Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Bag-1M Accelerates Nucleotide Release for Human Hsc70 and Hsp70 and Can Act Concentration-dependent as Positive and Negative Cofactor

2001; Elsevier BV; Volume: 276; Issue: 35 Linguagem: Inglês

10.1074/jbc.m105328200

ISSN

1083-351X

Autores

Claudia S. Gässler, Thomas Wiederkehr, Dirk Brehmer, Bernd Bukau, Matthias P. Mayer,

Tópico(s)

Toxin Mechanisms and Immunotoxins

Resumo

The cytosol of mammalian cells contains several Hsp70 chaperones and an arsenal of cochaperones, including the anti-apoptotic Bag-1M protein, which regulate the activities of Hsp70s by controlling their ATPase cycles. To elucidate the regulatory function of Bag-1M, we determined its influence on nucleotide exchange, substrate release, ATPase rate, and chaperone activity of the housekeeping Hsc70 and stress-inducible Hsp70 homologs of humans. Bag-1M and a C-terminal fragment of it are potent nucleotide exchange factors as they stimulated the ADP dissociation rate of Hsc70 and Hsp70 up to 900-fold. The N-terminal domain of Bag-1M decreased the affinity of Bag-1M for Hsc70/Hsp70 by 4-fold, indicating a modulating role of the N terminus in Bag-1M action as nucleotide exchange factor. Bag-1M inhibited Hsc70/Hsp70-dependent refolding of luciferase in the absence of Pi. Surprisingly, under physiological conditions, i.e. low Bag-1M concentrations and presence of Pi, Bag-1M activates the chaperone action of Hsc70/Hsp70 in luciferase refolding. Bag-1M accelerated ATP-triggered substrate release by Hsc70/Hsp70. We propose that Bag-1M acts as substrate discharging factor for Hsc70 and Hsp70. The cytosol of mammalian cells contains several Hsp70 chaperones and an arsenal of cochaperones, including the anti-apoptotic Bag-1M protein, which regulate the activities of Hsp70s by controlling their ATPase cycles. To elucidate the regulatory function of Bag-1M, we determined its influence on nucleotide exchange, substrate release, ATPase rate, and chaperone activity of the housekeeping Hsc70 and stress-inducible Hsp70 homologs of humans. Bag-1M and a C-terminal fragment of it are potent nucleotide exchange factors as they stimulated the ADP dissociation rate of Hsc70 and Hsp70 up to 900-fold. The N-terminal domain of Bag-1M decreased the affinity of Bag-1M for Hsc70/Hsp70 by 4-fold, indicating a modulating role of the N terminus in Bag-1M action as nucleotide exchange factor. Bag-1M inhibited Hsc70/Hsp70-dependent refolding of luciferase in the absence of Pi. Surprisingly, under physiological conditions, i.e. low Bag-1M concentrations and presence of Pi, Bag-1M activates the chaperone action of Hsc70/Hsp70 in luciferase refolding. Bag-1M accelerated ATP-triggered substrate release by Hsc70/Hsp70. We propose that Bag-1M acts as substrate discharging factor for Hsc70 and Hsp70. N 8-(4-N′-methylanthraniloylaminobutyl)-ADP/ATP dansyl-NRLLLTGC 5-dimethylaminonaphthalene-1-sulfonyl tritiated, reduced carboxymethylated α-lactalbumin The Hsp70 proteins are involved in folding processes throughout the entire life span of proteins. The molecular basis of their functions is the transient interaction with substrates through ATP-controlled cycles (1Bukau B. Horwich A.L. Cell. 1998; 92: 351-366Abstract Full Text Full Text PDF PubMed Scopus (2435) Google Scholar, 2Hartl F.U. Nature. 1996; 381: 571-580Crossref PubMed Scopus (3130) Google Scholar). In the ATP state Hsp70s have a low affinity for substrates, and in the ADP state the affinity for substrates is high (3Schmid D. Baici A. Gehring H. Christen P. Science. 1994; 263: 971-973Crossref PubMed Scopus (422) Google Scholar, 4Takeda S. McKay D.B. Biochemistry. 