©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Dissociation of ATP from hsp70 of Saccharomyces cerevisiae Is Stimulated by Both Ydj1p and Peptide Substrates (*)

Thomas Ziegelhoffer (§) , Pascual Lopez-Buesa (¶) , Elizabeth A. Craig(**)

From the (1) Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

hsp70 proteins of both eukaryotes and prokaryotes possess both ATPase and peptide binding activities. These two activities are crucial for the chaperone activity of hsp70 proteins. The activity of DnaK, the primary hsp70 of Escherichia coli, is modulated by the GrpE and DnaJ proteins. In the yeast Saccharomyces cerevisiae, the predominant cytosolic hsp70, Ssa1p, interacts with a DnaJ homologue, Ydj1p. In order to better understand the function of the Ssa1p/Ydj1p chaperone, the effects of polypeptide substrates and Ydj1p on Ssa1p ATPase activity were assessed using a combination of steady-state kinetic analysis and single turnover substrate hydrolysis experiments. Polypeptide substrates and Ydj1p both serve to stimulate ATPase activity of Ssa1p. The two types of effector are biochemically distinct, each conferring a characteristic K dependence on Ssa1p ATPase activity. However, in single turnover ATP hydrolysis experiments, both polypeptide substrates and Ydj1p destabilized the ATPSsa1p complex through a combination of accelerated hydrolysis of bound ATP and accelerated release of ATP from Ssa1p. The acceleration of ATP release by Ydj1p is a previously unidentified function of a DnaJ homologue. In the case of Ydj1p-stimulated Ssa1p, steady-state ATPase activity is increased less than 2-fold at physiological K concentrations, despite a 15-fold increase in the hydrolysis of bound ATP. The primary effect of Ydj1p appears to be to disfavor an ATP form of Ssa1p. On the other hand, peptide stimulation of Ssa1p ATPase activity was enhanced at physiological K concentrations, supporting the idea that cycles of ATP hydrolysis play an important role in the interaction of hsp70 with polypeptide substrates. The enhanced ATP dissociation caused by both polypeptide substrates and Ydj1p may play a role in the regulation of Ssa1p chaperone activity by altering the relative abundance of ATP- and ADP-bound forms.


INTRODUCTION

All cells respond to certain environmental stresses by inducing the expression of a stereotypical set of proteins known as heat shock proteins (1) . The most evolutionarily conserved of these proteins is known generically as hsp70. hsp70 is a multifunctional protein with a highly conserved N-terminal ATPase domain of 44 kDa followed by a somewhat less conserved peptide binding domain of 18 kDa and a variable domain at its extreme C terminus (2, 3, 4) . Genetic and biochemical experiments in Escherichia coli established the existence of two ``cohort'' proteins which modulate the activity of hsp70 in that organism: DnaJ and GrpE (5) . Numerous homologues of DnaJ and GrpE have since been found in other organisms. All DnaJ homologues are defined by the presence of a conserved motif homologous to the N-terminal domain of DnaJ in E. coli, but as a group, they are considerably more heterogeneous in both size and primary sequence than are hsp70s (6) . More divergent still are GrpE homologues, which have only been identified in prokaryotes (7) and mitochondria (8) to date.

Through the use of the genetic techniques available for the study of Saccharomyces cerevisiae, at least 10 hsp70 genes have been identified and studied in this simple eukaryote. Of these, six encode confirmed cytosolic proteins, products of two genetic subfamilies: SSA1-4 and SSB1-2(4) . The SSA subfamily is essential. At least one of the SSA gene products must be present in significant quantity to ensure viability. Loss of SSA function has pleiotropic effects, including perturbations in heat shock regulation and defects in protein localization (9, 10) . The SSB subfamily consists of two highly homologous genes whose products are part of the translation apparatus and appear to facilitate protein synthesis (11) . The SSE subfamily ( SSE1 is identical to the recently isolated MSI3 gene) is a recently identified set of two closely related genes whose products appear to be involved in calmodulin- and cAMP-dependent signaling pathways (12, 13) and may represent another cytosolic hsp70 subfamily. There is every reason to expect that similarly extensive hsp70 families also exist in other eukaryotes.

Recent work in several laboratories has established hsp70 as a molecular chaperone, a member of an expanding family of proteins whose function is to assist in the proper folding of polypeptides. The most complete understanding of hsp70 function has been achieved in studies of E. coli, whose principal hsp70 (encoded by the dnaK gene) has been shown to interact with both DnaJ and GrpE. Although some functions, DNA replication for example, require all three proteins (14, 15) , others such as bacteriophage P1 replication require only DnaK and DnaJ (16) . The ATPase and peptide binding activities of hsp70 have been studied by several groups. In general, hsp70s possess an intrinsic ATPase activity of 0.01-0.03 (mol of ATP hydrolyzed/mol of hsp70/min) which, in many cases, is stimulated by the presence of certain peptides or unfolded proteins (17) . Peptide substrates appear to be bound in an extended conformation (18) and are usually enriched for certain hydrophobic groups (19, 20, 21) . Peptide binding and release is controlled by the ATPase domain (22, 23) . The structure of the ATPase domain has been solved to 2.4 angstrom resolution, revealing a striking similarity to actin and the nucleotide binding sites of hexokinase and glycerol kinase (2, 24) . The presence of ATP in the nucleotide binding site generally disfavors stable binding of peptide or unfolded protein substrates, whereas ADP tends to stabilize such interactions. This effect has been shown to be K-dependent (25) . Stimulation of ATPase activity has also been shown for DnaJ and GrpE, which can act synergistically in vitro to increase ATPase activity by up to 50-fold. DnaJ acts primarily to increase the rate of ATP hydrolysis while GrpE increases the rate of ATP and ADP release (5) . Although no cytosolic GrpE homologue has been identified in yeast, Ydj1p, a cytosolic DnaJ homologue, has been shown to interact genetically with Ssa1p, the most abundant cytosolic hsp70 in yeast.() Furthermore, purified Ydj1p has been shown to stimulate the ATPase activity of Ssa1p in vitro. Unlike DnaJ, which confers only 2-fold stimulation on DnaK ATPase activity, Ydj1p can stimulate Ssa1p ATPase activity more than 10-fold (27, 28) .

