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• W2089148676 abstract "In addition to promoting protein folding and translocation, molecular chaperones of Hsp70/DnaJ families are essential for the selective breakdown of many unfolded proteins. It has been proposed that chaperones function in degradation to maintain the substrates in a soluble form. In Escherichia coli, a nonsecreted alkaline phosphatase mutant that lacks its signal sequence (PhoAΔ2–22) fails to fold in the cytosol and is rapidly degraded at 37 °C. We show that PhoAΔ2–22 is degraded by two ATP-dependent proteases, La (Lon) and ClpAP, and breakdown by both is blocked in a dnaJ259-ts mutant at 37 °C. Both proteases could be immunoprecipitated with PhoA, but to a much lesser extent in the dnaJ mutant. Therefore, DnaJ appears to promote formation of protease-substrate complexes. DnaJ could be coimmunoprecipitated with PhoA, and the extent of this association directly correlated with its rate of degradation. Although PhoA was not degraded when DnaJ was inactivated, 50% or more of the PhoA remained soluble. PhoA breakdown and solubility did not require ClpB. PhoA degradation was reduced in a thioredoxin-reductase mutant (trxB), which allowed PhoAΔ2–22 to fold into an active form in the cytosol. Introduction of thednaJ mutation into trxB cells further stabilized PhoA, increased enzyme activity, and left PhoA completely soluble. Thus, DnaJ, although not necessary for folding (or preventing PhoA aggregation), is required for PhoA degradation and must play an active role in this process beyond maintaining the substrate in a soluble form. In addition to promoting protein folding and translocation, molecular chaperones of Hsp70/DnaJ families are essential for the selective breakdown of many unfolded proteins. It has been proposed that chaperones function in degradation to maintain the substrates in a soluble form. In Escherichia coli, a nonsecreted alkaline phosphatase mutant that lacks its signal sequence (PhoAΔ2–22) fails to fold in the cytosol and is rapidly degraded at 37 °C. We show that PhoAΔ2–22 is degraded by two ATP-dependent proteases, La (Lon) and ClpAP, and breakdown by both is blocked in a dnaJ259-ts mutant at 37 °C. Both proteases could be immunoprecipitated with PhoA, but to a much lesser extent in the dnaJ mutant. Therefore, DnaJ appears to promote formation of protease-substrate complexes. DnaJ could be coimmunoprecipitated with PhoA, and the extent of this association directly correlated with its rate of degradation. Although PhoA was not degraded when DnaJ was inactivated, 50% or more of the PhoA remained soluble. PhoA breakdown and solubility did not require ClpB. PhoA degradation was reduced in a thioredoxin-reductase mutant (trxB), which allowed PhoAΔ2–22 to fold into an active form in the cytosol. Introduction of thednaJ mutation into trxB cells further stabilized PhoA, increased enzyme activity, and left PhoA completely soluble. Thus, DnaJ, although not necessary for folding (or preventing PhoA aggregation), is required for PhoA degradation and must play an active role in this process beyond maintaining the substrate in a soluble form. isopropyl-1-thio-β-d-galactopyranoside polyacrylamide gel electrophoresis Trigger Factor All cells utilize energy-dependent proteolytic pathways to degrade selectively polypeptides with highly abnormal conformations (1Goldberg A.L. Eur. J. Biochem. 1992; 203: 9-23Crossref PubMed Scopus (415) Google Scholar, 2Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6793) Google Scholar). Such misfolded proteins may result from nonsense or missense mutations, mistakes in translation, genetic engineering (e.g. gene fusions), failure of a polypeptide subunit to associate with others, or intracellular denaturation (3Gottesman S. Maurizi M.R. Microbiol. Rev. 1992; 56: 592-621Crossref PubMed Google Scholar, 4Wickner S. Maurizi M.R. Gottesman S. Science. 1999; 286: 1888-1893Crossref PubMed Scopus (907) Google Scholar). Another way such abnormal proteins may appear in cells is through incorrect localization; for example, if a secretory protein is retained in the cytosol, where it cannot form the correct disulfide bonds due to the reducing environment (5Derman A.I. Beckwith J. J. Bacteriol. 