1996; 35: 4636-4644Crossref PubMed Scopus (75) Google Scholar). ATP hydrolysis was shown to be stimulated synergistically by the simultaneous interaction with a substrate and a cochaperone of the DnaJ family (5Barouch W. Prasad K. Greene L. Eisenberg E. Biochemistry. 1997; 36: 4303-4308Crossref PubMed Scopus (58) Google Scholar, 6Karzai A.W. McMacken R. J. Biol. Chem. 1996; 271: 11236-11246Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 7Laufen T. Mayer M.P. Beisel C. Klostermeier D. Reinstein J. Bukau B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5452-5457Crossref PubMed Scopus (475) Google Scholar, 8Misselwitz B. Staeck O. Rapoport T.A. Mol. Cell. 1998; 2: 593-603Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). Under physiological conditions nucleotide exchange is rate-limiting for substrate release (3Schmid D. Baici A. Gehring H. Christen P. Science. 1994; 263: 971-973Crossref PubMed Scopus (422) Google Scholar, 9Palleros D.R. Reid K.L. Shi L. Welch W.J. Fink A.L. Nature. 1993; 365: 664-666Crossref PubMed Scopus (347) Google Scholar, 10Theyssen H. Schuster H.-P. Bukau B. Reinstein J. J. Mol. Biol. 1996; 263: 657-670Crossref PubMed Scopus (199) Google Scholar). For this functional cycle the regulation by cochaperones is an important issue. How do they act in the ATPase cycle and do they exhibit specificity for a given Hsp70 partner? Significant influence on the ATPase cycle of Hsp70s was shown for members of the families of DnaJ and Bag proteins. The Bag family of proteins consists of six members that share a conserved sequence stretch of 40–50 residues at the C terminus (11Takayama S. Xie Z. Reed J.C. J. Biol. Chem. 1999; 274: 781-786Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar, 12Thress K. Song J. Morimoto R.I. Kornbluth S. EMBO J. 2001; 20: 1033-1041Crossref PubMed Scopus (89) Google Scholar). The C terminus of Bag-1 was also shown to be responsible for the interaction with the ATPase domain of Hsp70 proteins (11Takayama S. Xie Z. Reed J.C. J. Biol. Chem. 1999; 274: 781-786Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar, 13Höhfeld J. Jentsch S. EMBO J. 1997; 16: 6209-6216Crossref PubMed Scopus (339) Google Scholar, 14Lüders J. Demand J. Schönfelder S. Frien M. Zimmermann R. Höhfeld J. Biol. Chem. 1998; 379: 1217-1226Crossref PubMed Scopus (34) Google Scholar, 15Takayama S. Bimston D.N. Matsuzawa S.-I. Freeman B.C. Aime-Sempe C. Xie Z. Morimoto R.I. Reed J.C. EMBO J. 1997; 16: 4887-4896Crossref PubMed Scopus (439) Google Scholar, 16Zeiner M. Gebauer M. Gehring U. EMBO J. 1997; 16: 5483-5490Crossref PubMed Scopus (148) Google Scholar). The anti-apoptotic protein Bag-1 that exists in at least three translational initiation variants, Bag-1L, Bag-1M, and Bag-1S, received the most attention. Bag-1M was reported to induce ADP release by Hsc70 (13Höhfeld J. Jentsch S. EMBO J. 1997; 16: 6209-6216Crossref PubMed Scopus (339) Google Scholar) similar to GrpE, the nucleotide exchange factor for the DnaK-type Hsp70 homologs of bacteria. However, such an analogous function for GrpE was discussed controversially as Bag-1M did not accelerate the dissociation of Hsp70·ADP or Hsp70·ATP complexes (17Bimston D. Song J. Winchester D. Takayama S. Reed J.C. Morimoto R.I. EMBO J. 1998; 17: 6871-6878Crossref PubMed Scopus (154) Google Scholar). Whether Bag-1M inhibited or stimulated the refolding of denatured substrates by Hsp70s in vitro andin vivo was also controversial (15Takayama S. Bimston D.N. Matsuzawa S.