It is not clear to what degree conclusions drawn from the analysis of the hsp70 system of E. coli can be extended to eukaryotic hsp70s and their cohort proteins. In order to better understand the function of hsp70 in the eukaryotic cytosol, we undertook a biochemical study of Ssa1p, the major heat-inducible species in S. cerevisiae. We compared the effects of Ydj1p and polypeptide substrates on the ATPase activity of Ssa1p through a combination of steady-state kinetic approaches and single turnover substrate hydrolysis experiments. The focus of these experiments was to determine the effect of polypeptide substrates and Ydj1p on the regulation of Ssa1p activity by ATP and ADP.


MATERIALS AND METHODS

Proteins and Peptides

Peptides A5 (APRLRFTSL) and A7 (RRLIEDAETAARG) were obtained from Sigma (catalog numbers A5308 and A7433, respectively) and used as 5 mg/ml stock solutions. Both peptides were devoid of measurable ATPase activity. Reduced carboxymethylated -lactalbumin (CMLA,() prepared as a 10 mg/ml stock solution) was also obtained from Sigma. This preparation was boiled to remove contaminating ATPase activity. Acetylated bovine serum albumin (BSA) was obtained from New England Biolabs. Protein concentrations were measured using the Bradford assay (Bio-Rad protein assay) and ovalbumin (Sigma) as a standard. Ssa1p was purified essentially as described previously (27) from a strain containing deletions in SSA2, SSB1, and SSB2 (kindly provided by B. Diane Gambill, this laboratory) and was stored at -70 °C at a concentration of 8 µM in a buffer containing 20 mM Tris HCl (pH 7.5), 20 mM NaCl, 5 mM MgCl, 5 mM 2-mercaptoethanol, and 10% (v/v) glycerol. DnaK was purified essentially as reported by others (5) . For Ydj1p purification, a T7 expression construct in E. coli, analogous to that described by Caplan et al.(29) , was constructed using a genomic YDJ1 clone (kindly provided by Wei Yan, this laboratory). Purification of recombinant Ydj1p was as described previously (27) , yielding a preparation of >95% purity. The final concentration of Ydj1p was 130 µM in a buffer containing 20 mM Tris HCl (pH 7.5), 20 mM NaCl, 5 mM 2-mercaptoethanol, and 10% (v/v) glycerol.

ATPase Assays

Assays were carried out at 33 °C in buffer containing 10 µM ATP (1 µCi [-P]ATP per reaction), 50 mM Tris-HCl (pH 7.5), 50 mM KCl, 2 mM MgCl, and 5 mM 2-mercaptoethanol unless otherwise specified. The final concentration of Ssa1p was between 0.18 and 0.33 µM, as noted in the figure legends. 40-µl reaction mixtures were pre-equilibrated for 10 min at the assay temperature prior to addition of substrate to initiate the reaction. At several time points, aliquots were removed and applied to polyethyleneimine TLC plates and developed essentially as published elsewhere (5) . Either liquid scintillation counting or PhosphorImager analysis (Molecular Dynamics, Inc.) was used to quantitate the fraction of ATP hydrolyzed to ADP at each time point. Slopes were determined by linear regression analysis (Cricketgraph software). For the K determinations, assays were carried out in the presence of 200 mM KCl and 0 µM ADP (0.125, 0.25, 0.5, 1.0, and 2.5 µM ATP), 15 µM ADP (0.5, 1.0, 3.0, 5.0, and 10 µM ATP), and 40 µM ADP (1.0, 3.0, 5.0, 10, and 17.5 µM ATP). Ssa1p was present at 0.34 µM and 0.07 µM, depending on whether it was assayed alone or in the presence of effector (peptide or Ydj1p), respectively. Peptide A7 and Ydj1p were present at 145 and 0.8 µM, respectively. The data were analyzed using a nonlinear regression routine (30) . [-P]ATPSsa1p Complex Formation-In a slight modification of the procedure used previously in the study of DnaK (5) , 60 µg of Ssa1p was incubated for 5 min at 21 °C with 100 µCi of [-P]ATP in a 100-µl reaction containing 200 mM KCl, 10 mM MgCl, 20 µM ATP, and 10% (v/v) glycerol. The reaction was chilled on ice and the [-P]ATPSsa1p complex was purified away from free ATP by fractionation over a 8 0.5-cm Sephadex G50 column equilibrated with standard buffer (150 or 200 mM KCl, 25 mM Tris-HCl (pH 7.5), 2.5 mM MgCl, and 5 mM 2-mercaptoethanol) at a flow rate of about 0.2 ml/min. Fractions containing the protein peak (as determined by amido black staining of column fractions spotted onto nitrocellulose) were pooled and frozen on dry ice prior to storage at -70 °C. Two such preparations were used in this study: preparation 1 is 0.8 µM Ssa1p, 150 mM KCl and 2.3 nCi/µl and was used in the experiments shown in Figs. 4 and 6 and , preparation 2 is 3 µM Ssa1p, 200 mM KCl, and 6.8 nCi/µl and was used in the experiment shown in Fig. 5. In assays of the hydrolysis of bound ATP, 8 µl of complex was added to a pre-equilibrated microcentrifuge tube containing 2 µl of additional reaction components. 2-µl aliquots were removed at the indicated time points and added to a tube containing 1 µl of 4 M formic acid to terminate the reaction. Samples were then applied to TLC plates as described for ATPase assays. Isolation of the -P-labeled nucleotideDnaK complex was as described by Liberek et al.(5) , yielding a preparation which was 8.3 µM DnaK and 2.5 nCi/µl.