1991; 173: 7719-7722Crossref PubMed Scopus (160) Google Scholar), the protein is rapidly degraded (6Sherman M. Goldberg A.L. EMBO J. 1992; 11: 71-77Crossref PubMed Scopus (173) Google Scholar). It remains unclear how the cell's proteolytic apparatus recognizes and destroys such abnormal polypeptides while sparing normal cell constituents.Molecular chaperones have been implicated in this degradative process in both bacterial and eukaryotic cells (7Sherman M.Y. Goldberg A.L. Exper. Suppl. (Basel). 1996; 77: 57-78PubMed Google Scholar, 8Hayes S.A. Dice J.F. J. Cell Biol. 1996; 132: 255-258Crossref PubMed Scopus (163) Google Scholar). In addition to participating in the folding of nascent polypeptides, their translocation across membranes, and the assembly of oligomeric complexes (9Gething M.J. Sambrook J. Nature. 1992; 355: 33-45Crossref PubMed Scopus (3575) Google Scholar, 10Hartl F.U. Nature. 1996; 381: 571-579Crossref PubMed Scopus (3090) Google Scholar), molecular chaperones of the Hsp70/DnaK family and the cofactors of the DnaJ families have recently been shown to play an essential role in protein degradation (6Sherman M. Goldberg A.L. EMBO J. 1992; 11: 71-77Crossref PubMed Scopus (173) Google Scholar, 11Wagner I. Arlt H. van Dyck L. Langer T. Neupert W. EMBO J. 1994; 13: 5135-5145Crossref PubMed Scopus (207) Google Scholar, 12Lee D.H. Sherman M.Y. Goldberg A.L. Mol. Cell. Biol. 1996; 16: 4773-4781Crossref PubMed Scopus (125) Google Scholar). A characteristic feature of Hsp70/DnaK is its ability to bind selectively to hydrophobic oligopeptides or unfolded proteins in extended conformations, where such domains may be exposed (13Flynn G.C. Pohl J. Flocco M.T. Rothman J.E. Nature. 1991; 353: 726-730Crossref PubMed Scopus (631) Google Scholar, 14Rudiger S. Buchberger A. Bukau B. Nat. Struct. Biol. 1997; 4: 342-349Crossref PubMed Scopus (294) Google Scholar, 15Fink A.L. Physiol. Rev. 1999; 79: 425-449Crossref PubMed Scopus (858) Google Scholar). DnaJ stimulates the ATPase activity of DnaK (16Laufen T. Mayer M.P. Beisel C. Klostermeier D. Mogk A. Reinstein J. Bukau B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5452-5457Crossref PubMed Scopus (464) Google Scholar, 17Russell R. Wali Karzai A. Mehl A.F. McMacken R. Biochemistry. 1999; 38: 4165-4176Crossref PubMed Scopus (90) Google Scholar) and by itself can also bind to certain unfolded proteins (18Hendrick J.P. Langer T. Davis T.A. Hartl F.U. Wiedmann M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10216-10220Crossref PubMed Scopus (145) Google Scholar, 19Greene M.K. Maskos K. Landry S.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6108-6113Crossref PubMed Scopus (245) Google Scholar, 20Langer T. Lu C. Echols H. Flanagan J. Hayer M.K. Hartl F.U. Nature. 1992; 356: 683-689Crossref PubMed Scopus (780) Google Scholar, 21Cheetham M.E. Caplan A.J. Cell Stress Chaperones. 1998; 3: 28-36Crossref PubMed Scopus (480) Google Scholar). Both components can thus possibly facilitate the recognition of substrate conformations or act as cofactors in the degradative process.Among the first examples of the involvement of DnaJ/DnaK in the degradation of a specific abnormal protein was alkaline phosphatase (PhoA61), which is not secreted from the cytosol due to a missense mutation in its signal sequence (6Sherman M. Goldberg A.L. EMBO J. 1992; 11: 71-77Crossref PubMed Scopus (173) Google Scholar). Its rapid degradation at 37 °C is mediated in part by the ATP-dependent protease La (Lon) and also requires DnaK, because a dnaK- null mutant completely prevents its breakdown. Interestingly, dnaK756, a missense mutation that fails to release substrates in response to ATP, was found to enhance PhoA61 degradation, whereas a dnaJ mutant, which reduced PhoA61 association with DnaK, slowed its turnover. Thus, complex formation between PhoA61 and DnaK appears essential for its rapid degradation. Presumably, the successful folding or secretion of a normal polypeptide involves only transient association with DnaK. We therefore proposed that the chaperone, through its prolonged association with a nonfoldable protein, may trigger proteolysis, perhaps by helping to stabilize an abnormal polypeptide in an unfolded, extended conformation that favors recognition or digestion by the protease.One characteristic feature of unfolded proteins is their tendency to aggregate, and an important function of molecular chaperones of the Hsp70 and DnaJ families is to prevent this process in vivo(15Fink A.L. Physiol. Rev. 1999; 79: 425-449Crossref PubMed Scopus (858) Google Scholar, 22Gragerov 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). In addition, DnaK, DnaJ, and GrpE, together with ClpB, can promote the solubilization of aggregated proteins (23Goloubinoff P. Mogk A. Zvi A.P. Tomoyasu T. Bukau B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13732-13737Crossref PubMed Scopus (499) Google Scholar, 24Motohashi K. Watanabe Y. Yohda M. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7184-7189Crossref PubMed Scopus (221) Google Scholar) inEscherichia coli, as can their homologs in yeast. It has been suggested that the primary function of DnaJ/DnaK in proteolysis is to help maintain unfolded proteins in a soluble form, which should render them more susceptible to proteolytic attack (25Gottesman S. Wickner S. Maurizi M.R. Genes Dev. 1997; 11: 815-823Crossref PubMed Scopus (468) Google Scholar). In several cases, undegraded abnormal proteins have been found to accumulate as intracellular aggregates. For example, in E. coli, the degradation of the short-lived transcription factor, RcsA, by protease La was retarded in a dnaJ mutant, where a significant fraction of RcsA was found in particulate form (26Jubete Y. Maurizi M.R. Gottesman S. J. Biol. Chem. 1996; 271: 30798-30803Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Similarly, in yeast mitochondria, the degradation by the ATP-dependent protease, Pim1 (a homolog of La), of two fusion proteins, bovine lactalbumin and cytochrome b 2-DHFR, requires the DnaK homolog mt-Hsp70 and the DnaJ homolog Mdj1 (11Wagner I. Arlt H. van Dyck L. Langer T. Neupert W. EMBO J. 1994; 13: 5135-5145Crossref PubMed Scopus (207) Google Scholar). In the absence of Mdj1, these fusion proteins formed insoluble aggregates. However, in these chaperone-deficient cells, there was no evidence that the aggregation of these substrates actually caused the block in proteolysis and was not a consequence of the reduced degradation. In other words, if the chaperones are required directly in the degradative process, when degradation is blocked in the chaperone mutants, the unfolded polypeptides that accumulate may then tend to aggregate. Thus, establishing direct involvement of DnaK/DnaJ (Hsp70/40) in the proteolytic pathway is difficult, because the association of these chaperones with the substrate may be important both for facilitating proteolytic attack and for promoting the proper folding of the substrate.To distinguish these possibilities and to clarify the role of the chaperones in the breakdown of unfolded proteins, we have studied the cytosolic degradation of a nonsecreted alkaline phosphatase lacking the entire signal sequence (PhoAΔ2–22) (27Derman A.I. Puziss J.W. Bassford Jr., P.J. Beckwith J. EMBO J. 1993; 12: 879-888Crossref PubMed Scopus (175) Google Scholar). The present studies have focused on the role(s) of DnaJ, because this chaperone has been suggested to bind directly to certain denatured proteins and favor DnaK binding to the substrate (20Langer T. Lu C. Echols H. Flanagan J. Hayer M.K. Hartl F.U. Nature. 1992; 356: 683-689Crossref PubMed Scopus (780) Google Scholar) and also to play a crucial role in preventing aggregation of abnormal proteins (28Schroder H. Langer T. Hartl F.U. Bukau B. EMBO J. 1993; 12: 4137-4144Crossref PubMed Scopus (497) Google Scholar). In the reducing environment of E. coli cytosol, the normally periplasmic PhoA cannot form the disulfide bonds necessary for the protein to acquire an active conformation (5Derman A.I. Beckwith J. J. Bacteriol. 1991; 173: 7719-7722Crossref PubMed Scopus (160) Google Scholar). However, in a mutant strain lacking thioredoxin reductase (trxB), the cytosol has less reducing potential, and some PhoA(Δ2–22) has been shown to fold into an active enzyme (29Derman A.I. Prinz W.A. Belin D. Beckwith J. Science. 