-I. Freeman B.C. Aime-Sempe C. Xie Z. Morimoto R.I. Reed J.C. EMBO J. 1997; 16: 4887-4896Crossref PubMed Scopus (439) Google Scholar, 17Bimston D. Song J. Winchester D. Takayama S. Reed J.C. Morimoto R.I. EMBO J. 1998; 17: 6871-6878Crossref PubMed Scopus (154) Google Scholar, 18Nollen E.A.A. Brunsting J.F. Song J. Kampinga H.H. Morimoto R.I. Mol. Cell. Biol. 2000; 20: 1083-1088Crossref PubMed Scopus (113) Google Scholar, 19Terada K. Mori M. J. Biol. Chem. 2000; 275: 24728-24734Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). All studies agree that Bag-1M stimulates the steady-state ATPase activity of Hsp70 and Hsc70. However, the extent of the stimulation varied largely (2–40-fold), acting synergistic or additive to Hdj-1, the major heat-inducible DnaJ-protein in mammalian cytosol (13Höhfeld J. Jentsch S. EMBO J. 1997; 16: 6209-6216Crossref PubMed Scopus (339) Google Scholar, 16Zeiner M. Gebauer M. Gehring U. EMBO J. 1997; 16: 5483-5490Crossref PubMed Scopus (148) Google Scholar, 17Bimston D. Song J. Winchester D. Takayama S. Reed J.C. Morimoto R.I. EMBO J. 1998; 17: 6871-6878Crossref PubMed Scopus (154) Google Scholar). In order to resolve these contradictions we investigated the influence of Bag-1M on nucleotide exchange, substrate release, ATPase, and chaperone activity of two cytosolic and nuclear Hsp70 homologs of human cells, Hsc70 and Hsp70, under identical conditions. Hsc70, Hsp70, Bag-1M, and Bag-1M-(151–274) were recombinantly expressed inΔdnaK52 Escherichia coli cells. Hsc70 and Hsp70 were purified according to the DnaK protocol (20Buchberger A. Schröder H. Büttner M. Valencia A. Bukau B. Nat. Struct. Biol. 1994; 1: 95-101Crossref PubMed Scopus (113) Google Scholar). Bag-1M was purified as described (13Höhfeld J. Jentsch S. EMBO J. 1997; 16: 6209-6216Crossref PubMed Scopus (339) Google Scholar); Bag-1M-(151–274) was purified via DEAE-cellulose and SP-Sepharose columns (Amersham Pharmacia Biotech). Hdj-1 was expressed in E. coli and purified according to the DnaJ protocol (21Schönfeld H.-J. Schmidt D. Zulauf M. Progr. Colloid Polym. Sci. 1995; 99: 7-10Crossref Google Scholar). Steady-state ATP hydrolysis rates were determined as described for DnaK (22Liberek K. Marszalek J. Ang D. Georgopoulos C. Zylicz M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2874-2878Crossref PubMed Scopus (693) Google Scholar, 23Mayer M.P. Laufen T. Paal K. McCarty J.S. Bukau B. J. Mol. Biol. 1999; 289: 1131-1144Crossref PubMed Scopus (115) Google Scholar, 24McCarty J.S. Buchberger A. Reinstein J. Bukau B. J. Mol. Biol. 1995; 249: 126-137Crossref PubMed Scopus (349) Google Scholar). Reactions were performed at 30 °C in mixtures containing buffer HKM (25 mm HEPES-KOH, pH 7.6, 50 mm KCl, 5 mm MgCl2), 1 µm Hsc70 or Hsp70, 2 µm Hdj-1, 0–17.5 µm Bag-1M, 250 µm ATP, and 0.1 µCi of [α-32P]ATP (Amersham Pharmacia Biotech). Quantification was performed as described (23Mayer M.P. Laufen T. Paal K. McCarty J.S. Bukau B. J. Mol. Biol. 1999; 289: 1131-1144Crossref PubMed Scopus (115) Google Scholar). Hsc70 (1 µm) and cochaperones (Hdj-1, 0.2 µm; Bag-1 as indicated) were incubated in HKM buffer supplemented with 5 mmdithiothreitol and 2 mm ATP for 5 min at 20 °C. Firefly luciferase (70 µm in 1 m glycylglycine, pH 7.4; Roche Molecular Biochemicals) was added to 0.1 µmand denatured for 20 min at 42 °C. The refolding reaction (20 µl) was started by shifting back the temperature to 30 °C and addition of 5% rabbit reticulocyte lysate (Promega) and 2.5 mm ATP. At the indicated times, 1-µl aliquots were diluted into 125 µl of assay buffer and analyzed for bioluminescence activity in a Biolumat (Berthold, Bad Wildbad, Germany) as described (25Schröder H. Langer T. Hartl F.-U. Bukau B. EMBO J. 1993; 12: 4137-4144Crossref PubMed Scopus (501) Google Scholar). The enzymatic activity of native luciferase diluted in refolding buffer containing Hsc70 (1 µm) and Hdj-1 (0.2 µm) and incubated at 30 °C was set to 100%. To determine the dissociation rates (k off) of ADP/ATP, the fluorescent labeled ADP/ATP analogsN 8-(4-N′-methylanthraniloylaminobutyl)-ADP/ATP (MABA-ADP/ATP)1 (10Theyssen H. Schuster H.