Figure 5: Quenching of bound ATP hydrolysis by excess unlabeled ADP in the presence and absence of CMLA and Ydj1p. The rate of hydrolysis of bound ATP was measured at 30 °C as in Fig. 3 using purified [-P]ATPSsa1p to which was added either BSA ( A), CMLA ( B), or Ydj1p ( C). Duplicate reaction tubes were prepared, one of which contained a large molar excess of unlabeled ADP (100 µM final concentration, filled triangles), the other contained no added nucleotide ( open squares). The final concentrations of the other components were as described in the legend to Fig. 3. The data for hydrolysis in the absence of ADP are taken from Table II, experiment 1.




RESULTS

Intrinsic Ssa1p ATPase Activity Is K-dependent

As a first step in characterizing the effects of peptide and Ydj1p on Ssa1p ATPase activity, we extended the characterization of the intrinsic ATPase activity of Ssa1p. The K dependence of Ssa1p ATPase activity was measured as a function of K concentration and ionic strength. The intrinsic Ssa1p ATPase activity was K-dependent, with maximum activity occurring at about 10 mM KCl (Fig. 1 A). Activity dropped approximately 8-fold as the KCl concentration was lowered to below 1 mM. This may be an underestimate of the K dependence, since, in the absence of added KCl, Ssa1p ATPase activity is near the limit of detection in these assays. At KCl concentrations above 20 mM, activity was reduced slightly. This dependence is clearly not simply an ionic strength effect, as the presence of compensating levels of NaCl does not abolish the K dependence ( closed circles). This experiment also shows that the concentration of the counterion, Cl, has no appreciable effect on Ssa1p ATPase activity. Since full activity of Ssa1p ATPase requires K, it is likely that the enzyme has one or more K binding sites.


Figure 1: K dependence of intrinsic and stimulated Ssa1p ATPase. ATPase activity was measured as a function of KCl concentration either alone ( A) or in the presence of 150 µM peptide A7 ( B) or 0.50 µM Ydj1p ( C). The concentration of Ssa1p was 0.30 µM ( A and B) or 0.18 µM ( C). Assays were done at the indicated concentrations of KCl either in the absence ( open squares) or presence of compensating concentrations of NaCl such that the total monovalent cation concentration was 300 mM ( closed circles). Note that the y axis differs substantially between A, B, and C. Similar experiments showed analogous KCl dependencies for intrinsic and peptide- or Ydj1p-stimulated activity.



The K dependence of Ssa1p ATPase activity is due in part to the dependence of the Kfor ATP on K concentration. The dependence of intrinsic ATPase activity on substrate concentration was measured at three different KCl concentrations and the data were analyzed using a nonlinear regression routine (30) . A dramatic decrease in the Kfor ATP from 4.2 µM at 2.5 mM KCl to 0.19 µM at 200 mM KCl was observed. V also increased significantly as KCl concentration was increased from 2.5 to 15 mM (). The Kvalues suggest that binding of substrate may be facilitated by the presence of K ions. The increase in V/ Kwith increasing KCl concentration suggests that the efficiency of catalysis is highest at physiological (>100 mM) K concentrations.

