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• W2070291809 abstract "ClpA and ClpX function both as molecular chaperones and as the regulatory components of ClpAP and ClpXP proteases, respectively. ClpA and ClpX bind substrate proteins through specific recognition signals, catalyze ATP-dependent protein unfolding of the substrate, and when in complexes with ClpP translocate the unfolded polypeptide into the cavity of the ClpP peptidase for degradation. To examine the mechanism of interaction of ClpAP with dimeric substrates, single round binding and degradation experiments were performed, revealing that ClpAP degraded both subunits of a RepA homodimer in one cycle of binding. Furthermore, ClpAP was able to degrade both protomers of a RepA heterodimer in which only one subunit contained the ClpA recognition signal. In contrast, ClpXP degraded both subunits of a dimeric substrate only when both protomers contained a recognition signal. These data suggest that ClpAP and ClpXP may recognize and bind substrates in significantly different ways. ClpA and ClpX function both as molecular chaperones and as the regulatory components of ClpAP and ClpXP proteases, respectively. ClpA and ClpX bind substrate proteins through specific recognition signals, catalyze ATP-dependent protein unfolding of the substrate, and when in complexes with ClpP translocate the unfolded polypeptide into the cavity of the ClpP peptidase for degradation. To examine the mechanism of interaction of ClpAP with dimeric substrates, single round binding and degradation experiments were performed, revealing that ClpAP degraded both subunits of a RepA homodimer in one cycle of binding. Furthermore, ClpAP was able to degrade both protomers of a RepA heterodimer in which only one subunit contained the ClpA recognition signal. In contrast, ClpXP degraded both subunits of a dimeric substrate only when both protomers contained a recognition signal. These data suggest that ClpAP and ClpXP may recognize and bind substrates in significantly different ways. Clp/Hsp100 chaperones, members of the AAA+ ATPase superfamily (1Neuwald A.F. Aravind L. Spouge J.L. Koonin E.V. Genome Res. 1999; 9: 27-43Crossref PubMed Google Scholar), participate in protein unfolding and remodeling necessary for many cellular functions, including DNA replication, tolerance to heat stress, control of gene expression, and solubilization of aggregated protein (2Parsell D.A. Kowal A.S. Singer M.A. Lindquist S. Nature. 1994; 372: 475-478Crossref PubMed Scopus (726) Google Scholar, 3Wickner 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 (320) Google Scholar, 4Levchenko I. Luo L. Baker T.A. Genes Dev. 1995; 9: 2399-2408Crossref PubMed Scopus (245) Google Scholar, 5Wawrzynow A. Wojtkowiak D. Marszalek J. Banecki B. Jonsen M. Graves B. Georgopoulos C. Zylicz M. EMBO J. 1995; 14: 1867-1877Crossref PubMed Scopus (208) Google Scholar, 6Hoskins J.R. Sharma S. Sathyanarayana B.K. Wickner S. Adv. Protein Chem. 2001; 59: 413-429Crossref PubMed Scopus (35) Google Scholar, 7Sauer R.T. Bolon D.N. Burton B.M. Burton R.E. Flynn J.M. Grant R.A. Hersch G.L. Joshi S.A. Kenniston J.A. Levchenko I. Neher S.B. Oakes E.S. Siddiqui S.M. Wah D.A. Baker T.A. Cell. 2004; 119: 9-18Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). Some Clp/Hsp100 proteins associate with proteolytic components to form energy-dependent proteases (6Hoskins J.R. Sharma S. Sathyanarayana B.K. Wickner S. Adv. Protein Chem. 2001; 59: 413-429Crossref PubMed Scopus (35) Google Scholar, 7Sauer R.T. Bolon D.N. Burton B.M. Burton R.E. Flynn J.M. Grant R.A. Hersch G.L. Joshi S.A. Kenniston J.A. Levchenko I. Neher S.B. Oakes E.S. Siddiqui S.M. Wah D.A. Baker T.A. Cell. 2004; 119: 9-18Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, 8Wickner S. Maurizi M.R. Gottesman S. Science. 1999; 286: 1888-1893Crossref PubMed Scopus (907) Google Scholar, 9Horwich A.L. Weber-Ban E.U. Finley D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11033-11040Crossref PubMed Scopus (165) Google Scholar). In Escherichia coli, ClpA and ClpX ATPases associate with ClpP peptidase, a serine peptidase unrelated to Clp ATPases, to form the ClpAP and ClpXP proteases. Likewise, the HslUV protease consists of the HslU ATPase and the HslV peptidase. In the presence of ATP, Clp ATPases self-assemble into oligomeric rings. The individual subunits are markedly similar in structure to the subunits of classic AAA+ ATPases (10Sousa M.C. Trame C.B. Tsuruta H. Wilbanks S.M. Reddy V.S. McKay D.B. Cell. 2000; 103: 633-643Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 11Guo F. Maurizi M.R. Esser L. Xia D. J. Biol. Chem. 2002; 277: 46743-46752Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 12Lee S. Sowa M.E. Choi J.M. Tsai F.T. J. Struct. Biol. 2004; 146: 99-105Crossref PubMed Scopus (78) Google Scholar, 13Kim D.Y. Kim K.K. J. Biol. Chem. 2003; 278: 50664-50670Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). A Clp protease