-P. Bukau B. Reinstein J. J. Mol. Biol. 1996; 263: 657-670Crossref PubMed Scopus (199) Google Scholar) were used. Hsc70/Hsp70 (0.5 µm) and MABA-ADP (0.5 µm) in HKM buffer were preincubated for 30 min at 30 °C and mixed 1:1 in a stopped-flow device (SX-18M Applied Photophysics, Surrey, UK) with a solution of 250 µmADP ± Bag-1M (0–90 µm). Fluorescence (excitation, 360 nm; cut-off filter at 420 nm) was measured for 1–200 s at 30 °C, and the time-dependent decrease was fitted to a single exponential decay. Where indicated 10 mmPi was added. ATP association was measured by mixing 0.25 µm MABA-ATP with different amounts of Hsc70/Hsp70. For the determination of ATP dissociation, MABA-ATP was mixed with Hsc70/Hsp70 for 30 s and subsequently mixed with unlabeled ATP ± Bag-1M in double-mixing experiments. To determine protein substrate release3H-labeled RCLMA was used. 10 µm Hsp70 or Hsc70 and 0.3 µm3H-RCLMA ± 20 µm Bag-1M were preincubated in 20 µl of T buffer (25 mm Tris, pH 7.8, 200 mm KCl, 5% glycerol, 0.05% Tween 20) for 1 h at 30 °C, ± 50 µmunlabeled RCLMA and ± 2.5 mm ATP were added, incubated for 1 min at 30 °C, and separated via a Superdex 200 gel filtration column (Amersham Pharmacia Biotech). To determine peptide substrate k off dansyl chloride (Molecular Probes)-labeled NR peptide (NRLLLTGC) was used (D-NR). Hsp70 (5 µm) and D-NR (2 µm) were preincubated in HKM buffer for 30 min at 30 °C and mixed 1:1 in a stopped-flow device (SX-18M Applied Photophysics, Surrey, UK) with 250 µm unlabeled NR ± 0.5 mm ATP ± 5 µm Bag-1M. Fluorescence (excitation, 334 nm; cut-off filter at 385 nm) was measured for 1–1000 s at 30 °C. To determine the rate constants a single or double exponential decay function was fitted to the time-dependent decrease in fluorescence. To determine the domain structure of Bag-1M a partial proteolytic digest with trypsin was performed. 18 µg of Bag-1M were incubated with 0.01 µg of trypsin at 30 °C for 0–30 min. The reaction was stopped by the addition of SDS sample buffer and incubation at 95 °C for 5 min. For analysis a 13% SDS gel was used. We wanted to investigate the effects of Bag-1M on nucleotide exchange of Hsc70 and Hsp70 by directly measuring nucleotide release. As nucleotide exchange is a rapid process for Hsc70, we intended to use the fluorescent ADP and ATP analogs MABA-ADP/ATP and stopped-flow instrumentation. These analogs were shown previously to have similar kinetic properties as their authentic counterparts in case of the E. coli homolog DnaK (10Theyssen H. Schuster H.-P. Bukau B. Reinstein J. J. Mol. Biol. 1996; 263: 657-670Crossref PubMed Scopus (199) Google Scholar). To verify the suitability of MABA-ADP and MABA-ATP for measuring Hsc70 and Hsp70 nucleotide exchange kinetics, we determined the mutual exchange rates of MABA-ADPversus ADP and MABA-ATP versus ATP. ADP and ATP exchange rates were independently measured using the intrinsic fluorescence of the single tryptophan that was shown to change in response to ATP binding (26Ha J.-H. McKay D.B. Biochemistry. 1995; 34: 11635-11644Crossref PubMed Scopus (74) Google Scholar, 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 (224) Google Scholar). The measured rates were very similar, and the fluorescent analogs were therefore suitable for the nucleotide exchange experiments. The MABA-ADP k off values determined for Hsc70 at 30 °C (0.24 s−1) were approximately 10-fold higher than published values for ADP release at 25 °C (TableI(28Ha J.-H. Hellman U. Johnson E.R. Li L. McKay D.B. Sousa M.C. Takeda S. Wernstedt C. Wilbanks S.M. J. Biol. Chem. 1997; 272: 27796-27803Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar)). Hsp70 had nearly identical MABA-ADP dissociation rates as Hsc70. For both chaperones, the addition of physiological concentrations of Pi (10 mm) decreased the dissociation rate by 5-fold (Table I).Table IAssociation and dissociation rates of MABA-ADP and MABA-ATPHsc70Hsp70k offADP(s−1)0.240.3k offADP+Pi(s−1)0.0460.05k off1ATP(s−1)0.20.78k off2ATP(s−1)0.010.06k onATP (m−1s−1)4.9 × 1051-aMeasured by tryptophan fluorescence at 18 °C, k on1 for MABA-ATP was 6.9 × 105m−1 s−1 at 30 °C.1.1 × 106k 2ATP(s−1)0.21-aMeasured by tryptophan fluorescence at 18 °C, k on1 for MABA-ATP was 6.9 × 105m−1 s−1 at 30 °C.ND1-bND, not determined.Dissociation of MABA-ADP was determined by rapidly mixing complexes of MABA-ADP and Hsc70 or Hsp70 with an excess of unlabeled ADP in a stopped-flow device. For the determination of the MABA-ATP dissociation rate, MABA-ATP was first mixed with the nucleotide-free Hsc70/Hsp70 for 30 s and subsequently with an excess of unlabeled ATP in double mixing experiments. Association of MABA-ATP was measured by mixing MABA-ATP or unlabeled ATP with different concentrations of nucleotide-free Hsc70 or Hsp70. Single and double exponential equations were fitted to the change of fluorescence.1-a Measured by tryptophan fluorescence at 18 °C, k on1 for MABA-ATP was 6.9 × 105m−1 s−1 at 30 °C.1-b ND, not determined. Open table in a new tab Dissociation of MABA-ADP was determined by rapidly mixing complexes of MABA-ADP and Hsc70 or Hsp70 with an excess of unlabeled ADP in a stopped-flow device. For the determination of the MABA-ATP dissociation rate, MABA-ATP was first mixed with the nucleotide-free Hsc70/Hsp70 for 30 s and subsequently with an excess of unlabeled ATP in double mixing experiments. Association of MABA-ATP was measured by mixing MABA-ATP or unlabeled ATP with different concentrations of nucleotide-free Hsc70 or Hsp70. Single and double exponential equations were fitted to the change of fluorescence. We used MABA-ADP to determine directly the effects of Bag-1M on nucleotide release from Hsc70 and Hsp70. As evidenced by the differential decrease in fluorescence signal, Bag-1M stimulated the off-rate of MABA-ADP for Hsc70 (Fig. 1A) and Hsp70 (not shown) in the absence of Pi. Titrating the Bag-1M concentration and plotting the measured off-rates against the Bag-1M concentration resulted in a hyperbolic curve with an apparentK d and a maximal rate of 1.8 µm and 23 s−1 for Hsp70 and 3.9 µm and 24 s−1 for Hsc70, respectively (Fig. 1, B andC). This clearly demonstrates that Bag-1M is a nucleotide exchange factor for both cytosolic Hsp70 proteins investigated, stimulating the off-rate of MABA-ADP by up to 100-fold. Surprisingly, higher concentrations of Bag-1M were needed to reach similar stimulatory effects in the presence of physiological concentrations of Pi (10 mm). Under these conditions we were not able to reach saturation with the Bag-1M concentrations used, and the data could not be fitted by a hyperbolic function. Due to the 5-fold decreased basal k offfor ADP in the presence of Pi, the highest stimulation factors reached were 690- and 460-fold over the basal rate for Hsc70 and Hsp70, respectively. In order to compare these data with the previously studied E. coli DnaK system (29Packschies L. Theyssen H. Buchberger A. Bukau B. Goody R.S. Reinstein J. Biochemistry. 1997; 36: 3417-3422Crossref PubMed Scopus (155) Google Scholar), we performed a similar titration using the E. coli GrpE to measure DnaK-MABA-ADP dissociation. The data obtained were almost identical to those published (29Packschies L. Theyssen H. Buchberger A. Bukau B. Goody R.S. Reinstein J. Biochemistry. 1997; 36: 3417-3422Crossref PubMed Scopus (155) Google Scholar) demonstrating that Pi does not affect the GrpE-stimulated ADP dissociation (not shown). We also investigated the association and dissociation of ATP using either MABA-ATP or tryptophan fluorescence. For both proteins the association and dissociation of ATP occurred in a two-step process as previously published for Hsc70 (26Ha J.