Distinct Interactions of Peptides and Ydj1p with Ssa1p

Since Ssa1p is K-dependent, we asked if either peptide (Fig. 1 B) or Ydj1p (Fig. 1 C) affect this dependence. In addition to their ability to stimulate ATPase activity (note differences in the y axes of the graphs), peptide A7 and Ydj1p also altered the salt dependence of Ssa1p ATPase activity. The presence of peptide had two effects: 1) optimum activity was shifted to higher K concentrations (50-100 mM KCl), and 2) ATPase activity was inhibited somewhat at concentrations of K below approximately 20 mM (Fig. 1 B). An unrelated stimulatory peptide, A5, had the same effect on Ssa1p ATPase activity, suggesting that this is a general response of Ssa1p to short peptide substrates (data not shown). Reduced CMLA, previously shown by others to be a polypeptide substrate for Ssa1p (27) , resulted in a similar shift in the K dependence, but did not exhibit the ATPase inhibition at low K concentrations seen with short peptides (data not shown). Unlike peptide, Ydj1p did not alter the concentration of K at which maximum activity was achieved. However, although the activity of both intrinsic and peptide-stimulated ATPase remained relatively high at physiological K concentrations, the Ydj1p-stimulated activity was greatly reduced at KCl concentrations above 100 mM, such that less than a 2-fold stimulation of ATPase activity was observed at 200 mM KCl. This inability of Ydj1p to stimulate ATPase activity did not depend specifically on K, as high concentrations of NaCl were able to substantially reduce stimulation even at ``optimal'' K concentrations (Fig. 1 C, filled circles). These data clearly show that while Ydj1p is capable of up to a 40-fold stimulation of ATPase activity at 10-20 mM K, at physiological K concentrations, only modest stimulation of Ssa1p ATPase activity can be achieved by Ydj1p.

Since the stimulation of Ssa1p ATPase activity by either peptide or Ydj1p changes its enzymatic properties depending upon the K concentration and the effector, the combined effect of peptide and Ydj1p was examined at several KCl concentrations in the hope that this would allow us to distinguish between the mechanisms of action of the two effectors. Concentrations of both Ydj1p and peptide A7 were chosen so as to give near maximal stimulation. KCl concentrations were chosen so as to separate the activities of peptides and Ydj1p, based on the results presented above. At 5 mM KCl, ATPase activity of Ssa1p increased nearly 20-fold in the presence of a 5-fold molar excess of Ydj1p. The addition of peptide A7 by itself had no effect on ATPase activity but strongly inhibited the stimulatory effect of Ydj1p, resulting in a 6-fold reduction in the Ydj1p-stimulated activity (Fig. 2 A). When similar assays were done at 50 mM KCl, intrinsic ATPase activity was about 3-fold higher than that seen at 5 mM KCl (Fig. 2 B). Ydj1p or peptide A7 further stimulated this activity by about 5- and 3-fold, respectively. In the presence of both effectors, activity was at a level comparable with that observed with Ydj1p alone. A further increase in the KCl concentration to 80 mM did not substantially alter the intrinsic ATPase activity (Fig. 2 C). Stimulation of ATPase activity by Ydj1p at 80 mM KCl was 4.3-fold, whereas peptide A7 stimulation was 3.7-fold. The presence of both effectors resulted in a 7-fold increase in ATPase activity, significantly greater than the stimulation observed with either Ydj1p or peptide alone, suggesting that under certain conditions, the effects of peptides and Ydj1p are cumulative. Analogous experiments in which peptide A5 and Ydj1p were combined also resulted in greater stimulation of Ssa1p ATPase than either effector by itself (data not shown), suggesting that the combinatorial stimulation seen at higher KCl concentrations does not depend on the identity of the stimulatory peptide.


Figure 2: Combined effects of peptide A7 and Ydj1p on Ssa1p ATPase at various KCl concentrations. ATPase activity was assayed under standard conditions with three different KCl concentrations, 5 mM ( A), 50 mM ( B), and 80 mM ( C). Ssa1p was 0.33 µM in all assays. Reactions contained the indicated concentrations of Ydj1p or peptide. In those reactions containing both effectors, the highest concentration (1600 nM Ydj1p and 200 µM peptide A7) was used. Assays were done in triplicate. Activity is expressed as moles of ATP hydrolyzed per mole of Ssa1p per min. Error bars denote the standard deviation for each activity determination.



Both Polypeptide Substrates and Ydj1p Accelerate the Hydrolysis of Bound ATP by Ssa1p

To further assess the roles of peptides and Ydj1p in modulating Ssa1p ATPase activity, their effects on the hydrolysis and stability of bound ATP were investigated. As described under ``Materials and Methods,'' a [-P]ATPSsa1p complex was allowed to form and then purified away from free ATP by size exclusion chromatography. Peptide A5 or A7, CMLA, acetylated BSA, or Ydj1p was added to the isolated complex, and the rate of hydrolysis of bound ATP was measured. BSA was used as a control for nonspecific protein effects and was found to have the same effect as buffer alone (data not shown). The ATP complex decayed to an ADP complex exponentially, characteristic of a first order reaction. Semi-log plots of the data yielded slopes which were used to determine the rate of hydrolysis of bound ATP in the presence of each effector. The addition of either peptide A5 or A7, CMLA, or Ydj1p to the ATPSsa1p complex resulted in a substantial increase in the rate of hydrolysis of bound ATP at 30 °C (Fig. 3, ). Ydj1p had the most dramatic effect on the hydrolysis of bound ATP, decreasing the half-life of the Ssa1pATP complex by up to 20-fold. Polypeptide substrates also accelerated the hydrolysis of bound ATP substantially, with t values approximately 3-fold lower than in the absence of effectors. It should be noted that the rate constants obtained for the hydrolysis of bound ATP in the absence of effectors are reasonably close to the value of k (4.5 10 min) derived from the data in , indicating that hydrolysis of bound ATP is probably the rate-limiting step in the ATPase reaction. Because of the very rapid hydrolysis of bound ATP by Ssa1p in the presence of Ydj1p, a set of analogous experiments was performed at 0 °C, the objective of which was to obtain more accurate values for the half-life of the complex in the presence and absence of effectors. While the hydrolysis of bound ATP by Ssa1p or CMLA-stimulated Ssa1p was approximately 30 times slower at 0 °C, the hydrolysis of bound ATP by Ydj1p-stimulated Ssa1p was reduced by less than 5-fold under the same conditions (), further suggesting that polypeptide substrates and Ydj1p differ mechanistically in their effects on Ssa1p.