resembling the eukaryotic 26 S proteasome is formed when the oligomeric ATPase rings associate with one or both ends of a proteolytic component (14Kessel M. Maurizi M.R. Kim B. Kocsis E. Trus B.L. Singh S.K. Steven A.C. J. Mol. Biol. 1995; 250: 587-594Crossref PubMed Scopus (262) Google Scholar). Crystal structures of the proteolytic components ClpP and HslV reveal that the proteolytic sites are within an internal chamber formed by two stacked rings of identical peptidase subunits, resembling the 20 S proteolytic core of the proteasome (10Sousa M.C. Trame C.B. Tsuruta H. Wilbanks S.M. Reddy V.S. McKay D.B. Cell. 2000; 103: 633-643Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 15Bochtler M. Ditzel L. Groll M. Huber R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6070-6074Crossref PubMed Scopus (166) Google Scholar, 16Wang J. Hartling J.A. Flanagan J.M. Cell. 1997; 91: 447-456Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar). Access to the proteolytic chamber appears to be limited to narrow pores at either end of the stacked rings, which are not large enough to allow passage of a native globular protein. The current mechanistic model proposes that to gain entry to the proteolytic chamber, native substrates are first specifically bound and unfolded by the ATPase components flanking the proteolytic core. The unfolded polypeptide is then translocated through the small pores into the proteolytic chamber (17Larsen C.N. Finley D. Cell. 1997; 91: 431-434Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 18Gottesman S. Maurizi M.R. Wickner S. Cell. 1997; 91: 435-438Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Studies demonstrating that ClpA and ClpX bind substrates specifically and catalyze ATP-dependent protein unfolding provide support for this model (19Weber-Ban E.U. Reid B.G. Miranker A.D. Horwich A.L. Nature. 1999; 401: 90-93Crossref PubMed Scopus (361) Google Scholar, 20Singh S.K. Grimaud R. Hoskins J.R. Wickner S. Maurizi M.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8898-8903Crossref PubMed Scopus (221) Google Scholar, 21Kim Y.I. Burton R.E. Burton B.M. Sauer R.T. Baker T.A. Mol. Cell. 2000; 5: 639-648Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Additionally, ClpA and ClpX translocate substrates in an ATP-dependent reaction from binding sites on the ATPase component to ClpP in a directional manner, providing further experimental evidence for this model (20Singh S.K. Grimaud R. Hoskins J.R. Wickner S. Maurizi M.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8898-8903Crossref PubMed Scopus (221) Google Scholar, 21Kim Y.I. Burton R.E. Burton B.M. Sauer R.T. Baker T.A. Mol. Cell. 2000; 5: 639-648Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 22Hoskins J.R. Pak M. Maurizi M.R. Wickner S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12135-12140Crossref PubMed Scopus (122) Google Scholar, 23Reid B.G. Fenton W.A. Horwich A.L. Weber-Ban E.U. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3768-3772Crossref PubMed Scopus (132) Google Scholar, 24Lee C. Schwartz M.P. Prakash S. Iwakura M. Matouschek A. Mol. Cell. 2001; 7: 627-637Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar). In some instances Clp proteases require specificity factors (or adaptor proteins) for recognition of particular substrates. These adaptors act by specifically facilitating the interaction of a Clp ATPase with a substrate that has low affinity for the Clp ATPase. For example, σS, the E. coli stationary phase sigma factor, is not detectably degraded by ClpXP alone. However, the RssB adaptor interacts with both ClpX and σS, thereby delivering σS to ClpXP for degradation, although not being degraded itself (25Zhou Y. Gottesman S. Hoskins J.R. Maurizi M.R. Wickner S. Genes Dev. 2001; 15: 627-637Crossref PubMed Scopus (228) Google Scholar). Two other ClpX specificity factors, SspB and UmuD, and one ClpA specificity factor, ClpS, have also been well characterized (26Levchenko I. Seidel M. Sauer R.T. Baker T.A. Science. 2000; 289: 2354-2356Crossref PubMed Scopus (248) Google Scholar, 27Bolon D.N. Wah D.A. Hersch G.L. Baker T.A. Sauer R.T. Mol. Cell. 2004; 13: 443-449Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 28Gonzalez M. Rasulova F. Maurizi M.R. Woodgate R. EMBO J. 2000; 19: 5251-5258Crossref PubMed Scopus (94) Google Scholar, 29Neher S.B. Sauer R.T. Baker T.