1993; 262: 1744-1747Crossref PubMed Scopus (370) Google Scholar). We have therefore also used the trxB strain to test whether the rapid degradation of PhoA is in fact due to its failure to achieve the native conformation and to determine what role DnaJ might play when folding and degradation of this protein can both occur.DISCUSSIONThe present studies indicate that the molecular chaperone, DnaJ, plays multiple roles in determining the fate in the cytosol of a nonsecreted alkaline phosphatase molecule, PhoA(Δ2–22). Normally, when a signal peptide is present, DnaJ, together with DnaK and GrpE, promotes translocation of this enzyme into the periplasm (36Wild J. Altman E. Yura T. Gross C.A. Genes Dev. 1992; 6: 1165-1172Crossref PubMed Scopus (167) Google Scholar, 37Wild J. Rossmeissl P. Walter W.A. Gross C.A. J. Bacteriol. 1996; 178: 3608-3613Crossref PubMed Scopus (92) Google Scholar). When export is impossible, DnaJ, apparently with DnaK and GrpE (6Sherman M. Goldberg A.L. EMBO J. 1992; 11: 71-77Crossref PubMed Scopus (173) Google Scholar), carries out some function essential for rapid degradation of this unfolded protein by the ATP-dependent proteases La (Lon) and ClpAP. Although DnaJ was found to help maintain this protein in a soluble form (for example, in the lon-ClpP mutant cell, where degradation is not possible), DnaJ clearly must play additional role(s) in PhoA degradation beyond preventing the substrate from forming insoluble aggregates. When DnaJ was inactivated, at least half of the PhoA molecules remained soluble at 100,000 × g, yet they were not degraded. Furthermore, when a favorable redox environment was provided (i.e. in the trxB mutant cells), in which some PhoA can fold into its native form, DnaJ was still required for its degradation, but was not necessary for the prevention of aggregation or for the proper folding of the active enzyme.Previous findings in E. coli (6Sherman M. Goldberg A.L. EMBO J. 1992; 11: 71-77Crossref PubMed Scopus (173) Google Scholar, 38Straus D.B. Walter W.A. Gross C.A. Genes Dev. 1988; 2: 1851-1858Crossref PubMed Scopus (136) Google Scholar) and yeast mitochondria (11Wagner I. Arlt H. van Dyck L. Langer T. Neupert W. EMBO J. 1994; 13: 5135-5145Crossref PubMed Scopus (207) Google Scholar) have implicated the DnaK/DnaJ/GrpE chaperones in the rapid breakdown of certain abnormal proteins by ATP-dependent proteases. Both the DnaK and DnaJ families of chaperones are known to bind selectively to unfolded proteins (9Gething M.J. Sambrook J. Nature. 1992; 355: 33-45Crossref PubMed Scopus (3575) Google Scholar, 15Fink A.L. Physiol. Rev. 1999; 79: 425-449Crossref PubMed Scopus (858) Google Scholar, 16Laufen T. Mayer M.P. Beisel C. Klostermeier D. Mogk A. Reinstein J. Bukau B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5452-5457Crossref PubMed Scopus (464) Google Scholar, 17Russell R. Wali Karzai A. Mehl A.F. McMacken R. Biochemistry. 1999; 38: 4165-4176Crossref PubMed Scopus (90) Google Scholar), and in this way, they may promote the degradative process by facilitating the recognition of unfolded proteins as substrates by the proteases. Alternatively, by maintaining the substrates in an unfolded conformation, these chaperones may function as cofactors that facilitate digestion by the proteases. Studies with another nonsecreted variant, PhoA61, which contains a point mutation in the signal sequence, had suggested a kinetic partitioning model for chaperone function, in which a prolonged association of the substrate with DnaK would favor proteolytic attack and help distinguish appropriate substrates for degradation from normal cell constituents (7Sherman M.Y. Goldberg A.L. Exper. Suppl. (Basel). 1996; 77: 57-78PubMed Google Scholar). Successful folding of a protein, as occurs normally for most proteins, would involve only a transient association with the chaperones, whereas an extended association, as may occur with a highly abnormal mutant protein or an irreversibly damaged polypeptide, would favor its proteolytic digestion.The present finding that the association of PhoA with DnaJ correlates with its rapid degradation is consistent with this proposed mechanism. At 30 °C, where PhoA was relatively stable, or at 37 °C in thednaJ259 mutant, where PhoA degradation was blocked, much less soluble PhoA was associated with DnaJ than in the wild-type at 37 °C. By contrast, in a strain carrying a nondissociating DnaK mutation (dnaK756), where the amount of DnaJ complexed with PhoA was at least 5-fold higher than in the wild-type, PhoA degradation was enhanced at least 2-fold. In our prior studies, we had observed that the extent of DnaK association with PhoA61 (in dnaK756 and dnaK deletion strains) also correlated with the rate of its hydrolysis (6Sherman M. Goldberg A.L. EMBO J. 1992; 11: 71-77Crossref PubMed Scopus (173) Google Scholar). (Those earlier studies, in contrast with the present ones, failed to demonstrate an association of PhoA with DnaJ because of the use of the detergent Triton X-100 in the buffers.) Thus, these findings together strongly suggest that prolonged association of this unfolded protein with DnaJ and DnaK facilitates its hydrolysis. However, these coimmunoprecipitation data only demonstrate correlations with the amount of PhoA present in complexes with the chaperones; the data do not distinguish whether rapid degradation may in fact be due to the presence of more molecules of the chaperone in complexes with the substrate or to a more prolonged or a tighter association of the substrate with the same number of chaperone molecules.Although not directly demonstrated in these experiments, it seems very likely that DnaJ and DnaK are present together in the same complexes with PhoA, as was found for certain other denatured proteins (16Laufen T. Mayer M.P. Beisel C. Klostermeier D. Mogk A. Reinstein J. Bukau B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5452-5457Crossref PubMed Scopus (464) Google Scholar, 17Russell R. Wali Karzai A. Mehl A.F. McMacken R. Biochemistry. 1999; 38: 4165-4176Crossref PubMed Scopus (90) Google Scholar,20Langer T. Lu C. Echols H. Flanagan J. Hayer M.K. Hartl F.U. Nature. 1992; 356: 683-689Crossref PubMed Scopus (780) Google Scholar, 21Cheetham M.E. Caplan A.J. Cell Stress Chaperones. 1998; 3: 28-36Crossref PubMed Scopus (480) Google Scholar). For example, in the dnaK756 mutant, which is defective in ATP-dependent dissociation from PhoA, more PhoA was associated with DnaJ than in the wild-type. Also, thednaJ259 mutation, which is located in the highly conserved “J” domain that interacts with DnaK and thus causes a defective association with DnaK (39Wall D. Zylicz M. Georgopoulos C. J. Biol. Chem. 1995; 270: 2139-2144Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) showed a dramatically reduced association with PhoA. This in vivo failure of dnaJ259 to bind to PhoA is noteworthy, because no deficiency had been observed previously in its binding to model substrates in vitro. It is not clear if DnaJ and DnaK influence degradation rates independently or if one chaperone acts by influencing binding of the other as suggested by the results with the dnaK756 mutant. Such mechanistic questions will be best analyzed with pure componentsin vitro.The present results, although indicating a requirement of DnaJ for proteolysis, also support the view that one function of this chaperone in cells is to prevent PhoA aggregation into insoluble inclusions, because 30–50% of the newly synthesized PhoA accumulated in rapidly sedimenting fractions in the dnaJ mutant. A role for DnaJ in maintaining denatured proteins in a soluble form has been established in several studies (28Schroder H. Langer T. Hartl F.U. Bukau B. EMBO J. 1993; 12: 4137-4144Crossref PubMed Scopus (497) Google Scholar, 40Prip-Buus C. Westerman B. Schmitt M. Langer T. Neupert W. Schwarz E. FEBS Lett. 1996; 380: 142-146Crossref PubMed Scopus (44) Google Scholar), especially in concert with DnaK (23Goloubinoff P. Mogk A. Zvi A.P. Tomoyasu T. Bukau B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13732-13737Crossref PubMed Scopus (499) Google Scholar, 24Motohashi K. Watanabe Y. Yohda M. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7184-7189Crossref PubMed Scopus (221) Google Scholar) and also ClpB (23Goloubinoff P. Mogk A. Zvi A.P. Tomoyasu T. Bukau B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13732-13737Crossref PubMed Scopus (499) Google Scholar). In mitochondria, the rapid breakdown of two fusion proteins by the Pim1 protease (a homolog of protease La) required the mitochondrial DnaJ homolog Mdj-1, and in its absence, some of these proteins accumulated as insoluble aggregates (11Wagner I. Arlt H. van Dyck L. Langer T. Neupert W. EMBO J. 1994; 13: 5135-5145Crossref PubMed Scopus (207) Google Scholar). Similarly, inE. coli, the rapidly degraded transcriptional activator RcsA was stabilized in a dnaJ mutant, and a large fraction of the RcsA was found in insoluble aggregates (26Jubete Y. Maurizi M.R. Gottesman S. J. Biol. Chem. 1996; 271: 