Purification and characterization of a 66-kDa protein from rabbit reticulocyte lysate which promotes the recycling of hsp 70.

We have purified to apparent homogeneity a 66-kDa protein from rabbit reticulocyte lysate which is associated with hsp 70. Our characterization of this 66-kDa protein demonstrates that its physiological role is to promote the recycling of hsp 70 by catalyzing the dissociation of hsp 70-bound ADP in exchange for ATP. We have therefore termed the 66-kDa protein RF-hsp 70, a recycling factor for hsp 70. RF-hsp 70 promotes stoichiometric binding of ATP to hsp 70, and it increases about 5-fold the rate of dissociation of hsp 70·ADP in the presence of ATP. This process represents adenine nucleotide exchange, since dissociation of ADP does not occur unless ATP is added; dATP, GTP, and ITP cannot substitute for ATP. The mechanism of action of RF-hsp 70 is to lower the KD of hsp 70 for ATP about 6-7-fold to a value that is close to the KD of hsp 70 for ADP. RF-hsp 70 also stimulates the ATPase activity of hsp 70, including the 42-kDa amino-terminal portion of hsp 70 generated by chymotrypsin, demonstrating that RF-hsp 70 interacts with that part of hsp 70 known to contain the ATP/ADP binding site. Confirming its recycling function, RF-hsp 70 stimulates about 7-10-fold the ability of hsp 70 to reactivate heat-denatured firefly luciferase. In addition, RF-hsp 70 acts catalytically to recycle hsp 70, since, at 0.2 times the molar concentration of hsp 70, RF-hsp 70 increases the rate of renaturation of luciferase by hsp 70 about 3-4-fold. The action of RF-hsp 70 is also partially species-specific since it is most effective with rabbit reticulocyte hsp 70, less effective with bovine brain hsp 70, even less effective with human hsp 70, and ineffective with broad bean hsp 70.

in this preparation consists of a prominent 90-kDa band (Fig. 1, lane 1). When this preparation is chromatographed on DEAE-cellulose as described (2), the 90-kDa band (hsp 90) is almost completely separated from hsp 70(R) and elutes at 0.23-0.30 M KCl, where it represents about 80 -90% of the total protein. This eluate was diluted to 100 mM KCl, concentrated by ultrafiltration, and then 9 mg was chromatographed at a flow rate of 1.0 ml/min on a 0.5 ϫ 5.0-cm column of Mono Q, equilibrated in buffer B. After washing for 3.0 min with buffer B, the column was brought linearly to buffer B containing 275 mM KCl in 3.0 min and then brought linearly to buffer B containing 550 mM KCl over the next 18 min. The 90-kDa protein, purified to apparent homogeneity (see Fig.  1, lane 7), eluted at 400 mM KCl. The pooled 90-kDa protein received a final concentration of 1.0 mM dithiothreitol and was stored in liquid nitrogen at a final concentration of 2.0 mg/ml. It was identified as hsp 90 based upon its abundance, molecular size, and its ability to stimulate the renaturation of firefly luciferase by hsp 70 (see Fig. 8), an activity shown previously to be characteristic of hsp 90 (18). Bovine brain hsp 70 and recombinant human hsp 70, denoted hsp 70(B) and hsp 70(H), respectively, were purchased from StressGen, and each appears homogeneous on gel electrophoretic analysis (Fig. 1, lanes 9 and 10, respectively). The hsp 70(H) migrates slightly farther on the gel than either hsp 70(B) or hsp 70(R), which migrate similarly. This is consistent with the fact that hsp 70(H) is a heat-inducible form of hsp 70 and is 10 amino acids shorter than hsp 70(B), a constitutive form of hsp 70. The similar migration of hsp 70(B) and hsp 70(R) suggests that the latter is also a constitutively expressed hsp 70. The amino acid sequence of the carboxyl terminus of hsp 70(R), produced by limited chymotrypsin digestion, is identical to the carboxyl-terminal 35 resi-dues of the constitutive form of rat hsp 70 (19,20), 2 confirming that the hsp 70(R) we have purified is the constitutively expressed form of the protein. Broad bean hsp 70, termed hsp 70(bn), was generously provided by Matthew A. Harmey of The National University of Ireland. It shows a single broad band with an estimated size of 68 kDa on gel analysis (Fig. 1, lane 11).