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13219-13224Crossref PubMed Scopus (84) Google Scholar, 30Dougan D.A. Reid B.G. Horwich A.L. Bukau B. Mol. Cell. 2002; 9: 673-683Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 31Guo F. Esser L. Singh S.K. Maurizi M.R. Xia D. J. Biol. Chem. 2002; 277: 46753-46762Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 32Zeth K. Ravelli R.B. Paal K. Cusack S. Bukau B. Dougan D.A. Nat. Struct. Biol. 2002; 9: 906-911Crossref PubMed Scopus (108) Google Scholar). Substrate recognition and binding by the ATPase component is the initial step in protein remodeling and degradation by Clp chaperones and Clp proteases, respectively. Generally, although not exclusively (33Hoskins J.R. Yanagihara K. Mizuuchi K. Wickner S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11037-11042Crossref PubMed Scopus (68) Google Scholar), ClpA and ClpX recognize substrates through short signals located very near either the N or C terminus of the polypeptide. Systematic analysis of more than 50 ClpX substrates identified five signal motifs ranging in size from 3 to 6 amino acids (34Flynn J.M. Neher S.B. Kim Y.I. Sauer R.T. Baker T.A. Mol. Cell. 2003; 11: 671-683Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). Two of these motifs are C-terminal, and the other three are located at the N terminus of substrate proteins. Additionally, the specific interactions between substrate and Clp ATPase have been examined in great detail for several substrates. One well characterized ClpA substrate is RepA, the P1 plasmid initiator protein. ClpA binds inactive RepA dimers and, in a reaction requiring ATP-dependent unfolding, converts dimers into active monomers that can then bind oriP1 DNA (3Wickner 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 (320) Google Scholar). However, when ClpA is associated with ClpP, ClpA targets RepA for degradation. The recognition signal that directs RepA to ClpA is located within the first 15 amino acids of RepA. This signal is both necessary and sufficient to target a protein fused to the peptide for unfolding by ClpA and degradation by ClpAP (35Hoskins J.R. Kim S.Y. Wickner S. J. Biol. Chem. 2000; 275: 35361-35367Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Another well characterized ClpA and ClpX recognition signal is SsrA. A polypeptide is tagged for degradation by the addition of an 11-amino acid peptide encoded by a small RNA, ssrA, when an mRNA lacking an in-frame stop codon stalls on the ribosome (36Keiler K.C. Waller P.R. Sauer R.T. Science. 1996; 271: 990-993Crossref PubMed Scopus (891) Google Scholar, 37Karzai A.W. Roche E.D. Sauer R.T. Nat. Struct. Biol. 2000; 7: 449-455Crossref PubMed Scopus (339) Google Scholar). The SsrA peptide is added to the C terminus of a nascent polypeptide chain by cotranslational switching of the ribosome from the damaged mRNA to ssrA RNA. This signal then targets the abnormal protein for degradation. In E. coli, both ClpAP and ClpXP degrade SsrA-tagged proteins (36Keiler K.C. Waller P.R. Sauer R.T. Science. 1996; 271: 990-993Crossref PubMed Scopus (891) Google Scholar, 38Gottesman S. Roche E. Zhou Y. Sauer R.T. Genes Dev. 1998; 12: 1338-1347Crossref PubMed Scopus (635) Google Scholar, 39Herman C. Thevenet D. Bouloc P. Walker G.C. D'Ari R. Genes Dev. 1998; 12: 1348-1355Crossref PubMed Scopus (236) Google Scholar). A study comparing SsrA-tagged proteins of different known stabilities revealed that the efficiency of degradation by ClpXP is determined largely by the presence of the tag, not by the intrinsic stability of the attached protein (21Kim Y.I. Burton R.E. Burton B.M. Sauer R.T. Baker T.A. Mol. Cell. 2000; 5: 639-648Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 40Burton R.E. Siddiqui S.M. Kim Y.I. Baker T.A. Sauer R.T. EMBO J. 2001; 20: 3092-3100Crossref PubMed Scopus (118) Google Scholar, 41Kenniston J.A. Baker T.A. Fernandez J.M. Sauer R.T. Cell. 2003; 114: 511-520Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). It was shown further that when ClpXP interacts with heterodimeric substrates in which only one protomer is SsrA-tagged, only the subunit with the recognition signal is degraded (40Burton R.E. Siddiqui S.M. Kim Y.I. Baker T.A. Sauer R.T. EMBO J. 2001; 20: 3092-3100Crossref PubMed Scopus (118) Google Scholar). The goals of the present study were to elucidate further the mechanisms of Clp-substrate interactions. We investigated the fate of individual subunits of a dimeric substrate interacting with the ClpAP protease to determine whether both protomers may be degraded during one round of binding. The interactions of ClpAP and ClpXP with heterodimeric substrates were also examined. Our findings indicate that the closely related proteases ClpAP and ClpXP may have fundamentally different substrate recognition, binding, and processing mechanisms. Materials—ATP and ATPγS 1The abbreviations used are: ATPγS, adenosine 5′-3-O-(thio)triphosphate; GFP, green fluorescent protein. were obtained from Roche Applied Science. Restriction endonucleases and DNA-modifying enzymes were obtained from New England BioLabs. PCR reagents were obtained from PerkinElmer Life Sciences. Plasmids and Strains—To construct pET-RepA(Δ25), a repA PCR fragment corresponding to RepA amino acids 26–286, and flanked with NdeI and HindIII sites at the 5′- and 3′-end, respectively, was cloned between the NdeI and HindIII sites of pET24B (Novagen). pET-RepA(Δ25)SsrA was constructed by generating a repA PCR fragment flanked with NdeI and HindIII sites at the 5′- and 3′-ends, respectively, in which the 3′-oligonucleotide incorporated the coding region for the 11-amino acid SsrA tag followed by a TAA stop codon. This PCR fragment was cloned between the NdeI and HindIII sites of pET24B. pET-RepAhis was constructed by synthesizing a repA PCR fragment lacking the stop codon and flanked at the 5′- and 3′-ends by NdeI and HindIII sites, respectively. The PCR fragment was then cloned between the NdeI and HindIII sites of pET24b, such that a C-terminal His6 tag was added to RepA. All mutations were confirmed by DNA sequencing. Proteins and DNA—RepA(Δ25)SsrA, RepA(Δ25), and RepAhis were isolated using the procedure for the purification of wild-type RepA (42Wickner S.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2690-2694Crossref PubMed Scopus (70) Google Scholar). ClpA (43Maurizi M.R. Thompson M.W. Singh S.K. Kim S.H. Methods Enzymol. 1994; 244: 314-331Crossref PubMed Scopus (135) Google Scholar), ClpX (44Kruklitis R. Welty D.J. Nakai H. EMBO J. 1996; 15: 935-944Crossref PubMed Scopus (104) Google Scholar), and ClpP (43Maurizi M.R. Thompson M.W. Singh S.K. Kim S.H. Methods Enzymol. 1994; 244: 314-331Crossref PubMed Scopus (135) Google Scholar) were purified as described previously. RepA-GFP and RepA(1–70)-GFP were purified as described for the isolation of RepA(1–70)-GFP (45Hoskins J.R. Singh S.K. Maurizi M.R. Wickner S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8892-8897Crossref PubMed Scopus (126) Google Scholar). Protein concentrations are expressed as molar amounts of ClpA hexamers, ClpX hexamers, ClpP tetradecamers, RepAhis dimers, RepA(Δ25)SsrA dimers, RepA(Δ25) dimers, RepA-GFP dimers, and RepA(1–70)-GFP monomers. 3H-La-beled oriP1 plasmid DNA was prepared as described previously (46Wickner S. Hoskins J. McKenney K. Nature. 1991; 350: 165-167Crossref PubMed Scopus (158) Google Scholar). RepA Activation Assay—Reaction mixtures contained (in 20 μl) buffer A (20 mm Tris-HCl, pH 7.5, 100 mm KCl, 5 mm dithiothreitol, 0.1 mm EDTA, 0.005% Triton X-100 (v/v), 10% glycerol (v/v)), 1 mm ATP, 10 mm MgOAc, 50 μg/ml bovine serum albumin, 0.7 pmol of ClpX, and RepA(Δ25)SsrA or RepAhis as indicated in Fig. 6. After 10 min at 24 °C, the mixtures were chilled to 0 °C. Calf thymus DNA (1 μg) and 11 fmol of 3H-labeled oriP1 plasmid DNA (3,800 cpm/fmol) were added. After 5 min at 0 °C, the mixtures were filtered through nitrocellulose filters, and the retained radioactivity was measured. Preparation of RepAhis:RepAΔ25 and RepAhis:RepA(Δ25)SsrA Heterodimers—RepAhis and either RepA(Δ25) or RepA(Δ25)SsrA were mixed together in a 1:5 ratio in 1× phosphate-buffered saline, 100 mm NaCl, 10% glycerol (v/v), and 6 m guanidine HCl. After 15 min at 24 °C, the denatured protein mixture was dialyzed for four h at 4 °C against the same buffer but lacking guanidine HCl. Heterodimers were isolated by applying the mixture to an immobilized metal affinity chromatography column (Talon) following the manufacturer's procedure (Clontech). The columns were washed successively with 20, 40, 80, and 100 mm imidazole. The purified heterodimers, eluting with 80 and 100 mm imidazole, were stored at –70 °C. ClpAP and ClpXP Degradation Assays—Reaction mixtures were assembled in 100 μl of buffer A containing 10 mm MgOAc, 2 mm ATP, 3 pmol of ClpA or 3.6 pmol of ClpX, 7.7 pmol of ClpP and 0.1 pmol of RepAhis, RepA(Δ25), RepA(Δ25)SsrA, RepAhis:RepA(Δ25), RepAhis: RepA(Δ25)SsrA, or chemically denatured RepA(Δ25) as indicated in Figs. 3, 4, 7, and 8. The mixtures were incubated at 24 °C for 15 min in Fig. 8 and for the times indicated in Fig. 3, 4, and 7. Trichloroacetic acid was added to 20% (w/v). The trichloroacetic acid pellets were subjected to SDS-PAGE and transferred onto nitrocellulose membranes (Invitrogen) by electroblotting. RepA and its derivatives were detected with specific rabbit antiserum using a Western blot immunodetection kit (Novex Western Breeze, Invitrogen) and the results quantified by densitometry.Fig. 4Denatured RepA(Δ25) is not degraded by ClpAP. 100 nm RepA(Δ25) was denatured in 6 m guanidine HCl and diluted 100-fold into degradation reaction mixtures as described under “Experimental Procedures.” Degradation was measured by Western blot analysis and quantified (filled circles) as described under “Experimental Procedures.” The experiment was carried out three times, and a representative example is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 7Only the RepA subunit with an SsrA recognition signal is degraded by ClpXP. ClpXP degradation of RepA(Δ25)SsrA homodimers (A), RepAhis homodimers (B), and RepAhis:RepA(Δ25)SsrA heterodimers (C) was measured by Western blot analysis and quantified as described under “Experimental Procedures.” In A and B degradation of RepA(Δ25)SsrA and RepAhis is shown with filled triangles and filled squares, respectively. In C degradation of RepA(Δ25)SsrA and RepAhis is shown with open triangles and open squares, respectively. Experiments were repeated three or more times, and representative results are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 8The RepA subunit lacking the ClpX recognition signal is activated for DNA binding, whereas the RepA(Δ25)SsrA subunit is degraded by ClpXP. RepAhis homodimers (A), RepA(Δ25)SsrA homodimers (B), and RepAhis:RepA(Δ25)SsrA heterodimers (C) were incubated as indicated with ClpX, ClpP, and ATP as described under “Experimental Procedures.” oriP1 DNA binding was measured at 0 °C as described under “Experimental Procedures” by adding 10-μl portions of the reactions to 10 μl of buffer A containing 1 μg of calf thymus DNA and 11 fmol of 3H-labeled oriP1 plasmid DNA. Degradation was measured by trichloroacetic acid precipitating 90-μl portions of the reaction mixtures and subjecting the samples to SDS-PAGE followed by Western blot analysis as described under “Experimental Procedures.” Experiments were carried out three times, and the results shown for RepA activation are the averages of three experiments ± S.E. For each substrate one representative Western blot is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Single Round Degradation Assay—Reaction mixtures (100 μl) containing buffer A, 10 mm MgOAc, 2 mm ATPγS, 0.1 mg/ml bovine serum albumin, 2.4 μm ClpA, 5.5 μm ClpP, and 1 μm RepA-GFP or 4.6 μm RepA(1–70)-GFP were incubated for 15 min at 23 °C. Substrate-ClpAP complexes were isolated by gel filtration on a Sephacryl S-200 column (0.7 × 17 cm) equilibrated with 20 mm Tris-HCl, pH 7.5, 100 mm KCl, 1 mm dithiothreitol, 1 mm EDTA, 0.05 mg/ml bovine serum albumin, 5 mm MgOAc, and 0.25 mm ATPγS. Fractions containing substrate-Cl-pAP complexes were pooled (450 μl). α-Casein (120 μg), 6 mm ATP, 10 mm MgOAc, and an ATP-regenerating system (in 26 μl) were added to one aliquot of 150 μl, and 26 μl of buffer A was added to another aliquot. Degradation was measured by monitoring loss of GFP fluorescence at 23 °C for 5 min. To measure the effectiveness of α-casein in inhibiting RepA-GFP degradation by ClpAP, complexes of ClpAP were generated and isolated as above, but without substrate. RepA-GFP (2.5 μg, an amount equivalent to that present in the degradation reactions described above), 6 mm ATP, 10 mm MgOAc, and an ATP-regenerating system were added in the presence or absence of 120 μg of α-casein to 150-μl aliquots of ClpAP complexes. Degradation was measured by monitoring the loss of GFP fluorescence at 23 °C for 5 min. Isolation of Substrate-ClpA Complexes—ClpA (300 pmol) was mixed with 45 pmol of RepAhis homodimers or RepAhis:RepA(Δ25) heterodimers in 100 μl of 20 mm Tris-HCl, pH 7.5, 100 mm KCl, 1 mm dithiothreitol, 1 mm EDTA, 0.05 mg/ml bovine serum albumin, 2 mm ATPγS, 10 mm MgOAc and incubated for 15 min at 24 °C. The mixtures were then applied to a Sephacryl S-100 gel filtration column (0.7 × 17 cm) equilibrated with the same buffer but containing 0.5 mm ATPγS. Fractions containing substrate-ClpA complexes were pooled. ClpP (400 pmol) and 5 mm ATP were added to 200-μl aliquots as indicated in Fig. 5. After 5 min at 24 °C, mixtures were trichloroacetic acid precipitated and analyzed by SDS-PAGE followed by staining with Coomassie Blue. ClpAP Degrades Both Subunits of a Dimer in One Round of Binding and Proteolysis—According to the current mechanistic model of ClpAP, a recognition signal on the substrate is bound by ClpA. After binding, ClpA unfolds the substrate in an ATP-dependent reaction, then translocates it to ClpP in a second ATP-dependent reaction. The substrate is degraded upon translocation into the proteolytic chamber of ClpP. An aspect of the ClpAP mechanism that remains unclear is the fate of the individual subunits of a multimeric substrate. After binding a dimeric substrate, ClpAP may unfold and degrade one subunit while releasing the other, or both protomers of the substrate may be unfolded and degraded. RepA is a native, dimeric substrate of ClpAP, and previous work in our laboratory demonstrated that one RepA dimer is bound by one ClpA6ClpP14 complex (47Pak M. Wickner S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4901-4906Crossref PubMed Scopus (75) Google Scholar). To determine whether one or both RepA protomers were degraded after binding ClpAP, GFP fusion proteins were utilized. RepA-GFP contains the full-length RepA protein with GFP fused to the C terminus, and it exists as a dimer in solution (Fig. 1A and Ref. 33Hoskins J.R. Yanagihara K. Mizuuchi K. Wickner S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11037-11042Crossref PubMed Scopus (68) Google Scholar). RepA(1–70)-GFP contains the 70 N-terminal amino acids of RepA with GFP fused to the C-terminal amino acid, and it exists as a native monomer at the concentrations used here (Fig. 1A and data not shown). Both fusion proteins are substrates for ClpAP (33Hoskins J.R. Yanagihara K. Mizuuchi K. Wickner S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11037-11042Crossref PubMed Scopus (68) Google Scholar, 45Hoskins J.R. Singh S.K. Maurizi M.R. Wickner S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8892-8897Crossref PubMed Scopus (126) Google Scholar). Complexes of substrate-ClpAP were formed in the presence of ATPγS, a poorly hydrolyzed ATP analog, by incubating ClpAP with either [RepA-GFP]2 or RepA(1–70)-GFP. The substrate-ClpAP complexes were then isolated by gel filtration. After the addition of a large excess of α-casein to compete with substrate released from ClpAP, ATP was added, and substrate degradation was measured by monitoring the decrease in GFP fluorescence. If both protomers of the dimer were degraded there would be close to a 100% decrease in fluorescence, whereas if one protomer was degraded and one was released, there would be a 50% decrease in fluorescence. The addition of ATP to ClpAP·[RepA-GFP]2 complexes resulted in an 80% decrease in fluorescence (Fig. 1B). Separation of the reaction mixtures by SDS-PAGE followed by staining and densitometry confirmed that 80% of the RepA-GFP was degraded by ClpAP (data not shown). These data suggest that the binding of dimers to ClpAP results in the proteolysis of both protomers in the majority of complexes. By comparison the addition of ATP to ClpAP·RepA(1–70)-GFP complexes resulted in degradation of 94% of the substrate as determined by the decrease in fluorescence and by densitometry (Fig. 1C and data not shown). This indicates that very few bound monomeric substrates escape proteolysis by ClpAP. The effectiveness of α-casein in competing with free RepA was determined by isolating ClpAP complexes in the absence of substrate, then adding [RepA-GFP]2, excess α-casein, and ATP together and measuring the loss of fluorescence. The presence of excess α-casein resulted in greater than 90% inhibition of RepA-GFP degradation (Fig. 1D), revealing that if RepA-GFP was released from ClpAP, it was unlikely to rebind and be degraded. Taken together, these results demonstrate that interactions between ClpAP and a homodimeric substrate most often lead to the proteolysis of both subunits within a single round of binding. The Presence of a Recognition Signal on One Protomer of a Heterodimer Is Sufficient to Target Both Protomers for Degradation by ClpAP—After demonstrating that ClpAP can unfold and degrade both subunits of a dimer in one round of binding, we then wanted to determine whether degradation of the dimer required the presence of a recognition signal on both protomers. RepA heterodimers were constructed in which only one subunit contained the ClpA recognition signal (Fig. 2A). Previously, we demonstrated that ClpA does not recognize a truncated version of RepA in which the 25 N-terminal residues are removed (RepA(Δ25)), although this mutant protein is a dimer and can be activated for DNA binding both by the DnaK chaperone system and by treatment with chemical denaturants (Ref. 48Kim S.Y. Sharma S. Hoskins J.R. Wickner S. J. Biol. Chem. 2002; 277: 44778-44783Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar and data not shown). Heterodimers containing one RepA(Δ25) protomer and one full-length, His-tagged RepA protomer were prepared by mixing RepA(Δ25) and His-tagged full-length RepA in a 5:1 ratio in denaturant and then removing the denaturant. The renatured heterodimers were isolated by immobilized metal affinity chromatography (Fig. 2B, lane 3). To demonstrate that the heterodimers were stable, they were diluted 5-fold and mixed with a 5-fold molar excess of non-His-tagged RepA and subjected to another metal affinity purification. SDS-PAGE showed that the RepA(Δ25) subunit coeluted with the His-tagged RepA protomer, indicating that the heterodimeric complex was stable in solution (Fig. 2B, lane 4). We then measured degradation of the heterodimers by ClpAP by monitoring the disappearance of the substrate with time by SDS-PAGE followed by Western blot analysis and densitometry. We found that both protomers were degraded, although only one subunit contained the recognition signal (Fig. 3A). The protomer lacking the recognition signal was degraded significantly slower than the full-length RepA protomer (Fig. 3A); RepAhis and RepA(Δ25) were degraded 50% in 7.2 ± 0.4 and 20.8 ± 0.6 min, respectively (calculated from three separate experiments). A slower rate of degradation and a lag phase appear to contribute to the slower proteolysis of the untagged protomer. Both subunits were undetectable when the incubations with ClpAP were extended to 40 min (data not shown). With the same conditions homodimeric RepA(Δ25), which lacks the ClpA recognition signal, was not detectably degraded by ClpAP within 25 min (Fig. 3B). Under the same conditions, more than 80% of RepAhis was degraded by ClpAP (Fig. 3C). Why the subunit lacking the recognition signal was consistently degraded more slowly than the tagged subunit is not known. We demonstrated previously that ClpAP degrades unfolded proteins lacking recognition signals (45Hoskins J.R. Singh S.K. Maurizi M.R. Wickner S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8892-8897Crossref PubMed Scopus (126) Google Scholar). Therefore, one possible reason might be that the RepA(Δ25) subunit is released from ClpAP in an unfolded form and then rebound by another ClpAP molecule. This was tested for by denaturing RepA(Δ25) in guanidine HCl and then measuring degradation following the addition of the denatured substrate to reaction mixtures containing ClpAP (Fig. 4). There was no detectable degradation, implying that if the untagged subunit was released in an unfolded conformation, it was not likely to be rebound and degraded by ClpAP with these conditions. We also measured degradation of the RepAhis:RepA(Δ25) heterodimers after the isolation of ClpA-heterodimer complexes. First, ClpA and the heterodimers were incubated in the presence of ATPγS to generate stable complexes, and then the complexes were isolated by size exclusion chromatography. ClpP was added, either with or without ATP, and degradation of the bound substrate was measured by SDS-PAGE. The results showed that both subunits of the heterodimer were degraded (Fig. 5A). When complexes of ClpA·[RepAhis]2 were similarly isolated and degradation was measured, both subunits were degraded (Fig. 5B). Because RepA(Δ25) lacks a recognition signal, stable complexes between it and ClpA could not be isolated. Taken together, these data demonstrate that the presence of a recognition signal on one protomer of a dimeric substrate is sufficient to target both protomers for degradation by ClpAP. They suggest that the untagged subunit remains associated with ClpAP until it is eventually degraded. ClpXP Degrades Only the Protomer Containing the Recognition Signal When Interacting with Heterodimeric Substrates—In contrast to our observations for ClpAP, ClpXP was previously demonstrated to degrade only the protomer containing the recognition signal when interacting with a heterodimeric substrate (40Burton R.E. Siddiqui S.M. Kim Y.I. Baker T.A. Sauer R.T. EMBO J. 2001; 20: 3092-3100Crossref PubMed Scopus (118) Google Scholar). The ClpXP experiments utilized Arc repressor heterodimers in which only one protomer contained the SsrA recognition signal. To determine whether the contrasting observations with ClpAP reflect different mechanisms for ClpAP and ClpXP or are because of differences in the substrates utilized, we investigated the interactions between ClpXP and RepA heterodimers. RepA is not a natural substrate for ClpXP, so an SsrA recognition signal was added to the C-terminal end of the RepA(Δ25) construct utilized in the ClpAP experiments (Fig. 6A). Without the SsrA recognition signal, ClpX was not able to activate RepA(Δ25) for DNA binding (Fig. 6B). However, ClpX was able to activate the RepA(Δ25)SsrA fusion protein for DNA binding, indicating that RepA(Δ25)SsrA exists as an inactive dimer in solution and is converted to an active monomer by the chaperone activity of ClpX (Fig. 6B). Activation of RepA(Δ25)SsrA by ClpX was similar to activation of RepAhis by ClpA (data not shown). Additionally, ClpA was able to activate RepA(Δ25)SsrA through recognition of the SsrA tag, although ClpA is unable to recognize RepA(Δ25) (data not shown). These data demonstrate that the presence of an SsrA recognition signal on RepA directs the fusion protein to ClpX without affecting the DNA binding activity of RepA. To determine whether ClpXP acted similarly to ClpAP with respect to heterodimeric RepA substrates, RepAhis: RepA(Δ25)SsrA heterodimers were prepared and isolated as described above for RepAhis:RepA(Δ25). As with the ClpAP heterodimeric substrate, the RepAhis:RepA(Δ25)SsrA heterodimers were stable to subunit exchange in solution (data not shown). Incubation of ClpXP with RepA(Δ25)SsrA homodimers resulted in complete degradation of the substrate, demonstrating that SsrA-tagged RepA(Δ25) is proteolyzed efficiently by ClpXP (Fig. 7A). RepAhis homodimers, which lack the SsrA recognition signal, were not detectably degraded by ClpXP, showing that without the SsrA tag, RepA is not a substrate for ClpXP-dependent degradation (Fig. 7B). Importantly, when ClpXP was incubated with the heterodimeric substrate, only the SsrA-tagged subunit was degraded (Fig. 7C). This demonstrates that, unlike ClpAP, ClpXP requires both subunits of a dimeric substrate to contain the ClpX recognition signal for both protomers to be degraded. Taken with the ClpAP data from above, these results suggest that despite their extensive structural and functional homology, ClpAP and ClpXP process substrates in different ways. ClpXP Releases the Nondegraded Subunit of a Heterodimeric Substrate as an Active Monomer—Unlike ClpAP, ClpXP did not degrade both subunits of a heterodimer. To determine whether the protomer that lacked the recognition signal was released as an active monomer or not, the DNA binding activity of the nondegraded subunit was measured. ClpX and ClpXP were incubated with RepAhis homodimers, RepA(Δ25)SsrA homodimers, and RepA:RepA(Δ25)SsrA heterodimers. A portion of each reaction was subjected to SDS-PAGE followed by Western blot analysis and densitometry. Another portion was used to measure DNA binding. RepAhis homodimers were neither activated by ClpX nor degraded by ClpXP because of the absence of the SsrA tag, as anticipated (Fig. 8A). The presence of the recognition signal on RepA(Δ25)SsrA homodimers resulted in activation by ClpX and proteolysis by ClpXP (Fig. 8B). In contrast, RepAhis:RepA(Δ25)SsrA heterodimers were activated for DNA binding by both ClpX and ClpXP. As expected, activation was reduced ∼50% in the samples incubated with ClpXP compared with those incubated with ClpX because of degradation of the tagged protomer by ClpXP (Fig. 8C). Western blot analysis confirmed that ClpXP degraded the SsrA-tagged subunit but not the subunit lacking SsrA (Fig. 8C). Together, these data show that when only one subunit of a dimeric substrate is degraded by ClpXP, the remaining protomer is released. The data presented here shed light on the mechanism of substrate recognition by Clp ATPases and degradation by Clp proteases. The results demonstrate that ClpAP degrades both subunits of a RepA heterodimer when only one subunit contains the N-terminal recognition signal. One possible mechanism for the action of ClpAP on heterodimers is that the interaction between ClpA and the tagged subunit brings the untagged subunit into close proximity to ClpA, where it is bound through low affinity secondary recognition signals. This model is consistent with previous results from our laboratory demonstrating that although the first 15 amino acids of RepA are sufficient to target a RepA-GFP fusion protein for degradation by ClpAP, fusion proteins containing larger portions of RepA (namely the first 46 or 70 amino acids) are bound by ClpA with higher affinity (35Hoskins J.R. Kim S.Y. Wickner S. J. Biol. Chem. 2000; 275: 35361-35367Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). These observations suggest that in addition to the essential Clp recognition signal located near the N or C terminus of the substrate, the presence of secondary recognition signals in the polypeptide may increase substantially the efficiency of substrate recognition and degradation by Clp proteases. The reason why the untagged subunit is degraded more slowly by ClpAP than the tagged subunit remains unexplained (Fig. 3A). It has been shown that protein unfolding is the rate-limiting step in degradation by Clp proteases (21Kim Y.I. Burton R.E. Burton B.M. Sauer R.T. Baker T.A. Mol. Cell. 2000; 5: 639-648Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Therefore one possibility is that more cycles of ATP hydrolysis are required to unfold and translocate an untagged subunit compared with a tagged subunit, and thus more time is required. It is possible that the lag in degradation of the untagged subunit seen in Fig. 3A may be caused by preferential unfolding and translocation of the tagged subunit prior to the processing of the untagged subunit. In contrast to ClpAP, ClpXP was only able to degrade the tagged subunit of a RepA heterodimer containing one tagged and one untagged subunit. Similar results were obtained by Burton et al. (40Burton R.E. Siddiqui S.M. Kim Y.I. Baker T.A. Sauer R.T. EMBO J. 2001; 20: 3092-3100Crossref PubMed Scopus (118) Google Scholar) using another substrate, Arc repressor. In their experiments, one Arc subunit was SsrA-tagged and the other was not; ClpXP only degraded the SsrA-tagged subunit. Thus, with two different substrates, the untagged subunit was not degraded by ClpXP. Our results also show that the RepA subunit lacking the SsrA tag that was not degraded by ClpXP was activated for oriP1 DNA binding. One possible explanation is that the untagged subunit is simply not recognized or bound by ClpXP and is activated as a consequence of the unfolding and degradation of the SsrA-containing subunit. Another possible interpretation is that the subunit without the SsrA tag is unfolded by ClpX coincidentally with the unfolding of the tagged subunit, but instead of being translocated to ClpP, is released and refolds spontaneously. This scenario is consistent with previous work demonstrating that ClpXP, unlike ClpAP, fails to degrade an unfolded substrate lacking a recognition signal (20Singh S.K. Grimaud R. Hoskins J.R. Wickner S. Maurizi M.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8898-8903Crossref PubMed Scopus (221) Google Scholar). These possibilities remain to be tested. In summary, our results demonstrate that ClpAP and ClpXP interact differently with heterodimeric substrates and suggest that there may be a significant difference in the mechanisms of substrate recognition and binding between ClpAP and ClpXP. We thank Matthew Chenoweth for many helpful discussions and comments on the manuscript." @default.
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