-H. McKay D.B. Biochemistry. 1995; 34: 11635-11644Crossref PubMed Scopus (74) Google Scholar). MABA-ATP associated with Hsp70 with a 2-fold higher rate than with Hsc70. ATP association measured via tryptophan fluorescence yielded within the error range identical values (Table I). MABA-ATP dissociated from Hsp70 also in a two-step mechanism with the faster rate being 0.78 s−1 which is four times faster than the dissociation rate for MABA-ATP from Hsc70 (Table I). The slower rate, which most likely corresponds to an isomerization step that precedes dissociation, was for Hsp70 (0.06 s−1) even six times faster than for Hsc70 (0.01 s−1). The presence of Bag-1M even at high concentrations did not increase the apparent association and dissociation rates of MABA-ATP for either Hsp70 or Hsc70. The potent nucleotide exchange factor Bag-1M therefore differs from GrpE in as much as Pi influences the concentration necessary for half-maximal stimulation, and that Bag-1M discriminates between bound ADP and bound ATP. To identify the domain within Bag-1M that is responsible for the observed stimulation of the ADP dissociation by Hsc70 and Hsp70, we attempted to determine the domain structure of Bag-1M biochemically using partial proteolytic digestion. Tryptic digestion of Bag-1M produced a very stable 20-kDa fragment consisting of the C-terminal 124 residues (151) as determined by N-terminal sequencing and molecular weight (Fig. 2). We cloned and purified the corresponding fragment (Bag-1M-(151–274)) and investigated its effect on ADP dissociation by Hsc70/Hsp70. Previous work used sequence homology criteria to derive a Bag-1 fragment that was slightly longer (Bag-1M-(129–274) (14Lüders J. Demand J. Schönfelder S. Frien M. Zimmermann R. Höhfeld J. Biol. Chem. 1998; 379: 1217-1226Crossref PubMed Scopus (34) Google Scholar)). The recently published crystal structure of a Bag-1 fragment in complex with Hsc70 as well as the recently published NMR structure of Bag-1 used fragments that were almost identical to our fragment (30Briknarova K. Takayama S. Brive L. Havert M.L. Knee D.A. Velasco J. Homma S. Cabezas E. Stuart J. Hoyt D.W. Satterthwait A.C. Llinas M. Reed J.C. Ely K.R. Nat. Struct. Biol. 2001; 8: 349-352Crossref PubMed Scopus (135) Google Scholar, 31Sondermann H. Scheufler C. Scheider C. Höhfeld J. Hartl F.-U. Moarefi I. Science. 2001; 291: 1553-1557Crossref PubMed Scopus (364) Google Scholar). Bag-1M-(151–274) catalyzed the ADP release by both Hsp70 proteins even more efficiently than the full-length Bag-1M protein. This was particularly apparent in the presence of Pi as demonstrated in Fig. 1 D for Hsc70. A hyperbolic function could be fitted to the data yielding K d values of 1 and 17 µm and a maximal rate of 20 and 42 s−1 (83- and 913-fold stimulation over basal dissociation rate) in the absence and presence of Pi, respectively. Comparison of theK d values with the value for full-length Bag-1M in absence of Pi (3.9 µm) revealed that the apparent affinity of the Bag-1M fragment was almost 4-fold higher. If the same were valid in the presence of Pi theK d for the full-length Bag-1M protein would be in the order of 70 µm. This would explain why these data points (see Fig. 1 B) seem to follow a linear dependence. The titration curves for Hsp70 were very similar (not shown). Taken together, the Bag-1 fragment stimulates nucleotide release by Hsp70 and Hsc70 even more efficiently than full-length Bag-1M. After having analyzed the influence of Bag-1M on nucleotide association and dissociation, we investigated its effect on the ATPase cycle. Measurement of the intrinsic ATPase rates yielded 2.48·10−3 s−1 and 4.5·10−4s−1 for Hsc70 and Hsp70, respectively. This is much slower than the ADP dissociation and the ATP association rates indicating that for both proteins ATP hydrolysis is rate-limiting for the ATPase cycle consistent with earlier publications for Hsc70 (28Ha J.-H. Hellman U. Johnson E.R. Li L. McKay D.B. Sousa M.C. Takeda S. Wernstedt C. Wilbanks S.M. J. Biol. Chem. 1997; 272: 27796-27803Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Under these conditions Bag-1M should not have any effect on the steady-state ATPase rate. The DnaJ-protein Hdj-1 stimulated both Hsp70 proteins up to a maximal steady-state ATPase rate of 0.008 and 0.003 s−1for Hsc70 and Hsp70, respectively. Under single turnover conditions increasing concentrations of Hdj-1 stimulated the ATPase activity of both Hsp70 proteins to much higher rates of more than 0.03 s−1 (not shown), demonstrating that in the presence of Hdj-1 ATP hydrolysis is not more rate-limiting for the ATPase cycle. Therefore, in the presence of Hdj-1, acceleration of nucleotide exchange by Bag-1M should stimulate the overall steady-state ATPase rate. We therefore determined the steady-state ATPase rates for Hsc70 and Hsp70 in the presence of high concentrations of Hdj-1 (2 µm) and titrated the Bag-1M concentration (1–15 µm). In the case of Hsc70, Bag-1M stimulated the ATPase rate as expected up to 2-fold over the rate in the absence of Bag-1M which is 20-fold over the basal rate (Fig. 3). These data are consistent with previously published data (13Höhfeld J. Jentsch S. EMBO J. 1997; 16: 6209-6216Crossref PubMed Scopus (339) Google Scholar). Surprisingly, in the case of Hsp70 a cooperative stimulation of the ATPase activity by Hdj-1 and Bag-1 was only observed at very high concentrations of Bag-1M despite the fact that nucleotide release from Hsp70 was stimulated by Bag-1M at least as efficiently as for Hsc70 (Fig. 3). Experiments using the C-terminal fragment of Bag-1M yielded similar results except that lower concentrations of the fragment compared with the full-length protein were necessary for the stimulation and that higher maximal rates were observed (not shown). These experiments demonstrate that Hdj-1 and Bag-1M cooperatively stimulate the ATPase cycles of Hsc70 and Hsp70 with marked differences in the required Bag-1M concentrations for the two Hsp70 homologs. The activity of Bag-1M to stimulate nucleotide dissociation from Hsc70 and Hsp70 conceivably allows ATP to bind to the nucleotide-free form of the chaperone and, consequently, is expected to trigger the release of any bound substrate. However, based on native gel electrophoresis data, Bag-1M was proposed to uncouple nucleotide exchange and substrate release (17Bimston D. Song J. Winchester D. Takayama S. Reed J.C. Morimoto R.I. EMBO J. 1998; 17: 6871-6878Crossref PubMed Scopus (154) Google Scholar). We used gel filtration to determine the amount of complex of Hsc70 or Hsp70 with tritiated, reduced carboxymethylated α-lactalbumin ([3H]RCMLA) as substrate after addition of ATP in the absence or presence of Bag-1M. Unlabeled RCMLA was added together with ATP to prevent rebinding of the tritiated substrate. 1 min after the addition of ATP the amount of Hsc70- or Hsp70-bound [3H]RCMLA was significantly reduced (Fig. 4). This reduction was enhanced and not prevented by Bag-1M. However, when the quench RCMLA was omitted a significant amount of [3H]RCMLA was found in complex with Hsc70 and Hsp70, indicating rebinding of the released substrate. To resolve the influence of Bag-1M on the dissociation of the Hsc70·substrate complex kinetically, we used the fluorescent labeled peptide substrate D-NR (dansyl-NRLLLTGC). This peptide bound to Hsp70·ADP with high affinity, and binding was detectable by an increase of the dansyl fluorescence at 526 nm. Mixing the complex of D-NR and Hsp70·ADP with an excess of unlabeled peptide led to a decrease in fluorescence at a rate of 3.9·10−3s−1 which is consistent with substrate dissociation rates measured for peptides and other Hsp70 homologs in the ADP state (3Schmid D. Baici A. Gehring H. Christen P. Science. 1994; 263: 971-973Crossref PubMed Scopus (422) Google Scholar, 4Takeda S. McKay D.B. Biochemistry. 1996; 35: 4636-4644Crossref PubMed Scopus (75) Google Scholar,32Pierpaoli E.V. Gisler S.M. Christen P. Biochemistry. 1998; 37: 16741-16748Crossref PubMed Scopus (61) Google Scholar, 33Mayer M.P. Schröder H. Rüdiger S. Paal K. Laufen T. Bukau B. Nat. Struct. Biol. 2000; 7: 586-593Crossref PubMed Scopus (311) Google Scholar). The addition of ATP to the Hsp70·ADP·D-NR complex accelerated the dissociation of D-NR to 0.43 s–1 which is similar to the ADP dissociation rate, indicating that nucleotide exchange is rate-limiting for substrate release under these conditions. Addition of Bag-1M in the absence of ATP did not influence the dissociation rate of the peptide. However, in the presence of ATP, Bag-1M accelerated the dissociation of the peptide significantly to 2.48 s–1 (Fig. 5). This value is identical to the dissociation rate of the Hsp70·D-NR complex in the presence of ATP when nucleotide-free preparations of Hsp70 are used (Table II). This clearly demonstrates that Bag-1M accelerates substrate release by accelerating nucleotide exchange. Our data suggest that Bag-1M acts as a substrate unloading factor for Hsc70 and Hsp70.Table IIEffect of Bag-1M on the dissociation of the Hsp70·substrate complexHsp70/D-NRk offs−1Hsp70ADP2-aHsp70ADP is Hsp70 with bound ADP.3.9 × 10−32-bKinetics followed a single exponential curve.Hsp70ADP + Bag-13.85 × 10−32-bKinetics followed a single exponential curve.Hsp70ADP + ATP0.43/0.042-cKinetics followed a double exponential curve; both rates are given.Hsp70ADP + ATP/Bag-12.48/0.232-cKinetics followed a double exponential curve; both rates are given.Hsp70Nu2-dHsp70Nu is nucleotide-free Hsp70. + ATP2.46/0.202-cKinetics followed a double exponential curve; both rates are given.Hsc70·D-NR complexes were rapidly mixed in a stopped-flow instrument with unlabeled NR and Bag-1 ± ATP as indicated. Single and double exponential equations were fitted to the decrease of fluorescence.2-a Hsp70ADP is Hsp70 with bound ADP.2-b Kinetics followed a single exponential curve.2-c Kinetics followed a double exponential curve; both rates are given.2-d Hsp70Nu is nucleotide-free Hsp70. Open table in a new tab Hsc70·D-NR complexes were rapidly mixed in a stopped-flow instrument with unlabeled NR and Bag-1 ± ATP as indicated. Single and double exponential equations were fitted to the decrease of fluorescence. To analyze the regulatory influence of Hdj-1 and Bag-1M on the chaperone activity of Hsc70 and Hsp70, we measured refolding of heat-denatured firefly luciferase. For Hsc70 and Hsp70 we first titrated Hdj-1 to determine the optimal ratios for luciferase refolding (not shown). For both systems, Hsp70/Hdj-1 and Hsc70/Hdj-1, we then titrated Bag-1M in the absence and in the presence of Piand measured the initial rate of luciferase refolding. In the absence of Pi, Hsp70 and Hsc70 in combination with Hdj-1 refolded up to 60% of denatured luciferase within 1 h (Fig.6A). Addition of increasing concentrations of Bag-1M or Bag-1M-(151–274) decreased significantly the rate and the yield of luciferase refolding as previously observed by different laboratories (13Höhfeld J. Jentsch S. EMBO J. 1997; 16: 6209-6216Crossref PubMed Scopus (339) Google Scholar, 15Takayama S.

Referência(s)