Preparation of Polypeptide A-Amino acid residues 41-54 of eIF-2␣, representing the sequence IEGRILLSELSRRR and comprising the phosphorylation site of HCR (21), was synthesized on a Applied Biosystems 430A peptide synthesizer and kindly provided by Stephen Meredith at The University of Chicago. This product, termed polypeptide A, was purified by reverse-phase chromatography on an Aquapore RP-300 column (4.6 ϫ 250 mm; Brownlee Laboratories) using a 0 -50% acetonitrile gradient in 0.05% (v/v) trifluoroacetic acid in water at a flow rate of 1.0 ml/min over a 60-min period.
Gel Electrophoresis and Immunoblotting-Electrophoresis of protein samples on 7% polyacrylamide-SDS slab gels (22) followed by either silver staining (23) or electrophoretic transfer to nitrocellulose sheets (at 60 V for 7 h in a Hoefer Transphor) and then immunoblotting (24) has been described previously.
Sucrose Gradient Centrifugation-Protein samples were layered over 4.4-ml 5-20% (w/v) linear sucrose gradients and centrifuged in a Beckman L5-65 ultracentrifuge for 17.5 h at 54,000 rpm (300,000 ϫ g av ) and 2°C in the SW 60-Ti rotor. Gradients were pumped out by upward displacement and divided into 35 0.13-ml fractions. Gradient buffer for the samples indicated in Fig. 7 contained 10 mM Tris-HCl, pH 7.5, 50 mM KCl, 1.5 mM MgCl 2 , 1 mM dithiothreitol, and 0.1 mM EDTA. Gradient buffer used in the purification of RF-hsp 70 was similar, but MgCl 2 was omitted.
ATP Binding and ATPase Determinations-Specific conditions are indicated in the figure and table legends. Samples labeled with [␣-32 P]ATP were Millipore filtered as described previously (25). Radiolabeled ATP and ADP were separated by chromatography on polyethyleneimine-cellulose sheets (Macherey-Nagel) in 0.75 M Tris-HCl, pH 8.0, 0.50 M LiCl (24,26), located by autoradiography, and quantitated by excision and counting with 7 ml of Aquasol (DuPont NEN).
Renaturation of Luciferase-The ability of hsp 70, hsp 90, and RFhsp 70 to promote the renaturation of luciferase was assessed as described by Schumacher et al. (18). Firefly luciferase (Sigma) was diluted in 25 mM Tricine, pH 7.8, 10 mM MgCl 2 , 1 mM dithiothreitol, 0.1 mM EDTA, 10%(v/v) glycerol, and 10 mg/ml bovine serum albumin (buffer C) to a final concentration of 0.065 nM. Duplicate aliquots were removed for determination of the initial activity, and the remainder was heated at 40°C for 20 min, reducing luciferase activity to an average of 5-8% of its initial activity. When samples were renatured for a single period of time, renaturation reactions, in duplicate, contained 2.7 l of heated luciferase, 0.50 mM ATP, 15 mM creatine phosphate, 45 units/ml creatine phosphokinase, and the protein additions indicated in the figure and table legends in a final volume of 27 l of 10 mM Tris-HCl, pH 7.5, 100 mM KCl, 3 mM MgCl 2 , 1 mM dithiothreitol, 0.1 mM EDTA (buffer D). When renaturation was followed with time, samples were similar but scaled up to 80 l. Incubation was at 32°C, and 25 l was removed at the indicated times and assayed for luciferase activity by mixing with 100 l of 25 mM Tricine, pH 7.8, 5 mM MgCl 2 , 1.7 mM dithiothreitol, 0.1 mM EDTA, 0.1 mM D-luciferin, 0.25 mM coenzyme A, and 0.5 mM ATP in a 1.5-ml polypropylene tube and counting immediately in a glass vial for 1.0 min in a TM-Analytic, model 6895 liquid scintillation spectrometer at the setting that is optimal for the determination of 3 H radioactivity. Aliquots of unheated and heated luciferase, which were not incubated, were diluted similarly with buffer D and assayed. Results are expressed as the percentage of the initial activity of unheated luciferase.
