. Moerschell R. Goldberg A.L. J. Biol. Chem. 1997; 272: 1730-1734Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), it is most likely that GroES does not bind to the substrate together with GroEL but joins the complex only transiently to catalyze substrate dissociation. The protein A domain of the CRAG molecule must protrude from the GroEL cavity, because CRAG in complex GroEL was still able to associate with IgG on the affinity column (9Kandror O. Sherman M. Rhode M. Goldberg A.L. EMBO J. 1995; 14: 6021-6027Crossref PubMed Scopus (90) Google Scholar). Because GroEL can be partially eluted from these complexes by addition of GroES and ATP (8Kandror O. Busconi L. Sherman M. Goldberg A.L. J. Biol. Chem. 1994; 269: 23575-23582Abstract Full Text PDF PubMed Google Scholar), GroES probably binds to the distal GroEL ring, causing GroEL dissociation from CRAG.This model for the role of GroEL in protein degradation (Fig. 7) resembles and is consistent with the type of reiterative mechanism proposed earlier for GroEL-assisted protein folding (12Weissman J.S. Kashi Y. Fenton W.A. Horwich A.L. Cell. 1994; 78: 693-702Abstract Full Text PDF PubMed Scopus (328) Google Scholar, 17Xu Z. Horwich A.L. Sigler P.B. Nature. 1997; 388: 741-750Crossref PubMed Scopus (1031) Google Scholar, 18Lorimer G. Nature. 1997; 388: 720-723Crossref PubMed Scopus (79) Google Scholar). Accordingly, the initial binding of unfolded proteins by GroEL depends on interactions between the hydrophobic inner surface of GroEL and exposed hydrophobic domains on the substrate (19Viitanen P.V. Gatenby A.A. Lorimer G.H. Protein Sci. 1992; 1: 363-369Crossref PubMed Scopus (191) Google Scholar). The subsequent binding of ATP and GroES to the cis GroEL ring creates an enlarged cavity in which the substrate can fold in an isolated environment (20Weissman J.S. Hohl C.M. Kovalenko O. Kashi Y. Chen S. Braig K. Saibil H.R. Fenton W.A. Horwich A.L. Cell. 1995; 83: 577-587Abstract Full Text PDF PubMed Scopus (387) Google Scholar, 21Todd M.J. Viitanen P.V. Lorimer G.H. Science. 1994; 265: 659-666Crossref PubMed Scopus (424) Google Scholar, 22Sparrer H. Rutkat K. Buchner J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1096-1100Crossref PubMed Scopus (72) Google Scholar). When ATP binds to the trans GroEL ring, the GroES cap dissociates from the GroEL ring (23Rye H.S. Burston S.G. Fenton W.A. Beechem J.M. Xu Z. Sigler P.B. Horwich A.L. Nature. 1997; 388: 792-798Crossref PubMed Scopus (351) Google Scholar), and the protein is ejected from the cavity, whether or not it has folded successfully (12Weissman J.S. Kashi Y. Fenton W.A. Horwich A.L. Cell. 1994; 78: 693-702Abstract Full Text PDF PubMed Scopus (328) Google Scholar).Apparently, some substrates are able to refold within the central cavity of GroEL; however, other polypeptides, especially mutant or damaged proteins, such as CRAG, are unable to reach a stable, native conformation, by the time of GroES-induced release (12Weissman J.S. Kashi Y. Fenton W.A. Horwich A.L. Cell. 1994; 78: 693-702Abstract Full Text PDF PubMed Scopus (328) Google Scholar, 24Martin J. Langer T. Boteva R. Schramel A. Horwich A.L. Hartl F.U. Nature. 1991; 352: 36-42Crossref PubMed Scopus (718) Google Scholar, 25Mendoza J.A. Rogers E. Lorimer G.H. Horowitz P.M. J. Biol. Chem. 1991; 266: 13044-13049Abstract Full Text PDF PubMed Google Scholar). Such polypeptides appear to dissociate from the chaperonin in an unfolded form that allows refolding or in the case of CRAG, degradation. The kinetic partitioning between these alternative fates must depend on the structural properties of the polypeptide and its fragments, which determine whether they fold, are susceptible to digestion, or rebind to GroEL for another folding attempt. Because CRAG, like many abnormal proteins (and presumably a fraction of normal gene products), never achieves a properly folded structure, it is temporarily unfolded by GroEL and released in a form that can be hydrolyzed by cytosolic proteases, primarily by ClpP, or at a slower rate by protease La.After release from GroEL, CRAG is cleaved by the ClpP (or La) protease in its C-terminal region, because all the fragments isolated from the cell contain the normal N-terminal sequence. To be degraded by ClpP, a polypeptide must enter within the degradative chamber of ClpP, formed by its two rings. Presumably, the role of GroEL/GroES is to release the CRAG molecule in a conformation capable of entering into the ClpP complex. It seems likely that GroEL unfolds CRAG, including its tight protein A domains, which