Figure 3: Effect of peptide A7, A5, CMLA, and Ydj1p on the hydrolysis of bound ATP. [-P]ATPSsa1p was combined with either BSA (1 mg/ml final concentration), peptide A5 (316 µM final concentration), peptide A7 (289 µM final concentration), CMLA (73 µM final concentration), or Ydj1p (4.3 µM final concentration) and incubated at 31 °C. Aliquots were removed at the indicated times and quantitated for ATP hydrolysis as described under ``Materials and Methods.'' The final concentration of Ssa1p was 0.64 µM.



Polypeptide Substrates and Ydj1p Increase the Rate of Dissociation of ATP from an ATPSsa1p Complex

To assess the stability of the nucleotideSsa1p complex, reactions were allowed to proceed to about 50% ATP hydrolysis and then rapidly subjected to another round of chromatography to separate free nucleotide from the remaining complex. In the control reaction, more than 95% of the nucleotide in the remaining complex was ATP, whereas most of the free nucleotide was ADP, suggesting that ATP was stably bound and that ADP release was complete within minutes of ATP hydrolysis (Fig. 5, open squares). However, in the presence of peptide A7, ADP represented about 40% of the bound nucleotide. Of the free nucleotide, 40% was ATP, indicating that the stability of the ATPSsa1p complex was reduced in the presence of peptide A7 (Fig. 4, filled squares). The stability of the ATPSsa1p complex in the absence of peptide is particularly striking when one considers that the control reaction was incubated at 30 °C 12 times longer than the A7-stimulated reaction to achieve a similar degree of ATP hydrolysis, thereby allowing more time for the ATPSsa1p complex to dissociate. Given the extremely rapid hydrolysis of bound ATP in the presence of Ydj1p at both 30 °C and 0 °C, it was not possible to recover a comparable nucleotideSsa1p complex in the presence of Ydj1p to perform similar experiments.


Figure 4: Stability of ATPSsa1p complex in the presence and absence of peptide A7. A 25-µl aliquot of isolated [-P]ATPSsa1p complex (3 µM Ssa1p, 170 nCi/aliquot) was incubated at 33 °C for either 6 min (unstimulated, open squares) or 30 s (peptide A7-stimulated, 97 µM final concentration, filled squares) to achieve approximately 50% ATP hydrolysis. The tubes were then placed on ice and 5 µl of a 50% (v/v) glycerol solution was added. The samples were applied to a 8 0.5-cm Sephadex G50 column equilibrated with standard buffer (see ``Materials and Methods'') and four-drop fractions (approximately 120 µl) were collected. The quantity of P in each fraction was determined by liquid scintillation counting. Aliquots of fractions 6 and 16 (corresponding to bound and free nucleotide, respectively) were applied to polyethyleneimine TLC plates and processed as described under ``Materials and Methods'' in order to determine the ratio of ATP to ADP present.



In order to further investigate the effects of polypeptide substrates and Ydj1p on [-P]ATPSsa1p complex stability, the ability of excess unlabeled ADP to quench the hydrolysis of bound [-P]ATP was determined. The quenching of [-P]ATP hydrolysis by ADP depends on the ability of ADP to competitively inhibit ATP hydrolysis under steady-state conditions (as described below). Although the presence of 100-fold excess unlabeled ADP should have no effect on the hydrolysis of bound ATP, [-P]ATP, which dissociates from Ssa1p, will remain unhydrolyzed, because it must compete with the large pool of free ADP for binding to Ssa1p. When excess unlabeled ADP (Fig. 5 A, triangles) was added to the [-P]ATPSsa1p reaction mixture, hydrolysis of ATP to ADP no longer followed first order kinetics. The extent to which ATP hydrolysis was inhibited provided a measure of the dissociation of the ATPSsa1p complex, a process which is also expected to occur exponentially. An approximation of the rate of ATP dissociation was obtained by plotting 1 - fraction ATP hydrolyzed - fraction ATP hydrolyzed as a function of time on a semi-log plot and determining the slope of the line describing the data points. In two separate experiments, the apparent half-life of the complex was 32 and 48 min with respect to ATP dissociation. The addition of excess unlabeled ADP to reactions containing Ydj1p or CMLA resulted in complete quenching of [-P]ATP hydrolysis within several minutes (Fig. 5, B and C), leading us to conclude that both CMLA and Ydj1p accelerate the rate of ATP dissociation from Ssa1p by at least an order of magnitude. A similar degree of quenching occurred in the presence of peptides A5 and A7 (data not shown), indicating that destabilization of ATP binding is a general property of polypeptide substrates of Ssa1p. When unlabeled ATP was substituted for ADP in analogous quenching experiments, similar results were obtained (data not shown), indicating that the observed effects of peptides and Ydj1p do not depend on the identity of the unlabeled nucleotide.

The ATPase Domains of Ssa1p and DnaK Respond Differently to the Binding of CMLA

Single turnover experiments carried out with DnaK have clearly shown that GrpE can function to dissociate nucleotideDnaK complexes (5) . However, no such activity has been attributed to polypeptide substrates of DnaK. In order to test this possibility, we determined the effect of CMLA, which has previously been shown to form an ATP-sensitive complex with DnaK (25) , on the stability of an ATPDnaK complex. While we were able to isolate a nucleotideSsa1p complex with as little as 3% ADP, the purified nucleotideDnaK complex always contained a substantial fraction of ADP, varying from 50 to 80% of the total bound nucleotide in several similar experiments. A similar high percentage of ADP in the nucleotideDnaK complex was observed by Liberek et al.(5) and is probably due to the stability of the ADP-DnaK complex. Compared with the control reaction (Fig. 6, squares), a 9-fold stimulation of hydrolysis of bound ATP was observed in the presence of CMLA ( circles). However, no quenching of the hydrolysis reaction occurred when a large excess of unlabeled ADP was present in the reaction ( filled symbols), clearly indicating that CMLA binding to DnaK did not increase the ability of ADP to quench ATP hydrolysis and, hence, the rate of ATP release from DnaK. Thus, a comparison of the effect of CMLA binding on these two hsp70 proteins (Fig. 5 B versusFig. 6 ) shows that DnaK, unlike Ssa1p, does not release bound ATP appreciably upon the binding of peptide ligands.


Figure 6: Effect of CMLA on the stability of an ATPDnaK complex. The [-P]ATPDnaK complex was isolated as described under ``Materials and Methods'' and incubated with BSA (control, squares) or CMLA ( circles). ADP was added to 400 µM final concentration in two of the reactions ( filled symbols). The final concentration of proteins was 1 mg/ml BSA or 73 µM CMLA and 5 µM DnaK. The addition of ADP to the nucleotideDnaK complex in the presence or absence of CMLA reproducibly increased the rate of ATP hydrolysis about 2-fold.



Both Peptide A7 and Ydj1p Favor an ADP Form of Ssa1p

Several models for hsp70 function have been proposed. A prevalent model is based on models for G protein function, in which modulators of GTPase activity act to favor either the GTP or GDP bound form of the enzyme (31) . By analogy, Ssa1p might switch between two different conformations depending upon whether ATP or ADP is bound at the ATPase domain. Since both peptides and Ydj1p appear to have effects on the stability of an ATPSsa1p complex, we asked if either effector changed the stability of the ADP form of the enzyme. Because of the instability of the ADPSsa1p complex, the effects of Ydj1p and polypeptide substrates on this complex were difficult to study directly. However, we found that either peptide or Ydj1p increased the potency of ADP as an inhibitor of Ssa1p ATPase activity. The effect of Ydj1p and peptide A7 on the prevalence of an ADPSsa1p complex was measured by determining the inhibition constant ( K) for ADP in the presence of either effector. ATPase assays were performed in the presence of two different ADP concentrations and in the absence of ADP. In each of these three experimental conditions, the ATP concentration dependence of the ATPase activity was determined. Inhibition by ADP was competitive (data not shown), consistent with a single nucleotide binding site. The intrinsic ATPase had a K for ADP of 2.8 ± 0.52 µM under the assay conditions used. In the presence of peptide A7 or Ydj1p, the K was reduced to 0.79 ± 0.18 µM or 1.2 ± 0.64 µM, respectively. No significant change in the Kfor ATP was observed in the presence of peptide or Ydj1p. Clearly, both Ydj1p and peptide A7 significantly decrease the K for ADP, supporting the conclusion that they serve to increase the prevalence of an ADP form of Ssa1p.


DISCUSSION

It is clear from the work presented here that K concentration is important for Ssa1p ATPase activity, since the ATPase activity of Ssa1p increases nearly 10-fold between 0 and 20 mM KCl. Such a K dependence is not unique, as several other ATPases are also known to be K-dependent (32) . This dependence may not be characteristic of all hsp70s, since DnaK (33) and the yeast mitochondrial hsp70, Ssc1p, show less than a 2-fold increase in activity as KCl concentration is increased from 0 to 100 mM. ()

The distinct effects of Ydj1p and peptide substrates on Ssa1p ATPase activity were evident in studies of the K dependence of this activity. Ydj1p did not change the K optimum for Ssa1p ATPase, although stimulation was much weaker at high ionic strength, such that approximately 1.5-fold stimulation was observed at physiological K concentrations (150-200 mM KCl). In contrast, peptide binding shifted the [K] optimum of Ssa1p ATPase to between 50 and 100 mM KCl. Unlike Ydj1p, peptide stimulation of Ssa1p ATPase persists at physiological K concentrations. It should also be noted that maximal stimulation of Ssa1p ATPase by peptide requires at least a 100-fold higher molar ratio of peptide to Ssa1p than is required for Ydj1p.

ATPase assays containing both peptide and Ydj1p at several KCl concentrations further showed that peptide and Ydj1p interact differently with Ssa1p. At 5 mM KCl, a concentration which prevented peptide stimulation of ATPase activity, the presence of peptide was able to interfere with Ydj1p dependent stimulation. This result by itself could lead one to conclude that peptide and Ydj1p compete for the same binding site. However, when both effectors were present in a reaction containing 80 mM KCl, a concentration which allowed modest stimulation of ATPase by either Ydj1p or peptide A7 (4.3- and 3.7-fold, respectively), the resulting 7-fold increase in activity was greater than that obtained with either effector alone. The combinatorial effect of Ydj1p and peptide A7 suggests that they interact with distinct sites on Ssa1p or that binding of the two effectors is separated temporally. Since these experiments were done at near saturating levels of both Ydj1p and peptide A7, the latter possibility would require that either Ydj1p or peptide act transiently to change the biochemical characteristics of Ssa1p. The available data do not allow us to distinguish between these models at present.

Although Ydj1p and peptides have distinct effects on Ssa1p ATPase activity, the two effectors share both the capacity to accelerate the hydrolysis of bound ATP and to destabilize the binding of ATP to Ssa1p. Studies of the fate of a [-P]ATPSsa1p complex showed that in the absence of Ydj1p or polypeptide substrates, the conversion of ATPSsa1p to ADPSsa1p is the rate-limiting step in the reaction, resulting in the accumulation of ATPSsa1p (Fig. 7, k). Both effectors greatly stimulated the hydrolysis of bound ATP, by at least 15-fold for Ydj1p and 3-fold for polypeptide substrates. The addition of polypeptide substrate or Ydj1p to the complex also resulted in its rapid dissociation, as evidenced by the ability of excess unlabeled ADP to quench the ATPase reaction. We estimate that the rate of ATP release from Ssa1p increased by more than 10-fold in the presence of either Ydj1p or CMLA. Therefore, polypeptide substrates and Ydj1p destabilize the ATPSsa1p state by increasing both the k and k terms (Fig. 7). The increased ATP dissociation we observed in the presence of CMLA is similar to the stimulation of nucleotide exchange by unfolded proteins which has been reported in studies of bovine hsc73 (31) , also known as hsc70 or bovine brain-uncoating ATPase. However, the destabilization of nucleotide binding by a DnaJ homologue has not been reported previously, raising the possibility that this activity might be a general feature of DnaJ homologues of the eukaryotic cytosol. In a sense, both peptides and Ydj1p act as ``exchange factors,'' a role proposed for GrpE in its interaction with DnaK.


Figure 7: Diagram of the hsp70 ATPase reaction.



Unlike GrpE, however, Ydj1p and peptide substrates of Ssa1p also accelerate the rate of hydrolysis of bound ATP. The degree of stimulation of hydrolysis of bound ATP by polypeptide substrates is similar to the degree of stimulation of steady state ATPase activity under similar conditions. In other words, the change in k is proportional to the change in steady state ATP turnover ( k), indicating that k probably remains rate-limiting in the presence of peptide substrates. The similarity of k and k exists in spite of the increase in the dissociation of ATP from Ssa1p in the presence of peptides or CMLA. Indeed, our results suggest that, in the presence of peptide, bound ATP is probably released from the active site more rapidly than it is hydrolyzed to ADP. Despite this fact, we observe first order kinetics in the absence of excess unlabeled ADP, suggesting that the rebinding of ATP (Fig. 7, k) to the enzyme is not a rate-limiting step in the reaction. The decreased K ( e.g. increased affinity) for ADP in the presence of peptide A7 is consistent with our observation that ATP release is enhanced by polypeptide substrates and suggests that binding of peptide substrates would tend to favor an ADP form of Ssa1p.

Ydj1p accelerated the hydrolysis of bound ATP by as much as 20-fold at 150 mM KCl. This acceleration is in contrast to the steady-state ATPase activity, which is virtually unaffected under the same conditions, suggesting that ADP release (Fig. 7, k) from Ssa1p may be rate-limiting in the presence of Ydj1p. In this respect, Ydj1p appears to function like DnaJ of E. coli, primarily serving to accelerate the hydrolysis of bound ATP to ADP. The increased inhibition of ATPase by ADP in the presence of Ydj1p also suggests that the role of Ydj1p is to favor an ADP form of Ssa1p. As in the case of peptide-stimulated Ssa1p, the increased rate of ATP dissociation ( k), relative to ADP dissociation ( k), from Ydj1p-stimulated Ssa1p should also serve to drive equilibrium toward an ADP form of the enzyme.

The DnaK/DnaJ/GrpE system of E. coli is often cited as a paradigm for hsp70 function. Although the study of the E. coli system has been very informative, our results suggest that there may be important differences between the hsp70s of prokaryotes and those of the eukaryotic cytosol. Upon incubation with [-P]ATP, both Ssa1p and DnaK were readily recovered as P-labeled nucleotidehsp70 complexes. In contrast to the DnaK complex, the P-labeled nucleotideSsa1p complex was virtually devoid of ADP (Fig. 4); indeed, we were unable to isolate an ADPSsa1p complex. However, the nucleotideDnaK complex generally contained at least 50% ADP, despite our efforts to minimize the hydrolysis of ATP during recovery of the complex. Therefore, a primary difference between the Ssa1p and DnaK ATPase reaction appears to be the rate of ADP dissociation from the active site ( k, Fig. 7). Preliminary experiments indicate that the half-life of the ADPDnaK complex is approximately 1-2 min ( k 0.3-0.7 min) at 23 °C.() The apparent instability of ADPSsa1p suggests to us that Ssa1p may not require a GrpE-like nucleotide exchange factor, since the apparent function of such proteins, by analogy with nucleotide exchange factors of G proteins (34) , is to facilitate the release of ADP from the ATPase domain and permit the binding of ATP. In support of this idea, preliminary studies indicate that Ssa1p and Ydj1p can together facilitate the folding of denatured luciferase in vitro.()

Because the ATPSsa1p complex is intrinsically quite stable, one might expect that stabilization of an ADP form of the enzyme would be an important function of some modulators of Ssa1p activity. As discussed above, this appears to be a function of both polypeptide substrates and Ydj1p. In contrast, the release of ATP from DnaK was not affected by the presence of CMLA. In the case of Ssa1p, the ATP release activity of polypeptide substrates and Ydj1p may be required to compensate for the relative instability of the ADP form of the enzyme. Another function of the ATP release activity could be to couple chaperone activity to the cellular ADP:ATP ratio, which reflects the physiologically important ``energy charge'' (35) of the cell. This ratio is reported to vary between about 0.4 and 2 in S. cerevisiae(36) , depending upon whether growth occurs under anaerobic or aerobic conditions, respectively. Furthermore, some evidence suggests that the cellular ADP:ATP ratio may increase upon heat shock (37) . It is tempting to speculate that increases in the intracellular ADP:ATP ratio might tend to favor an ADP form of Ssa1p and, consequently, the stable binding of substrate polypeptides.

Despite the differences between Ssa1p and DnaK, the results presented above are generally in good agreement with recent models of hsp70 function. It has been suggested that hsp70 is regulated by occupancy of the ATPase domain by either ATP, which promotes rapid but unstable binding of polypeptide substrates, or ADP, which favors stable binding of polypeptide substrates (38, 39) . Indeed, recent results from other laboratories suggest that ATP hydrolysis is not stoichiometrically coupled to cycles of peptide binding and release (25, 38) . In such a model, GrpE would serve to convert hsp70 from an ADP- to an ATP-bound form through the exchange of bound ADP for ATP. DnaJ would have the opposite effect, favoring an ADP-bound form through the hydrolysis of bound ATP to ADP (40) . Our results suggest that the requirement for modulators of hsp70 activity may be determined by the enzymatic properties of that particular hsp70 protein. In E. coli, DnaJ appears to interact with certain polypeptide substrates prior to DnaK, possibly to target these substrates for DnaK action (16, 26, 41, 42, 43) . Preliminary evidence indicates that Ydj1p prevents the aggregation of unfolded rhodanese, suggesting that Ydj1p may bind to unfolded or partially folded polypeptides. Perhaps Ydj1p enhances the ability of Ssa1p to ``capture'' polypeptide substrates by targeting Ssa1p to those substrates and then stabilizing the ADPSsa1ppolypeptide complex.

  
Table: Values of K and V for Ssalp ATPase at several KCl concentrations

The data were analyzed using a nonlinear regression routine as described under ``Materials and Methods.'' Units are moles of ATP hydrolyzed per mole of Ssalp min ( V) and µM ATP ( K).


  
Table: Stability of the ATPSsalp complex in the presence and absence of effectors

Reaction mixtures were prepared and reactions carried out at 0 °C (on ice) or 30 °C as described in the legend to Fig. 3. Half-life ( t) in minutes was calculated using the formula t = 0.301 slope. These values were used to calculate rate constants (min) using the formula k = 0.693 t. Experiment 2 corresponds to the data presented in Fig. 3. Experiments 1 and 2 were done on separate days using freshly prepared or frozen [-P]ATPSsalp complex, respectively. Exp, experiment; ND, not determined.



FOOTNOTES

*
This work was supported in part by United States Public Health Service Grant NIH 5 R01 GM31107 (to E. A. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by National Institutes of Health Postdoctoral Fellowship Award 5 F32 GM14394.

Present address: Facultad De Veterinaria, Universidad De Zaragoza, 50013 Zaragoza, Spain.

**
To whom correspondence should be addressed. Tel.: 608-263-7105; Fax: 608-262-5253; E-mail: bcraig@macc.wisc.edu.

J. Becker, W. Yan, and E. A. Craig, submitted for publication.

The abbreviations used are: CMLA, carboxymethylated -lactalbumin; BSA, bovine serum albumin.

B. Miao and E. A. Craig, unpublished data.

T. Ziegelhoffer, P. Lopez-Buesa, and E. A. Craig, unpublished observations.

B. A. Schilke and E. A. Craig, unpublished data.


ACKNOWLEDGEMENTS

We thank Paul Bertics for helpful discussions and critical reading of the manuscript. We also thank Jill Johnson, Shikha Laloraya, Ed Maryon, and Eva Ziegelhoffer for helpful comments on the manuscript.


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