Materials-Reduced and carboxymethylated ␣-lactalbumin (RCM-␣LB) and reagents for the renaturation and assay of firefly luciferase were all from Sigma, except that dithiothreitol was purchased from CalBiochem. [␣-32 P]ATP was obtained from ICN. Reagents for gel electrophoresis were bought from Bio-Rad. Electrophoresis, gel transfer, and gel drying equipment were from Hoefer, and ultrafiltration supplies were from Amicon.

RESULTS
Purification of RF-hsp 70 -The purification of RF-hsp 70 from rabbit reticulocyte lysate, as described under "Experimental Procedures," is shown in Table I. Activity is based upon stimulation of the renaturation of luciferase by hsp 70(R) and is 2 M. Gross, unpublished observation.   Table IV). Based upon this activity, RF-hsp 70 was purified approximately 840-fold with a yield of about 2%. One explanation for the relatively low yield of RF-hsp 70 in the purification is that appreciable 66-kDa protein and activity separate into side fractions at steps 2, 3, and 5. In addition, using immunoblot analysis with polyclonal antibody raised to hsp 70(R), which reacts only with the 72-kDa band of hsp 70(R) and the 66-kDa band of RF-hsp 70 (1), we estimate that the concentration of the 66and 72-kDa proteins in rabbit reticulocyte lysate is about 0.09 and 0.24 mg/ml, respectively (data not shown). This indicates that the recovery of the 66-kDa protein is approximately 3.6%, and its purification is about 1,600-fold. This suggests that one or more proteins in the lysate, separate from the 66-kDa protein (RF-hsp 70), also promote the renaturation of luciferase in the presence of hsp 70(R) and could account for up to 50% of such activity. One possibility is hsp 90, which has been shown previously to have such activity (Ref. 18 and see Fig. 8) and is separated from the 66-kDa protein at the DEAE-cellulose step (see Fig. 1). Another possibility that has been suggested (27) is the large hetero-oligomeric ring complex TCP-1. Alternatively, RF-hsp 70 may undergo partial inactivation (up to 50%) in the first several steps of its purification. In either case, we have observed that RF-hsp 70 activity, measured as indicated in Table I, copurifies with the 66-kDa protein from steps 3-6 in the purification (see Fig. 1).
Effect of RF-hsp 70 on the Binding of ATP to Hsp 70 and on Adenine Nucleotide Exchange-Our observation that RF-hsp 70 and hsp 70(R) appear to be associated with each other during their purification prompted us to test whether this association is physiologically significant. Therefore, we examined the ability of hsp 70(R) to bind [␣- 32  shows no significant ATP binding at concentrations of ATP up to 105 M (data not shown), indicating that RF-hsp 70 does not have a strong ATP binding site. This confirms our earlier conclusion that the adsorption of RF-hsp 70 to ATP-agarose ( Fig. 1) is likely due to its association with hsp 70(R) and not direct binding to ATP-agarose. The potentiation of ATP binding to hsp 70(R) and hsp 70(B) due to RF-hsp 70 (Fig. 2) suggests that the association of RF-hsp 70 with hsp 70(R) is physiologically relevant and that RF-hsp 70 may function to promote the dissociation of ADP from hsp 70(R) and hsp 70(B), permitting stoichiometric binding of ATP. This explanation would be consistent with previous studies demonstrating that a considerable fraction of hsp 70, isolated by chromatography on ATPagarose, contains bound adenine nucleotide (primarily ADP) which prevents stoichiometric binding of ATP (28). This explanation was tested by determining the effect of RF-hsp 70 on the rate of dissociation of radioactivity from hsp 70(R), prelabeled with [␣-32 P]ATP, upon subsequent incubation in the presence of excess unlabeled ATP. As shown in Table II, experiment A, there is a slow rate of dissociation of radiolabel from hsp 70(R) in the presence of unlabeled ATP which is increased approximately 6-fold by an equimolar concentration of RF-hsp 70. Even one-third as much RF-hsp 70 stimulated the rate of dissociation 3-fold, suggesting that RF-hsp 70 action on hsp 70(R) is catalytic rather than stoichiometric. Analysis of the radioactivity bound to hsp 70(R) after the prelabeling with [␣-32 P]ATP showed that 76% is ADP (presumably due to the ATPase activity of hsp 70(R), which is demonstrated in Figs. 4 -6). In addition, the extent of dissociation of the radioactivity bound to hsp 70(R) in the presence of RF-hsp 70 in the experiments in Table II is   RF-hsp 70 was purified as described under "Experimental Procedures." One unit of activity is defined as the amount required to renature luciferase (denatured to 5-8% of its initial activity by heating at 40°C for 20 min) to 50% of its initial activity when incubated for 60 min under renaturation conditions (see "Experimental Procedures") and in the presence of 0.07 mg/ml hsp 70(R) in a final volume of 27 l.
Step rebinding of ATP shown in Fig. 3), demonstrating that a majority of the dissociated nucleotide is ADP. The dissociation of ADP from hsp 70(R) promoted by RF-hsp 70 is strictly dependent upon the addition of ATP, since no dissociation occurs in the presence of GTP, ITP, or dATP (Table II,  To verify that the dissociation of adenine nucleotide (primarily ADP) from hsp 70 in Table II is associated with binding of ATP (i.e. adenine nucleotide exchange), hsp 70(R) and hsp 70(B) were preincubated with ATP and MgCl 2 to produce maximal binding of unlabeled nucleotide. We then determined the effect of RF-hsp 70 on the rate of binding of [␣-32 P]ATP to each hsp 70, which would be dependent upon dissociation of bound nucleotide. Binding of labeled ATP to each hsp 70 in the absence of RF-hsp 70 is relatively slow (Fig. 3), presumably because of the slow rate of dissociation of bound adenine nucleotide shown in Table II. In contrast, initial [␣-32 P]ATP binding to each hsp 70 in the presence of RF-hsp 70 is about six times faster (Fig. 3), similar to the increased rate of dissociation of bound nucleotide induced by RF-hsp 70 (Table II). This result confirms that the dissociation of adenine nucleotide from hsp 70 promoted by RF-hsp 70 represents adenine nucleotide exchange. The extent of binding of [␣-32 P]ATP to hsp 70(R) is approximately 1.75 times that to hsp 70(B) (Fig. 3), presumably because the concentration of ATP in the binding reaction, 20 M, permits maximal binding to hsp 70(R) but not hsp 70(B) (Fig. 2).
Effect of RF-hsp 70 on the ATPase Activity of Hsp 70 -If RF-hsp 70 promotes the recycling of hsp 70 by increasing the rate of dissociation of ADP and binding of ATP, as indicated by the results in Table II and Fig. 3, one would expect the rate of hydrolysis of ATP to ADP by hsp 70 to be increased similarly by RF-hsp 70. This is confirmed experimentally, since the ATPase activity of hsp 70(R) (0.035 nmol/min/ml) is increased 6-fold (to 0.21 nmol/min/ml) when incubated with an equimolar (and saturating) concentration of RF-hsp 70 (Fig. 4). RF-hsp 70 by   32 P]ATP (12 ⅐ 10 4 cpm/pmol), were preincubated for 8 min at 34°C. Reactions were constituted as described in the legend to Fig. 2, but creatine phosphate was omitted from those samples receiving added ADP. Samples in experiment A contained 0.7 g of hsp 70(R), ATP (0.50 mM), and the indicated amount of RF-hsp 70. Samples in experiment B contained 0.7 g of hsp 70(R), 0.7 g of RF-hsp 70 (where added), and the indicated nucleotide (0.50 mM). Samples in experiment C contained 0.7 g of hsp 70, ATP (0.50 mM), and 0.7 g of RF-hsp 70 (where added). The final volume was 23 l, and incubation was at 34°C. The rate of dissociation (nmol/min/ml) of radioactivity, prebound to hsp 70, was determined by Millipore filtration of 4.0-l aliquots (five total) removed from each sample at 20-s intervals. Thin layer chromatography on polyethyleneimine-cellulose of 5% (w/v) trichloroacetic acid extracts of Millipore filters indicated that the radioactivity bound to hsp 70(R) after the 8-min preincubation was 76% ADP and 24% ATP and that this proportion was not altered appreciably by 1.0 min of further incubation in the presence of creatine kinase and creatine phosphate. The results are an average of two separate determinations. itself has no significant ATPase activity. RF-hsp 70 appears to act catalytically on hsp 70(R), since one-fourth as much RF-hsp 70 still has close to a saturating effect on its ATPase activity. This effect of RF-hsp 70 is partially specific to hsp 70(R), since RF-hsp 70 has progressively less ability to stimulate the ATPase activity of hsp 70(B) and hsp 70(H) (Fig. 4), and it is unable to stimulate the ATPase activity of hsp 70(bn) (Fig. 4, inset). In contrast, the endogenous ATPase activity of hsp 70(R), hsp 70(B), and hsp 70(H) is similar, and each is stimulated to the same degree (about 6-fold) by polypeptide A. The ATPase activity of hsp 70(bn) is much greater than that of the mammalian hsp 70s, but it is also stimulated (about 2-fold) by polypeptide A (Fig. 4, inset). Previous studies by Chappell et al. (29) demonstrated that limited chymotrypsin digestion of hsp 70(B) produces a 60-kDa fragment that is converted subsequently to a 44-kDa component. Conversion to the 44-kDa, but not the 60-kDa, component is associated with a loss of stimulation of ATPase activity by clathrin (29). In addition, the 44-kDa component was localized to the amino-terminal portion of hsp 70(B) by site-specific antibodies (29). We have employed similar chymotryptic digestion (Fig. 6) to convert both hsp 70(R) and hsp 70(B) almost entirely to a product with an estimated molecular mass of 42 kDa (as judged by SDS-polyacrylamide gel electrophoresis), which appears to be slightly smaller than the 44-kDa component characterized by Chappell et al. (29). As noted by Chappell et al. (29), the ATPase activity of the 42-kDa component derived from hsp 70(B) is considerably greater (about 8-fold more) than that of the native hsp 70 (Fig. 6B). Conversion to the 42-kDa component also increases the ATPase activity of hsp 70(R) (Fig.  6A), but not to the same degree. In addition, whereas the ATPase activity of hsp 70(R) (Fig. 6A) and hsp 70(B) (Fig. 6B) is stimulated (about 8-fold and 5-fold, respectively) by polypeptide A, the ATPase activity of the 42-kDa component derived from each hsp 70 is not stimulated by polypeptide A. This result is consistent with the findings of Chappell et al. (29) and supports the belief that a carboxyl-terminal domain in hsp 70 is required for its interaction with unfolded protein or polypeptide. In contrast, RF-hsp 70 increases the ATPase activity of each 42-kDa component and the activity of the corresponding native hsp 70 to about the same degree (Fig. 6). This result indicates that unlike unfolded protein or polypeptide, the interaction of RF-hsp 70 with hsp 70 is localized to the aminoterminal, 42-kDa domain of the latter, and it suggests that RF-hsp 70 may bind to hsp 70 near its ATP binding site, which is located within the amino-terminal domain.
Mechanism of Action of RF-hsp 70 -To examine the mechanism by which RF-hsp 70 increases the rate of dissociation of hsp 70⅐ADP and the binding of ATP (Table II and (Table III), are in close agreement with the values (9.5 and 1.6 M, respectively) reported by Palleros et al. (14) which were determined by equilibrium dialysis. The finding that RF-hsp 70 does not alter the K D of hsp 70(R) for ADP correlates with our observation (Table II, experiment B) that RF-hsp 70 has no effect on the rate of exchange of radioactive for unlabeled ADP bound to hsp 70(R). This reinforces the belief that RF-hsp 70 promotes only the physiologically relevant exchange reaction where hsp 70-bound ADP is replaced by ATP.
To determine whether the action of RF-hsp 70 on hsp 70(R) involves stable association of these two proteins, RF-hsp 70 and hsp 70(R) were preincubated either separately or together and then fractionated on sucrose density gradients. Aliquots of gradient fractions were analyzed for RF-hsp 70 (66-kDa band) and hsp 70(R) (72-kDa band) by SDS-polyacrylamide gel electrophoresis, silver staining, and laser densitometry. The results ( Fig. 7) demonstrate that RF-hsp 70 alone sediments entirely as a single component with a peak at fraction 16, migrating somewhat slower than rabbit hemoglobin (peak at fractions 18 -19), and hsp 70(R) alone shows a major peak migrating at fraction 19 or slightly faster than rabbit hemoglobin. These findings are in reasonable agreement with the estimated molecular sizes of RF-hsp 70 and hsp 70(R) from SDSpolyacrylamide gel electrophoresis, and they indicate therefore that RF-hsp 70 is a single polypeptide chain. Gradient analysis of hsp 70(R) alone also shows a faster sedimenting component with a peak at fraction 26, which likely represents a homodimer, as we noted previously (1) and as has been observed with other hsp 70 species (14,20). Analysis of RF-hsp 70 preincubated with hsp 70(R) shows the same peaks seen with each protein alone as well as two additional peaks. One, sedimenting with a peak at fraction 24 hsp 70(B), which were incubated in the presence (ϩ) or absence (Ϫ) of 0.7 g of RF-hsp 70. Incubations were as indicated in the legend to Fig. 2, but both creatine phosphate and creatine kinase were omitted, and final volumes were 24 l. Final ATP concentrations varied between 0.4 and 4 M for hsp 70(R) and 2 and 20 M for hsp 70(B). Incubations varied from 5 to 30 min at 34°C in an attempt to produce similar conversion of ATP to ADP in all samples, and this varied between 20 and 60% ADP. Small aliquots were removed from each and brought to 5% (w/v) trichloroacetic acid. Each sample was then Millipore filtered, filters were extracted by shaking for 1 h in 0.50 ml of 5% (w/v) trichloroacetic acid at the bottom of glass vials, and the extracts (containing 80 -85% of total bound radioactivity) were pipetted off. Separation by chromatography on polyethyleneimine-cellulose sheets and then liquid scintillation counting of ATP and ADP in the initial aliquots of the intact samples and in aliquots of the trichloroacetic acid extracts of the Millipore filters provided a measure of the total and bound, ATP and ADP, respectively. Values represent an average of five (hsp 70(R)) or two (hsp 70(B)) determinations. Effect of RF-hsp 70 on the Renaturation of Luciferase by Hsp 70 -To demonstrate that RF-hsp 70 catalyzes the recycling of hsp 70 in a functional assay, we tested its effect on the renaturation of denatured firefly luciferase by hsp 70, as described recently (18,27). The data in Fig. 8 show the rate of renaturation of heat-denatured luciferase in an isolated reaction containing an ATP-regenerating system. Pretreatment of luciferase for 20 min at 40°C reduced its activity to 5% of the initial activity (zero time in Fig. 8), and reincubation at 32°C with only the ATP-regenerating system resulted in no renaturation over a 90-min period. Reincubation with 0.070 mg/ml hsp 70(R), a concentration that is near saturating (see Fig. 9B and Ref. 18), promotes only a doubling of luciferase activity after 90 min, whereas this concentration of hsp 70(R) plus 0.15 mg/ml hsp 90 produces a 3.2-fold increase in luciferase activity (Fig.  8). Reincubation with hsp 90 alone has almost no effect (data not shown). These effects are similar to those observed by Schumacher et al. (18). In contrast, reincubation with 0.070 mg/ml hsp 70(R) and an equimolar concentration of RF-hsp 70 increases the initial rate of reactivation of luciferase more than 7-fold relative to hsp 70(R) alone, leading to the restoration of 42% of the initial luciferase activity after 90 min (Fig. 8). The same concentration of RF-hsp 70 alone has almost no renaturing effect (Fig. 8), and RF-hsp 70 does not promote renaturation in the presence of hsp 90 (data not shown). Consistent with a catalytic effect, 0.012 mg/ml RF-hsp 70 increases the rate of renaturation of luciferase by 0.070 mg/ml hsp 70(R) about three times faster than hsp 70(R) alone, matching the effect of limiting (0.035 mg/ml) hsp 70(R) plus an equimolar concentration of RF-hsp 70 (Fig. 8). Although hsp 90 increases the rate of luciferase renaturation when incubated with hsp 70(R) alone, it does not increase the renaturation produced by hsp 70(R) plus RF-hsp 70 (Fig. 8). A saturating renaturation effect (see also Fig. 9A) is produced by reincubation with 0.12 mg/ml hsp 70(R) and an equimolar concentration of RF-hsp 70 (Fig. 8). This increases the initial rate of renaturation of luciferase 10-fold faster than hsp 70(R) alone, leading to the restoration of 55% of the initial activity after 90 min.
Having established the kinetics of renaturation of luciferase with selected concentrations of hsp 70(R) and RF-hsp 70 (Fig.  8), we examined the effect of different concentrations and molar ratios of these two proteins on renaturation after a 60-min incubation at 32°C (Fig. 9). Incubation with hsp 70(R) alone produces, at most, only a 2-fold restoration of luciferase activity with a saturating effect achieved at about 0.070 mg/ml (Fig.  9B). Incubation with hsp 70(R) plus a limiting concentration of RF-hsp 70 (added at one-half the molar concentration of hsp 70(R)) is much more effective, renaturing luciferase to 42% of its initial activity at 0.070 mg/ml hsp 70(R) and to a maximal 55-60% of initial activity at 0.12-0.18 mg/ml hsp 70(R). Interestingly, at concentrations below 0.070 mg/ml, hsp 70(R) (plus RF-hsp 70) is much less effective, producing, for example, only one-fourth the renaturation at one-half (0.035 mg/ml) the concentration (Fig. 9B). This sigmoidal concentration-dependence curve is independent of the RF-hsp 70 concentration, since hsp 70(R) produces almost four times the renaturation at 0.070, as at 0.035, mg/ml over a molar ratio of RF-hsp 70 to hsp 70(R) ranging from 0.20 to 1.6 (Fig. 9A). The data in Fig. 9A also demonstrate that, at limiting, near saturating, and saturating concentrations of hsp 70(R), a maximal effect of RF-hsp 70 is achieved when its concentration is equimolar to that of hsp 70(R). In addition, at saturating concentrations of hsp 70(R) and RF-hsp 70, luciferase is renatured to more than 70% of its initial activity. These data (Fig. 9A) also confirm the catalytic action of RF-hsp 70, since RF-hsp 70 increases the effect of 0.12 mg/ml hsp 70(R) 2.5-and 3.7-fold when added at 0.08 and 0.20 times, respectively, the molar concentration of hsp 70(R).
As noted by Schumacher et al. (18), the hsp 70(R) concentration of 0.070 mg/ml (or about 1 M) which we have found to be near saturating in the renaturation reaction ( Fig. 9) is almost 5 orders of magnitude greater than the luciferase concentration (0.013 nM). Consequently, we tested whether the same concentration of hsp 70(R) would be effective with much higher concentrations of denatured luciferase, since much higher concentrations of protein requiring chaperonin action are present in vivo. We found that in renaturation reactions (60 min at 32°C) containing 1 M hsp 70(R) and RF-hsp 70, heat-denatured luciferase, varying in concentration from 0.013 nM to 0.11 M or 4 orders of magnitude, was similarly renatured from 7-10% to 58 -71% of its initial activity (data not shown). This suggests that the RF-hsp 70-promoted chaperonin action of hsp 70(R) illustrated in Figs. 8 and 9 may apply under physiological conditions. It also should be noted that our estimate of the concentration of hsp 70(R) in rabbit reticulocyte lysate of 0.24 mg/ml is significantly above the concentration we have found to be saturating in an isolated luciferase renaturation assay.
We also compared the renaturation effect of different hsp 70s (at 0.070 mg/ml) in the absence or presence of an equimolar concentration of RF-hsp 70 (Table IV). Added alone, hsp 70(R), hsp 70(B), and hsp 70(H) each had about the same effect, increasing luciferase activity slightly less than 2-fold. In contrast, RF-hsp 70-stimulated renaturation is partly hsp 70-specific (Table IV), since RF-hsp 70 increases the renaturation by hsp 70(R) 7.4-fold, that by hsp 70(B) 4.0-fold, and that by hsp 70(H) only 2.8-fold. These results correlate with the differential stimulation by RF-hsp 70 of the ATPase activity of these hsp 70s (Fig. 4). They confirm the partial specificity of RF-hsp 70 action on different hsp 70s, and they strongly suggest that the promotion of adenine nucleotide exchange (resulting in enhanced ATPase activity) on hsp 70 by RF-hsp 70 underlies the enhanced chaperonin activity (renaturation of luciferase) of hsp 70 produced by RF-hsp 70. DISCUSSION Previous studies have demonstrated that the action of hsp 70 is mediated by binding to other proteins such as nascent polypeptides (7), immunoglobulin heavy chains, and unfolded, abnormally formed, or incorrectly folded proteins (8 -13). This protein-protein interaction with hsp 70 is believed to result in the proper folding or refolding of newly or abnormally formed or denatured proteins. Dissociation of hsp 70 can then occur in the presence of ATP/Mg 2ϩ and is associated with ATP hydrolysis by the hsp 70 ATPase (7)(8)(9)(10)(11)(12)(13). Recycling of hsp 70 is thought to involve release of hsp 70⅐ADP following ATP hydrolysis, rebinding to another polypeptide or protein, exchange of ATP for bound ADP, ATP hydrolysis, and dissociation. However, the precise sequence of events and whether the sequence of events varies or is the same for different hsp 70s and different protein-protein interactions are currently unclear (12). It is clear, however, that hsp 70 (bovine brain or rabbit reticulocyte) binds ADP 6 -10 times more tightly than ATP (Table III and Ref. 14), suggesting that dissociation of ADP and binding of ATP may limit the rate of hsp 70 recycling.
We believe that the characteristics of RF-hsp 70 action on hsp 70, summarized below, indicate that ADP/ATP exchange is likely to be rate-limiting for hsp 70 recycling and that the physiological role of RF-hsp 70 is to promote the recycling of hsp 70. RF-hsp 70 promotes the dissociation of ADP from hsp 70⅐ADP, but only in the presence of ATP, by lowering the K D of hsp 70 for ATP to a value that approaches the K D of hsp 70 for ADP. This effect of RF-hsp 70 is associated with the stimulation of hsp 70 ATPase activity and involves an interaction with the amino-terminal portion of hsp 70, in contrast to the effect of unfolded protein or polypeptide. RF-hsp 70 and hsp 70(R) are associated with each other in partially purified preparations from rabbit reticulocyte lysate, and the purified proteins reform a complex when incubated together. Finally, RF-hsp 70 acts catalytically to stimulate up to 10-fold the ability of hsp 70 to reactivate heat-denatured luciferase, providing a functional demonstration of its recycling activity.
Our findings are consistent with the following possible cycle: hsp 70⅐ADP, either free or associated with a protein substrate, binds RF-hsp 70, is converted to hsp 70⅐ATP, then dissociates from RF-hsp 70, and finally undergoes ATP hydrolysis with dissociation from a protein substrate and formation of free hsp 70⅐ADP. