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• W1999121105 abstract "Thermal stress might lead to protein aggregation in the cell. Reactivation of protein aggregates depends on Hsp100 and Hsp70 chaperones. We focus in this study on the ability of DnaK, the bacterial representative of the Hsp70 family, to interact with different aggregated model substrates. Our data indicate that DnaK binding to large protein aggregates is mediated by DnaJ, and therefore it depends on its affinity for the cochaperone. Mutations in the structural region of DnaK known as the “latch” decrease the affinity of the chaperone for DnaJ, resulting in a defective activity as protein aggregate-removing agent. As expected, the chaperone activity is recovered when DnaJ concentration is raised to overcome the lower affinity of the mutant for the cochaperone, suggesting that a minimum number of aggregate-bound DnaK molecules is necessary for its efficient reactivation. Our results provide the first experimental evidence of DnaJ-mediated recruiting of ATP-DnaK molecules to the aggregate surface. Thermal stress might lead to protein aggregation in the cell. Reactivation of protein aggregates depends on Hsp100 and Hsp70 chaperones. We focus in this study on the ability of DnaK, the bacterial representative of the Hsp70 family, to interact with different aggregated model substrates. Our data indicate that DnaK binding to large protein aggregates is mediated by DnaJ, and therefore it depends on its affinity for the cochaperone. Mutations in the structural region of DnaK known as the “latch” decrease the affinity of the chaperone for DnaJ, resulting in a defective activity as protein aggregate-removing agent. As expected, the chaperone activity is recovered when DnaJ concentration is raised to overcome the lower affinity of the mutant for the cochaperone, suggesting that a minimum number of aggregate-bound DnaK molecules is necessary for its efficient reactivation. Our results provide the first experimental evidence of DnaJ-mediated recruiting of ATP-DnaK molecules to the aggregate surface. Members of the Hsp70 protein family are involved in numerous cellular processes that include protein folding and refolding (1Bukau B. Deuerling E. Pfund C. Craig E. Cell. 2000; 101: 119-122Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 2Hartl F.-U. Hayer-Hartl M. Science. 2002; 295: 1852-1858Crossref PubMed Scopus (2766) Google Scholar), protein translocation across membranes (3Liu Q. D′Silva P. Walter W. Marszalek J. Craig E.A. Science. 2003; 300: 139-141Crossref PubMed Scopus (142) Google Scholar), and protein complex assembly and disassembly (4Liberek K. Georgopoulos C. Zylicz M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6632-6636Crossref PubMed Scopus (139) Google Scholar). To fulfill its functional role in protein (re)folding, the Hsp70 protein has to collaborate with co-chaperones that modulate its ATPase cycle and might also carry protein substrates (5Mayer M.P. Bukau B. Cell Mol. Life Sci. 2005; 62: 670-684Crossref PubMed Scopus (2019) Google Scholar). DnaK is the bacterial representative of the Hsp70 family and, in collaboration with GrpE (nucleotide exchange factor) and DnaJ (Hsp40), prevents protein aggregation and productively solubilizes protein aggregates either alone or together with ClpB (the Hsp100 representative in Escherichia coli) (6Weibezahn J. Schlieker C. Tessarz P. Mogk A. Bukau B. Biol. Chem. 2005; 386: 739-744Crossref PubMed Scopus (76) Google Scholar). DnaJ is involved in the transfer of substrates to DnaK (7Han W. Christen P. J. Biol. Chem. 2003; 278: 19038-19043Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 8Han W. Christen P. FEBS Lett. 2004; 563: 146-150Crossref PubMed Scopus (35) Google Scholar, 9Laufen 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, 10Gamer J. Multhaup G. Tomoyasu T. McCarty J.S. Rudiger S. Schonfeld H.-J. Schirra C. Bujard H. Bukau B. EMBO J. 1996; 15: 607-617Crossref PubMed Scopus (237) Google Scholar, 11Hartl F.U. Nature. 1996; 381: 571-579Crossref PubMed Scopus (3090) Google Scholar) and, together with GrpE, in controlling the time the chaperone spends in the low affinity and high affinity states for substrate proteins (5Mayer M.P. Bukau B. Cell Mol. Life Sci. 2005; 62: 670-684Crossref PubMed Scopus (2019) Google Scholar). These DnaJ roles have been found essential for almost all chaperone activities of Hsp70 proteins (12Laufen T. Zuber U. Buchberger A. Bukau B. Fink A.L. Goto Y. Molecular Chaperones in Proteins: Structure, Function, and Mode of Action. Marcel Dekker, New York1998: 241-274Google Scholar, 13Kelley W.L. Curr. Biol. 1999; 9: R305-R308Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar).The mechanism of Hsp100/Hsp70-dependent disaggregation of aggregated substrates is as yet poorly understood. The chaperone network that leads to productive aggregate reactivation depends on aggregate properties, which would include size, conformational properties of the denatured protein molecules, and number and nature of the intermolecular interactions between unfolded polypeptides. Several findings suggest that the activity of DnaK is required in the early stages of protein disaggregation to extract polypeptides from aggregates (14Zietkiewicz S. Lewandowska A. Stocki P. Liberek K. J. Biol. Chem. 2006; 281: 7022-7029Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Kinetic studies have identified the interaction of DnaK with aggregated proteins as the rate-limiting step of the disaggregation process (15Zietkiewicz S. Krewska J. Liberek K. J. Biol. Chem. 2004; 279: 44376-44383Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Analysis of the ClpB variant BAP has also revealed that DnaK is required for the initial substrate unfolding event that leads to aggregate processing (16Weibezahn J. Tessarz P. Schlieker C. Zahn R. Maglica Z. Lee S. Zentgraf H. Weber-Ban E.U. Dougan D.A. Tsai F.T. Mogk A. Bukau B. Cell. 2004; 119: 653-665Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar). The DnaK system might also be involved downstream of ClpB action, taking over the protein once it has been processed by ClpB, preventing its reassociation with the aggregate and therefore promoting substrate reactivation (16Weibezahn J. Tessarz P. Schlieker C. Zahn R. Maglica Z. Lee S. Zentgraf H. Weber-Ban E.U. Dougan D.A. Tsai F.T. Mogk A. Bukau B. Cell. 2004; 119: 653-665Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar).To understand the role of the DnaK system (K/J/E) at the initial stage of reactivation, it is necessary to study how the components of this system interact with client proteins and particularly with protein aggregates. It seems clear that the chaperone action is driven by ATP hydrolysis, which converts the ATP-bound, low affinity state into the ADP-bound high affinity state for substrate proteins (9Laufen 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, 17Karzai A.W. McMacken R. J. Biol. Chem. 1996; 271: 11236-11246Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 18Kelley W.L. Trends Biochem. Sci. 1998; 23: 222-227Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 19Pierpaoli E.V. Sandmeier E. Schonfeld H.-J. Christen P. J. Biol. Chem. 1998; 273: 6643-6649Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). This conversion is stimulated by DnaJ, which itself is also able to bind (partially) denatured proteins, avoiding their aggregation (10Gamer J. Multhaup G. Tomoyasu T. McCarty J.S. Rudiger S. Schonfeld H.-J. Schirra C. Bujard H. Bukau B. EMBO J. 1996; 15: 607-617Crossref PubMed Scopus (237) Google Scholar, 20Langer T. Lu C. Echlos H. Flanagan J. Hayer M.K. Hartl F.U. Nature. 1992; 356: 683-689Crossref PubMed Scopus (780) Google Scholar, 21Schroder H. Langer T. Hartl F.U. Bukau B. EMBO J. 1993; 12: 4137-4144Crossref PubMed Scopus (497) Google Scholar). Refolding of denatured proteins required both domains of DnaJ: the J domain, which stimulates the ATPase activity of DnaK (17Karzai A.W. McMacken R. J. Biol. Chem. 1996; 271: 11236-11246Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 22Misselwitz B. Staeck O. Rapoport T.A. Mol. Cell. 1998; 2: 593-603Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 23Wall D. Zylicz M. Georgopoulos C. J. Biol. Chem. 1995; 270: 2139-2144Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), and the binding substrate domain (23Wall D. Zylicz M. Georgopoulos C. J. Biol. Chem. 1995; 270: 2139-2144Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 24Szabo A. Koorszun R. Hartl F.U. Flanagan J. EMBO J. 1996; 15: 408-417Crossref PubMed Scopus (274) Google Scholar). Based on these observations, it was proposed that after the initial binding of the substrates to DnaJ, they were transferred to the peptide-binding site of DnaK (9Laufen 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, 10Gamer J. Multhaup G. Tomoyasu T. McCarty J.S. Rudiger S. Schonfeld H.-J. Schirra C. Bujard H. Bukau B. EMBO J. 1996; 15: 607-617Crossref PubMed Scopus (237) Google Scholar, 11Hartl F.U. Nature. 1996; 381: 571-579Crossref PubMed Scopus (3090) Google Scholar), which was also proposed to be activated by DnaJ for binding client proteins (22Misselwitz B. Staeck O. Rapoport T.A. Mol. Cell. 1998; 2: 593-603Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 25Wawryznow A. Banecki B. Wall D. Liberek K. Georgopoulos C. Zylicz M. J. Biol. Chem. 1995; 270: 19307-19311Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). More recently, a model has been put forward in which binding of DnaJ and ATP-DnaK to the same polypeptide chain leads to formation of ternary complexes. In these complexes, DnaJ, through a proximity effect, efficiently triggers the conversion of ATP-bound DnaK into the high affinity state that locks onto the substrate (7Han W. Christen P. J. Biol. Chem. 2003; 278: 19038-19043Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 8Han W. Christen P. FEBS Lett. 2004; 563: 146-150Crossref PubMed Scopus (35) Google Scholar). However, the question of how the components of the DnaK system interact with and assist in the reactivation of protein aggregates is not yet fully answered.To address this question, we explore in this work the association of DnaK and DnaJ with aggregates of different client proteins and find that the co-chaperone mediates DnaK binding to the aggregate. DnaK is unable by its own to significantly bind to the aggregate surface, a “catalytically” productive chaperone concentration on the aggregate surface being only achieved in the presence of DnaJ. In contrast, the co-chaperone binds the aggregate in a concentration-dependent manner, the association not requiring additional protein factors to occur. The use of a DnaK mutant in which specific ionic contacts at the substrate binding domain (SBD) 7The abbreviations used are: SBDsubstrate binding domainDnaK2ADnaK(D540A/K548A)SPRsurface plasmon resonanceDTTdithiothreitolMDHmalate dehydrogenaseG6PDHglucose-6-phosphate dehydrogenaseWTwild typeSPRsurface plasmon resonance spectroscopy. 7The abbreviations used are: SBDsubstrate binding domainDnaK2ADnaK(D540A/K548A)SPRsurface plasmon resonanceDTTdithiothreitolMDHmalate dehydrogenaseG6PDHglucose-6-phosphate dehydrogenaseWTwild typeSPRsurface plasmon resonance spectroscopy. (26Fernandez-Saiz V. Moro F. Arizmendi J.M. Acebron S.P. Muga A. J. Biol. Chem. 2006; 281: 7479-7488Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) were disrupted reinforces the above interpretation. This mutant holds two substitutions (D450A/K548A) at the “latch” and shows a reduced affinity for DnaJ. As a consequence, it is recruited less efficiently by the aggregate-DnaJ complex, which in turn compromises its aggregate reactivation ability. Taken together, our data indicate that DnaK binding to protein aggregates, and therefore their reactivation, depends on DnaJ.EXPERIMENTAL PROCEDURESProtein Expression and Purification—Mutant DnaK2A was generated as previously described (26Fernandez-Saiz V. Moro F. Arizmendi J.M. Acebron S.P. Muga A. J. Biol. Chem. 2006; 281: 7479-7488Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Wild type DnaK and the mutant were produced, purified, and extensively dialyzed to obtain nucleotide-free samples (27Buchberger A. Theyssen H. Schröder H. McCarty J.S. Virgallita G. Milkereit P. Reinstein J. Bukau B. J. Biol. Chem. 1995; 270: 16903-16910Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar).DnaJ and GrpE were expressed in BL21 cells and purified as described earlier (28Zylicz M. Yamamoto T. McKittrick N. Sell S. Geosgopoulos C. J. Biol. Chem. 1985; 260: 7591-7598Abstract Full Text PDF PubMed Google Scholar, 29Mehl A.F. Heskett L.D. Neal K.M. Biochem. Biophys. Res. Commun. 2001; 282: 562-569Crossref PubMed Scopus (25) Google Scholar). ClpB was obtained as previously reported (30Woos K.M. Rims K. Goldberg A.L. Has D.B. Chungs C.H. J. Biol. Chem. 1992; 267: 20429-20434Abstract Full Text PDF PubMed Google Scholar). Protein concentration was determined by the colorimetric Bradford assay (Bio-Rad).Surface Plasmon Resonance Spectroscopy (SPR) Binding Assays—SPR measurements were performed on a BIACORE 3000 system (BIACORE, Uppsala, Sweden). Neutravidin was coupled to Research grade CM5 sensor chips using the EDC/NHS cross-linking reagent (Amersham Biosciences). DnaJ was biotinylated (NHS-LC-Biotin; Pierce) and coupled to the chips to ∼2200 resonance units. Excess neutravidin was blocked with ethanolamine. For binding studies, 40 μl of DnaK or the mutant (0.75–6 μm) was incubated with a 10-fold higher ATP concentration in 25 mm Hepes, 50 mm KCl, 10 mm MgCl2, 0.005% (v/v) Tween 20, pH 7.6. Protein solution was injected at 30 °C in the above buffer over the chips at a flow rate of 20 μl min–1. Complete regeneration of the chip surfaces was achieved by two 10-μl injections of 1 m urea at a flow rate of 5 μl min–1 as described (31Mayer M.P. Laufen T. Paal K. McCarty J.S. Bukau B. J. Mol. Biol. 1999; 289: 1131-1144Crossref PubMed Scopus (115) Google Scholar). This treatment did not affect the interaction between immobilized DnaJ and DnaK or DmaK2A, as verified for each experimental series where a DnaK wild-type control was run at the beginning and the end of the series to rule out any possible inactivation of immobilized DnaJ. In all cases studied here, these controls were virtually identical. Background binding to neutravidin was subtracted from each signal to account for nonspecific binding.ATPase Activity—Steady-state ATPase activity was performed in 40 mm Hepes, pH 7.5, 50 mm KCl, 11 mm magnesium acetate buffer at 30 °C, as described previously (32Moro F. Fernandez V. Muga A. FEBS Lett. 2003; 533: 119-123Crossref PubMed Scopus (71) Google Scholar). DnaK or DnaK2A and ATP concentrations were 3 μm and 1 mm, respectively, and DnaJ was added at varying concentrations. Reactions were followed measuring the absorbance decay at 340 nm for 30 min in a Cary spectrophotometer (Varian).Refolding of Client Proteins—Luciferase (2.5 μm; Promega) was denatured for 45 min at 25 °C, in 6 m guanidinium hydrochloride, 100 mm Tris-HCl, pH 7.7, 10 mm DTT. Luciferase was diluted to 80 nm in 50 mm Tris-HCl, 55 mm KCl, 15 mm MgCl2, 5.5 mm DTT, 0.5 mg·ml–1 bovine serum albumin, pH 7.7, containing an ATP-regenerating system (4 mm phosphoenolpyruvate and 20 ng·ml–1 pyruvate kinase). The diluted sample was incubated 10 min, and afterward 1 μm DnaK or DnaK2A, 0.5 μm GrpE, and different DnaJ concentrations (0.1–10 μm) were added. Reactivation was initiated by the addition of 5 mm ATP. Luciferase activity was measured after a 90-min reactivation period at 25 °C, using the luciferase assay system (Promega E1500) in a Sinergy HT (Biotek) luminometer.Malate Dehydrogenase (MDH)—MDH (2 μm monomer; Sigma) was denatured and aggregated by incubating the protein for 30 min at 47 °C in 50 mm Tris-HCl, 150 mm KCl, 20 mm MgCl2, 2 mm DTT, pH 7.5. The sample was diluted (final MDH concentration 1 μm) in the presence of 1 μm DnaK or DnaK2A, 1.5 μm ClpB, 0.25 μm GrpE, and 0.05–10 μm DnaJ in the above buffer. Reactivation was started by the addition of 2 mm ATP and was measured at 30 °C in a medium containing the above ATP-regenerating system. MDH activity was recorded after 2 h of reactivation as previously described (33Schlieker C. Tews I. Bukau B. Mogk A. FEBS Lett. 2004; 578: 351-356Crossref PubMed Scopus (70) Google Scholar). Turbidity of luciferase and MDH aggregates was recorded under the same experimental conditions used in the refolding assays, on a SLM8100 spectrofluorimeter (Aminco) with both excitation and emission wavelengths set at 550 nm.Glucose-6-phosphate Dehydrogenase (G6PDH)—G6PDH (2.5 μm; Sigma) was incubated for 15 min at 52 °C in 100 mm Tris-HCl, 150 mm KCl, 20 mm MgCl2, 10 mm DTT, pH 7.5, to denature and aggregate the protein. The sample was then diluted (final protein concentration 1 μm) in the same buffer containing 6.4 μm DnaK, 0.25 μm GrpE, 0.05–10 μm DnaJ. Reactivation was measured at 25 °C in the presence of the ATP-regenerating system, as previously described (34Ben-Zvi A. De Los Rios P. Dietler G. Goloubinoff P.J. J. Biol. Chem. 2004; 279: 37298-37303Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar).Chaperone Binding Assays—Association of chaperones with protein aggregates was characterized by SDS-PAGE analysis of the pellets obtained after centrifugation of the protein complexes (33Schlieker C. Tews I. Bukau B. Mogk A. FEBS Lett. 2004; 578: 351-356Crossref PubMed Scopus (70) Google Scholar). Luciferase (80 μm) in 6 m guanidinium hydrochloride was diluted 50 times the absence of chaperones and incubated for 10 min at 25 °C to allow protein aggregation. After the addition of 1 μm DnaK or DnaK2A and DnaJ (0.1–1 μm), the mixture containing 1 μm luciferase and 3 mm ATP was incubated for 10 min at 25 °C in refolding buffer and centrifuged in a Beckman Optima ultracentrifuge at 95,600 × g for 30 min at 4 °C. MDH was denatured as described above and mixed with 1 μm DnaK or DnaK2A, 1.5 μm ClpB, and 0.05-5 μm DnaJ. The sample was incubated for 10 min at 30 °C in refolding buffer containing 3 mm ATP and centrifuged (76,000 × g, 30 min, 4 °C) (33Schlieker C. Tews I. Bukau B. Mogk A. FEBS Lett. 2004; 578: 351-356Crossref PubMed Scopus (70) Google Scholar). The procedure used with G6PDH was the same as described above for its refolding but in the absence of GrpE. Aggregated G6PDH (1 μm), DnaK (6.4 μm), and DnaJ (0.05–10 μm) were incubated for 10 min at 25 °C in refolding buffer containing 3 mm ATP. Protein complexes were centrifuged at 76,000 × g to separate aggregate-bound from free chaperones. The resulting pellets and controls, containing known amounts of native proteins, were analyzed by SDS-PAGE (12.5%). Commercial ADP was further purified by ion exchange chromatography to remove contaminating ATP (35Horst M. Oppliger W. Feifel B. Schatz G. Glick B.S. Protein Sci. 1996; 5: 759-767Crossref PubMed Scopus (77) Google Scholar).The amount of aggregate-bound proteins was quantitated by densitometry of the gel bands with a gel scanner G-800 and the Quantity One software from Bio-Rad. Each data point is an average of at least two experiments and was estimated by subtracting the amount of the corresponding protein in pellets of control experiments.RESULTSDnaJ Mediates DnaK Interaction with Protein Aggregates—It has been suggested that the DnaK system functions upstream of ClpB to reactivate protein aggregates. However, how the components of this system interact with protein aggregates remains as yet unknown. To address this question, the interaction of DnaK and DnaJ (the only components of the system that can bind client proteins) with aggregates of luciferase (Fig. 1A) and of MDH (Fig. 1B) has been analyzed. Experiments show that DnaK is unable on its own to significantly interact with aggregates of either protein substrates, but it binds in the presence of DnaJ and ATP. In contrast, the co-chaperone binds to the aggregates in the absence of DnaK, suggesting that the interaction of the chaperone with aggregate-bound DnaJ drives its association with protein aggregates. Since reactivation of MDH aggregates requires the combined action of the DnaK system and ClpB, we also tested whether ClpB had any influence on the way the DnaK system binds to aggregates. A similar analysis to that shown in Fig. 1B but carried out in the presence of ClpB indicates that the Hsp100 bacterial homolog does not modify the DnaJ-dependence of DnaK binding to protein aggregates (Fig. 1B). As expected for an interaction that depends on ATP to occur, DnaJ-mediated association of DnaK to protein aggregates is not observed for the ADP-bound or apo-forms of the chaperone (Fig. 1, C and D).Two factors might affect the association of chaperones with protein aggregates, namely the time the protein substrate spends under denaturing and reactivation conditions. The first one (i.e. the time at denaturing temperatures) determines both the size of the aggregate and the conformational properties of the denatured protein molecules within the aggregate. These properties have been related to the ability of different combination of chaperones to reactivate protein aggregates (36Ben Zvi A.P. Goloubinoff P. J. Struct. Biol. 2001; 135: 84-93Crossref PubMed Scopus (197) Google Scholar, 37Lewandowska A. Matuszewska M. Liberek K. J. Mol. Biol. 2007; 371: 800-811Crossref PubMed Scopus (22) Google Scholar) and might influence chaperone binding to the aggregate. Special care has been taken to denature and aggregate protein substrates under the same experimental conditions (protein concentration, temperature, and incubation time) to obtain samples that, after cooling or diluting them from denaturant-containing solutions, maintain homogeneous size and secondary structure (data not shown). Therefore, chaperones bind to stable protein aggregates that do not evolve with time. The second one (i.e. the effect of the incubation time on the association reaction) has been analyzed by performing binding experiments at different incubation times. The results (not shown) indicate that the system reaches a binding equilibrium within the first 10 min of incubation in the presence of ATP. It should be noted that less than 10% of the nucleotide is hydrolyzed during the initial incubation at 25 °C and subsequent centrifugation at 4 °C. Long incubation times might change the equilibrium as the amount of ATP hydrolyzed increases.If the above finding holds for DnaK and DnaJ binding to different protein aggregates, the amount of aggregate-bound chaperone should be sensitive to co-chaperone concentration. To test this hypothesis, binding experiments at constant DnaK and protein aggregate concentrations and increasing DnaJ amounts were carried out. When this type of experiments is performed with aggregates of MDH (Fig. 2) and of G6PDH (Fig. 3), the amount of aggregate-bound DnaK increases with DnaJ concentration, indicating that the cochaperone binds to the aggregate surface in a concentration-dependent manner and that association of DnaK depends on previous DnaJ binding. If DnaK could bind aggregates by itself, a constant aggregate-bound chaperone concentration should be observed, and this is clearly not the case. Data shown in Figs. 2 and 3 also reveal that: (i) the concentration of aggregate-associated DnaK increases exponentially with co-chaperone concentration up to 1 μm DnaJ and slightly decreases at higher co-chaperone concentrations (Figs. 2B and 3B); (ii) in contrast to DnaK and DnaJ, the interaction of ClpB with MDH aggregates is not sensitive to co-chaperone concentration (Fig. 2B); and (iii) the reactivation yield of both client proteins shows the same DnaJ concentration dependence observed for DnaK binding to their aggregates (Figs. 2C and 3C). These data indicate that DnaK binds to the client protein aggregate once the cochaperone is bound at the aggregate surface.FIGURE 2Interaction of chaperones with MDH aggregates. A, SDS-PAGE (12.5%) analysis of the aggregate-associated chaperones. DnaK (1 μm), ClpB (1.5 μm), and aggregated MDH (1 μm) were incubated with varying amounts of DnaJ (0.05–5 μm) in the presence of 3 mm ATP and centrifuged as described under “Experimental Procedures.” The resulting pellets were analyzed by electrophoresis. The lane marked with an asterisk contains 20% (DnaK), 100% (DnaJ; 1 μm), 10% (ClpB), and 50% (MDH) of the initially added protein. Lanes 0 show proteins that sediment in the presence of aggregate (A) or native (N) MDH and in the absence of DnaJ. B, estimation of the amount of aggregate-bound chaperones. Shown is intensity of the bands corresponding to DnaK (filled circles), DnaJ (empty circles), and ClpB (triangles) relative to that of aggregated MDH. The concentration of each protein was estimated by comparing the intensity of the bands in each lane with those of the control mentioned above. C, chaperone-mediated reactivation yields (squares) of the same samples analyzed in A and B, but in the presence of 0.25 μm GrpE, after a reactivation period of 2 h. The aggregate-bound DnaK/DnaJ molar ratios (filled triangles) were obtained from data shown in B.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Interaction of chaperones with G6PDH aggregates. A, SDS-PAGE (12.5%) analysis of aggregate-bound chaperones. DnaK (6.4 μm) and aggregated G6PDH (1 μm) were incubated with increasing amounts of DnaJ (0.05–5 μm) in the presence of 3 mm ATP and centrifuged as described under “Experimental Procedures.” The resulting pellets were analyzed by electrophoresis. The lane marked with an asterisk contains 10% (DnaJ; 1 μm), 20% (DnaK), and 100% (G6PDH) of the initially added protein. Lanes 0 show proteins that sediment in the presence of aggregate (A) or native (N) G6PDH and in the absence of DnaJ. B, estimation of the amount of aggregate-bound chaperones. DnaK (filled circles) and DnaJ (empty circles) concentrations were estimated as in Fig. 2. C, chaperone-mediated reactivation yields (squares) of the same samples analyzed in A and B but in the presence of 0.25 μm GrpE, measured after a reactivation period of 2 h. The aggregate-bound DnaK/DnaJ molar ratios (filled triangles) were estimated from data shown in B.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The estimated aggregate-bound DnaK/DnaJ molar ratio increases at DnaJ concentrations below 0.05 μm and exponentially decreases above this cochaperone concentration for both substrate proteins (Figs. 2C and 3C). If DnaK would interact only with DnaJ at the aggregate surface, this value could not be higher than 1, in contrast to what is experimentally observed. This suggests that during the functional cycle, DnaK molecules bound to the DnaJ-aggregate complex are transferred to the aggregate. The comparison of the reactivation yields and the estimated aggregate-bound DnaK/DnaJ molar ratios also indicates that the values that provide the highest reactivation yields are between 3 and 1 for MDH and between 4 and 2 for G6PDH. It should be noted that the percentage of aggregate-bound DnaK is at most 24 and 16% of the initially added protein for MDH and G6PDH, respectively.A DnaK Mutant with Impaired Affinity for DnaJ Shows Defective Binding to Protein Aggregates and Reactivation Yields—To further demonstrate that the ability of DnaK to interact with and reactivate protein aggregates depends on DnaJ, the association of WT DnaK and DnaK2A (Fig. 4A) with DnaJ and protein aggregates has been characterized. This DnaK mutant has two amino acid replacements (D540A and K548A) located in the “latch” region that have a small effect on (i) protein conformation, (ii) peptide association kinetics, (iii) protein and peptide-protein thermal stability, and (iv) interdomain communication. However, DnaK2A was unable to refold luciferase (26Fernandez-Saiz V. Moro F. Arizmendi J.M. Acebron S.P. Muga A. J. Biol. Chem. 2006; 281: 7479-7488Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar).FIGURE 4A, schematic diagram of the SBD of DnaK. The ionic contacts disrupted by the mutations and the bound substrate are indicated. B, stimulation of the ATPase activity of WT DnaK and DnaK2A by DnaJ. The steady-state ATPase activity of WT DnaK (filled circles) and DnaK2A (squares) was measured at 30 °C at increasing DnaJ concentrations (0.05–4 μm), as described under “Experimental Procedures.” C, interaction of DnaK and DnaK2A with DnaJ monitored by SPR. Experimental interaction curve of WT DnaK (solid line) and DnaK2A (broken line) with DnaJ. The interaction between Hsp70 (4 μm) and the co-chaperone was analyzed in the presence of 40 μm ATP in the running buffer. D, analysis of the interaction with DnaJ. Apparent association constants (Kon obs) for WT DnaK (filled circles) and DnaK2A (squares) as a function of Hsp70 concentration. Kinetic constants k+1 and k–1 are derived from the slopes and the y intercepts, respectively (shown in Table 1).View Large Image Fi" @default.
• W1999121105 created "2016-06-24" @default.
• W1999121105 creator A5003147872 @default.
• W1999121105 creator A5006643494 @default.
• W1999121105 creator A5025919450 @default.
• W1999121105 creator A5057805699 @default.
• W1999121105 creator A5065307612 @default.
• W1999121105 date "2008-01-01" @default.
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• W1999121105 title "DnaJ Recruits DnaK to Protein Aggregates" @default.
• W1999121105 cites W1525754348 @default.
• W1999121105 cites W1525893433 @default.
• W1999121105 cites W1541948779 @default.
• W1999121105 cites W1575935179 @default.
• W1999121105 cites W1601050661 @default.
• W1999121105 cites W1968729829 @default.
• W1999121105 cites W1971749357 @default.
• W1999121105 cites W1982245562 @default.
• W1999121105 cites W1989350796 @default.
• W1999121105 cites W1990568024 @default.
• W1999121105 cites W1991862791 @default.
• W1999121105 cites W1992687906 @default.
• W1999121105 cites W1996623614 @default.
• W1999121105 cites W1997718936 @default.
• W1999121105 cites W1998018574 @default.
• W1999121105 cites W2002074105 @default.
• W1999121105 cites W2012129187 @default.
• W1999121105 cites W2016396087 @default.
• W1999121105 cites W2016467855 @default.
• W1999121105 cites W2019012009 @default.
• W1999121105 cites W2020496041 @default.
• W1999121105 cites W2024243591 @default.
• W1999121105 cites W2032780814 @default.
• W1999121105 cites W2036629609 @default.
• W1999121105 cites W2041423372 @default.
• W1999121105 cites W2041660879 @default.
• W1999121105 cites W2044343350 @default.
• W1999121105 cites W2048399782 @default.
• W1999121105 cites W2051323334 @default.
• W1999121105 cites W2051563344 @default.
• W1999121105 cites W2051941413 @default.
• W1999121105 cites W2052843890 @default.
• W1999121105 cites W2055652827 @default.
• W1999121105 cites W2057945108 @default.
• W1999121105 cites W2060303895 @default.
• W1999121105 cites W2064123540 @default.
• W1999121105 cites W2068357721 @default.
• W1999121105 cites W2070028258 @default.
• W1999121105 cites W2072744250 @default.
• W1999121105 cites W2073580695 @default.
• W1999121105 cites W2076945013 @default.
• W1999121105 cites W2084420022 @default.
• W1999121105 cites W2088305244 @default.
• W1999121105 cites W2092431461 @default.
• W1999121105 cites W2093983285 @default.
• W1999121105 cites W2096966529 @default.
• W1999121105 cites W2102925080 @default.
• W1999121105 cites W2109936231 @default.
• W1999121105 cites W2110829900 @default.
• W1999121105 cites W2114245165 @default.
• W1999121105 cites W2119999468 @default.
• W1999121105 cites W2121142142 @default.
• W1999121105 cites W2125071622 @default.
• W1999121105 cites W2138158290 @default.
• W1999121105 cites W2156447960 @default.
• W1999121105 cites W2158546889 @default.
• W1999121105 cites W42076805 @default.
• W1999121105 cites W70515239 @default.
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