otherwise probably could not be digested by the ClpP protease. GroES then causes release of CRAG or its fragments in a conformation in which the C-terminal end is susceptible to proteolysis by ClpP (at least temporarily).Presumably, the degradative intermediates of CRAG, e.g. the 26,000 and 18,000 fragments, are relatively stable and can be isolated because their structure retards further digestion, unless they rebind to GroEL and undergo another round of ATP-GroES-mediated unfolding. This mechanism implies that there are probably two competing processes, in which digestion by ClpP competes with the tendency of CRAG (and its fragments) to quickly reacquire a tight globular conformation that prevents further digestion. Thus, these intermediates probably exist free in the cell until they are recaptured by GroEL and released in a more unfolded, more readily digested conformation. In this way, smaller and smaller N-terminal fragments are generated by ClpP (Fig. 7). The final steps in the degradative pathway are uncertain, because the present approach could not isolate smaller intermediates that have lost the protein A domain.The finding that CRAG degradation proceeds through the formation of relatively stable polypeptide intermediates that can be isolated from the cell was surprising. The major ATP-dependent proteases in E. coli, Lon, ClpAP, and HslUV (26Rohrwild M. Coux O. Huang H.-C. Moerschell R.P. Yoo S.J. Seol J.H. Chung C.H. Goldberg A.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5808-5813Crossref PubMed Scopus (212) Google Scholar) degrade model proteins in a highly processive manner without releasing partially digested proteins (27Edmunds T. Goldberg A.L. J. Cell. Biochem. 1986; 32: 187-191Crossref PubMed Scopus (16) Google Scholar, 28Thompson M.W. Singh S.K. Maurizi M.R. J. Biol. Chem. 1994; 269: 18209-18215Abstract Full Text PDF PubMed Google Scholar, 29Huang H.C. Mechanism of ATP-dependent Proteases and Role of Molecular Chaperones in Protein Degradation in Escherichia coli.Ph.D. thesis. Harvard University, Cambridge, Massachusetts1997Google Scholar). In eukaryotic cells and archaea, proteasomes also function in a highly processive way (30Akopian T.N. Kisselev A.F. Goldberg A.L. J. Biol. Chem. 1997; 272: 1791-1798Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 31Kisselev A.F. Akopian T.N. Woo K.M. Goldberg A.L. J. Biol. Chem. 1999; 274: 3363-3371Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar). This processive behavior must be advantageous for the cell because it prevents the appearance in the cytosol of partially digested fragments, which could interfere with normal metabolic regulation and protein-protein interactions. However, with many partially folded proteins, there may be internal features (e.g. tight globular domains in CRAG) that prevent rapid proteolysis and lead to substrate dissociation from the protease complex. Further degradation of the released fragments would thus require this unfolding by chaperones. Accordingly, when GroEL or GroES were inactivated, CRAG degradation was completely blocked (Fig. 3).Protein degradation by ClpP (8Kandror O. Busconi L. Sherman M. Goldberg A.L. J. Biol. Chem. 1994; 269: 23575-23582Abstract Full Text PDF PubMed Google Scholar) normally requires the function of an associated ATPase subunit (32Katayama-Fujimura Y. Gottesman S. Maurizi M.R. J. Biol. Chem. 1987; 262: 4477-4485Abstract Full Text PDF PubMed Google Scholar, 33Hwang B.J. Woo K.M. Goldberg A.L. Chung C.H. J. Biol. Chem. 1988; 263: 8727-8734Abstract Full Text PDF PubMed Google Scholar). Two ATPases, ClpA (32Katayama-Fujimura Y. Gottesman S. Maurizi M.R. J. Biol. Chem. 1987; 262: 4477-4485Abstract Full Text PDF PubMed Google Scholar, 33Hwang B.J. Woo K.M. Goldberg A.L. Chung C.H. J. Biol. Chem. 1988; 263: 8727-8734Abstract Full Text PDF PubMed Google Scholar) and ClpX (34Wojtkowiak D. Georgopoulos C. Zylicz M. J. Biol. Chem. 1993; 268: 22609-22617Abstract Full Text PDF PubMed Google Scholar, 35Gottesman S. Clark W.P. de Crecy-Lagard V. Maurizi M.R. J. Biol. Chem. 1993; 268: 22618-22626Abstract Full Text PDF PubMed Google Scholar), can function in the ClpP-dependent degradation of different proteins and confer substrate specificity on this process (34Wojtkowiak D. Georgopoulos C. Zylicz M. J. Biol. Chem. 1993; 268: 22609-22617Abstract Full Text PDF PubMed Google Scholar, 35Gottesman S. Clark W.P. de Crecy-Lagard V. Maurizi M.R. J. Biol. Chem. 1993; 268: 22618-22626Abstract Full Text PDF PubMed Google Scholar). Both ClpA and ClpX appear to possess some "chaperone-like activity," because by themselves they can promote the disassembly of specific protein complexes in vitro (36Wickner S. Gottesman S. Skowyra D. Hoskins J. McKenney K. Maurizi M.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12218-12222Crossref PubMed Scopus (323) Google Scholar, 37Wawrzynow A. Wojtkowiak D. Marszalek J. Banecki B. Jonsen M. Graves B. Georgopoulos C. Zylicz M. EMBO J. 1995; 14: 1867-1877Crossref PubMed Scopus (209) Google Scholar). Surprisingly, although CRAG degradation by ClpP is strictly ATP-dependent (8Kandror O. Busconi L. Sherman M. Goldberg A.L. J. Biol. Chem. 1994; 269: 23575-23582Abstract Full Text PDF PubMed Google Scholar), CRAG degradation is not reduced in the triple clpABXmutant strain (Fig. 4). Thus, ClpP functions in this process without any of these ATPases, and the energy requirement for CRAG breakdown appears, by exclusion, to be due to the involvement of GroEL/ES. Because ClpP by itself has been shown to degrade only oligopeptides, it seems likely that molecular chaperones GroEL/GroES substitute for the ATPases in CRAG degradation by unfolding the CRAG molecule or its fragments and presenting them in a form susceptible to the ClpP protease. Because ClpP was never found in these CRAG-GroEL complexes, the unfolding must be a distinct GroEL-mediated event taking place prior to the substrate's entry into the degradative chamber.It seems quite unlikely that CRAG degradation is a special mechanism. On the contrary, GroEL and GroES are likely to play a similar role in degradation of other proteins. Also, whereas GroEL and GroES are absolutely required for this process, some CRAG breakdown occurs, albeit 4–5-fold more slowly, in the clpP strain. This residual proteolysis, although catalyzed by the ATP-dependent protease La, still requires involvement of GroEL/ES and is not processive. Other cytosolic protease complexes (e.g. HslUV and 20S and 26S proteasomes) also have ring-like structures that require polypeptide unfolding for entry into their central degradative chambers. Therefore, it seems likely that the chaperones may function, as they seem to in CRAG degradation, to facilitate the hydrolysis of polypeptides or fragments that otherwise resist digestion by such proteases. In addition to their roles in protein folding and translocation (1Hartl F.U. Semin. Immun. 1991; 3: 5-16PubMed Google Scholar, 2Hartl F.U. Nature. 1996; 381: 571-580Crossref PubMed Scopus (3090) Google Scholar, 3Gething M.J. Sambrook J. Nature. 1992; 355: 33-45Crossref PubMed Scopus (3575) Google Scholar, 4Welch W.J. Curr. Opin. Cell Biol. 1991; 3: 1033-1038Crossref PubMed Scopus (121) Google Scholar), molecular chaperones are also necessary for the selective degradation of certain proteins with highly abnormal conformations. This role for the molecular chaperones is best documented inEscherichia coli but has also been demonstrated in yeast, animal cells, and mitochondria (5Sherman M. Goldberg A.L. Feige U. Morimoto R.I. Yahara I. Bolla B. Stress-inducible Cellular Responses. Birkhäuser Verlag, Basel, Switzerland1996: 57-78Crossref Google Scholar, 6Hayes S.A. Dice J.F. J. Cell Biol. 1996; 132: 255-258Crossref PubMed Scopus (163) Google Scholar). Moreover, the breakdown of different abnormal polypeptides appears to require different chaperones and distinct ATP-dependent proteases (7Sherman M. Goldberg A.L. EMBO J. 1992; 11: 71-77Crossref PubMed Scopus (173) Google Scholar, 8Kandror O. Busconi L. Sherman M. Goldberg A.L. J. Biol. Chem. 1994; 269: 23575-23582Abstract Full Text PDF PubMed Google Scholar). For example, if alkaline phosphatase fails to be secreted into the periplasm, it does not fold correctly and is rapidly degraded by protease La (lon) in a process requiring DnaK (the Hsp70 homolog in bacteria), and its cofactors, DnaJ and GrpE (7Sherman M. Goldberg A.L. EMBO J. 1992; 11: 71-77Crossref PubMed Scopus (173) Google Scholar). By contrast, the rapid breakdown of the recombinant fusion protein, CRAG, requires the ClpP protease as well as GroEL, GroES, and trigger factor (TF)1 (8Kandror O. Busconi L. Sherman M. Goldberg A.L. J. Biol. Chem. 1994; 269: 23575-23582Abstract Full Text PDF PubMed Google Scholar, 9Kandror O. Sherman M. Rhode M. Goldberg A.L. EMBO J. 1995; 14: 6021-6027Crossref PubMed Scopus (90) Google Scholar). The precise roles of chaperones in these degradative processes are still uncertain. One critical property of the molecular chaperones is that they selectively bind to unfolded proteins, and it has been suggested that association of an unfolded polypeptide with the chaperone may serve to promote substrate recognition by the cell's ATP-dependent proteases (5Sherman M. Goldberg A.L. Feige U. Morimoto R.I. Yahara I. Bolla B. Stress-inducible Cellular Responses. Birkhäuser Verlag, Basel, Switzerland1996: 57-78Crossref Google Scholar, 6Hayes S.A. Dice J.F. J. Cell Biol. 1996; 132: 255-258Crossref PubMed Scopus (163) Google Scholar). Alternatively, the chaperones may function together with proteases during the degradative process, preventing aggregation of the unfolded substrates, promoting their unfolding, or helping to maintain them in a conformation that can be readily digested by cellular proteases. The major goal of the present study was to clarify the role of the molecular chaperones, GroEL and GroES, in protein breakdown. The unique structure of the fusion protein CRAG provides many experimental advantages for such studies. This rapidly degraded protein contains at its N terminus an unfolded 12-amino acid domain of the Cro repressor, followed by an IgG-binding domain of protein A, and 14 amino acids derived from β-galactosidase at its C terminus (10Hellebust H. Uhlen M. Enfors S.O. J. Bacteriol. 1990; 172: 5030-5034Crossref PubMed Google Scholar). Because of the protein A domain, CRAG can be easily isolated from cell extracts together with associated proteins by affinity chromatography on an IgG-Sepharose column. By this approach, GroEL, TF, and CRAG were shown to form in vivo ternary complexes, containing one TF molecule, one GroEL dodecamer, and one CRAG molecule (or CRAG fragment) (11Kandror O. Sherman M. Moerschell R. Goldberg A.L. J. Biol. Chem. 1997; 272: 1730-1734Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The formation of these complexes appears to be an initial, rate-limiting step in CRAG degradation, and increased expression of GroEL/GroES promotes complex formation and CRAG degradation (8Kandror O. Busconi L. Sherman M. Goldberg A.L. J. Biol. Chem. 1994; 269: 23575-23582Abstract Full Text PDF PubMed Google Scholar, 9Kandror O. Sherman M. Rhode M. Goldberg A.L. EMBO J. 1995; 14: 6021-6027Crossref PubMed Scopus (90) Google Scholar). Even in cells not expressing CRAG, some TF forms complexes with GroEL (11Kandror O. Sherman M. Moerschell R. Goldberg A.L. J. Biol. Chem. 1997; 272: 1730-1734Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), and these GroEL-TF complexes show a higher affinity for CRAG and for various other unfolded proteins than does GroEL alone (11Kandror O. Sherman M. Moerschell R. Goldberg A.L. J. Biol. Chem. 1997; 272: 1730-1734Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). This capacity of TF to enhance that association of GroEL with CRAG can account for the finding that increased expression of TF also promotes CRAG degradation (9Kandror O. Sherman M. Rhode M. Goldberg A.L. EMBO J. 1995; 14: 6021-6027Crossref PubMed Scopus (90) Google Scholar). To elucidate the degradative pathway and the precise roles of GroEL and GroES in CRAG degradation, we have studied the fate of CRAG molecules after binding to GroEL. We show here that the digestion of CRAG occurs not by a single highly processive mechanism but through the formation of discrete short-lived polypeptide intermediates. Moreover, this process appears to involve multiple cycles of binding of these polypeptides to GroEL followed by GroES-mediated dissociation from GroEL, which seems to allow proteolytic digestion by ClpP. Degradation of CRAG, like that of most proteins, requires ATP, but the biochemical basis for this energy requirement is unclear (8Kandror O. Busconi L. Sherman M. Goldberg A.L. J. Biol. Chem. 1994; 269: 23575-23582Abstract Full Text PDF PubMed Google Scholar), because none of the ATPases that are known to associate with ClpP (ClpA, ClpB, or ClpX) are essential for this degradative process (8Kandror O. Busconi L. Sherman M. Goldberg A.L. J. Biol. Chem. 1994; 269: 23575-23582Abstract Full Text PDF PubMed Google Scholar). Data presented in this paper suggest that the energy required for GroEL/GroES function might account for the ATP utilized in the degradation of CRAG. DISCUSSIONSeveral mechanisms have been proposed to account for the requirement for molecular chaperones in protein degradation; for example, the chaperones have been proposed to maintain substrates in a soluble nonaggregated form (13Gragerov A. Nudler E. Komissarova N. Gaitanaris G.A. Gottesman M.E. Nikiforov V. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10341-10344Crossref PubMed Scopus (184) Google
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