30798-30803Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). It was therefore proposed that the primary role of DnaJ, and by extension DnaK and GrpE, in protein breakdown is to maintain the substrate soluble and therefore susceptible to the protease (25Gottesman S. Wickner S. Maurizi M.R. Genes Dev. 1997; 11: 815-823Crossref PubMed Scopus (468) Google Scholar). However, there is no evidence that maintenance of these molecules in a soluble form is actually essential for proteolysis. Although it is likely that proteolytic attack on aggregated substrates may be slower and less efficient than degradation of soluble proteins (at comparable concentrations), the automatic assumption (25Gottesman S. Wickner S. Maurizi M.R. Genes Dev. 1997; 11: 815-823Crossref PubMed Scopus (468) Google Scholar) that polypeptides in particulate fractions cannot be digested by soluble proteases seems unwarranted. Indeed, it has long been known that denatured proteins, once in large inclusion bodies, can still be rapidly hydrolyzed to amino acids (41Prouty W.F. Karnovsky M.J. Goldberg A.L. J. Biol. Chem. 1975; 250: 1112-1122Abstract Full Text PDF PubMed Google Scholar, 42Prouty W.F. Goldberg A.L. Nat. New Biol. 1972; 240: 147-150Crossref PubMed Scopus (44) Google Scholar) and that protease La can digest insoluble, membrane-associated proteins (43Goff S.A. Goldberg A.L. J. Biol. Chem. 1987; 262: 4508-4515Abstract Full Text PDF PubMed Google Scholar).Rapid digestion of such aggregated species may also require the involvement of molecular chaperones to promote substrate solubilization, and for this reason, we investigated the possible involvement in PhoA breakdown of ClpB, which can function with DnaK/DnaJ/GrpE to solubilize aggregated proteins (23Goloubinoff P. Mogk A. Zvi A.P. Tomoyasu T. Bukau B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13732-13737Crossref PubMed Scopus (499) Google Scholar). ClpB, unlike DnaK and DnaJ, was not necessary for proteolysis. However, theclpB strain did consistently show a small reduction in PhoA breakdown, perhaps because a minor portion of the substrate was particulate and may require this chaperone for solubilization prior to DnaJ-dependent degradation.Although earlier studies (26Jubete Y. Maurizi M.R. Gottesman S. J. Biol. Chem. 1996; 271: 30798-30803Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), like the present ones, have demonstrated a tendency of the stabilized abnormal proteins to aggregate, they did not show (but assumed) that aggregation is the cause of the stabilization. On the contrary, the present findings clearly indicate an important additional function of chaperones in proteolysis, because in the dnaJ mutant, degradation was abolished completely, although at least half of the nondegraded PhoA remained soluble at 100,000 × g. Perhaps the best evidence for a role of DnaJ in facilitating the degradative process came from thetrxB strains, where the dnaJ mutant completely blocked PhoA degradation, even though none of the substrate was aggregated.In these trxB mutants, where a large fraction of the PhoA was able to fold into the active conformation, the inactivation of DnaJ led to a 3-fold increase in PhoA enzymatic activity. This increase in activity was most likely due to the increase in the amount of soluble PhoA that resulted from blocking its degradation. In other words, by promoting PhoA degradation, DnaJ appears to suppress the folding process. Such a role for DnaJ is supported by the finding that overexpression of DnaK and DnaJ reduced the cytosolic accumulation of a cloned human polypeptide (SPARC), presumably by increasing its degradation, and it also reduced the yield of SPARC with correct disulfide bond formation in a trxB mutant (44Schneider E.L. Thomas J.G. Bassuk J.A. Sage E.H. Baneyx F. Nat. Bio/Technol. 1997; 15: 581-585Crossref PubMed Scopus (25) Google Scholar). Interestingly, DnaJ itself has been reported to possess disulfide oxidoreductase activity in vitro (45de Crouy-Chanel A. Kohiyama M. Richarme G. J. Biol. Chem. 1995; 270: 22669-22672Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), and it is conceivable that this activity may help maintain certain proteins, such as PhoA, in an unfolded conformation in vivo (e.g. in atrxB cell).These findings suggest that an essential function of DnaJ and DnaK in proteolysis is to facilitate the binding of substrates to the ATP-dependent proteases, ClpAP or La, or to stabilize these enzyme-substrate complexes once they are formed. The chaperones may promote the recognition of the partially unfolded protein by maintaining certain domains in a conformation that is particularly susceptible to association with the proteases. The critical finding here is that, when DnaJ was defective, very little La or ClpAP appeared to be associated with PhoA. These results indicate for the first time a requirement for DnaJ for the formation of degradative complexes between a substrate and the proteases that digest it. An analogous role of a DnaJ homolog in targeting a substrate to proteolysis has been proposed in yeast cytosol, based on the finding that binding of the DnaJ homolog Ydj1 was necessary for ubiquitin conjugation to a rapidly degraded protein (12Lee D.H. Sherman M.Y. Goldberg A.L. Mol. Cell. Biol. 1996; 16: 4773-4781Crossref PubMed Scopus (125) Google Scholar). An additional example of Ydj1 maintaining a substrate in a conformation that allows enzymatic attack is in the ubiquitination of the Cln3 cyclin. In this case, Ydj1 binding to Cln3 was required for Cln3 phosphorylation by p34Cdc28, which in turn triggers ubiquitin-dependent proteolysis (46Yaglom J.A. Goldberg A.L. Finley D. Sherman M.Y. Mol. Cell. Biol. 1996; 16: 3679-3684Crossref PubMed Scopus (60) Google Scholar). Other chaperone systems have also been implicated in “presenting” substrates for proteolysis or helping maintain them in conformations that allow proteolytic attack; for example, the rapid degradation of the fusion protein, CRAG, in E. coli requires a specific association between the substrate GroEL and Trigger Factor (TF) (31Kandror O. Busconi L. Sherman M. Goldberg A.L. J. Biol. Chem. 1994; 269: 23575-23582Abstract Full Text PDF PubMed Google Scholar, 47Kandror O. Sherman M. Rhode M. Goldberg A.L. EMBO J. 1995; 14: 6021-6027Crossref PubMed Scopus (90) Google Scholar, 48Kandror O. Sherman M.Y. Goldberg A.L. J. Biol. Chem. 1999; 274: 37743-37749Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar).It is very unlikely that the requirement for DnaJ in proteolysis is for the folding, assembly, or activation of these two ATP-dependent proteases. Inactivation of these enzymes seems unlikely in these experiments, because the cells were grown at 30 °C and switched to 37 °C for only 20 min, which was sufficient to prevent complex formation between the protease and the substrate. In addition, hydrolysis of specific fluorogenic peptide substrates by these proteases occurred at similar rates in extracts of the wild-type and dnaJ mutant strains, and complex formation between ClpA and ClpP occurred whether or not DnaJ was functional. Finally, Jubeteet al. (26Jubete Y. Maurizi M.R. Gottesman S. J. Biol. Chem. 1996; 271: 30798-30803Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) have noted that inactivation of DnaJ stabilized some protein substrates of protease La, but not others. Therefore, the chaperone must instead be necessary for substrate recognition or rapid digestion.An important feature of the degradation of PhoA shown here is that this process is catalyzed by two very different proteases, La and ClpAP. The two ATP-dependent proteases contribute about equallyin vivo, and each can degrade the substrate apparently without the involvement of the other. Degradation by both proteases is likely to occur in a highly processive fashion, because no intermediate fragments of PhoA were observed; also with each, relatively stable substrate-protease complexes could be captured by coimmunoprecipitation. Previous findings with PhoA61 (6Sherman M. Goldberg A.L. EMBO J. 1992; 11: 71-77Crossref PubMed Scopus (173) Google Scholar) and CRAG (31Kandror O. Busconi L. Sherman M. Goldberg A.L. J. Biol. Chem" @default.
• W2089148676 created "2016-06-24" @default.
• W2089148676 creator A5004934242 @default.
• W2089148676 creator A5006962945 @default.
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• W2089148676 date "2001-02-01" @default.
• W2089148676 modified "2023-09-29" @default.
• W2089148676 title "The Molecular Chaperone DnaJ Is Required for the Degradation of a Soluble Abnormal Protein in Escherichia coli" @default.
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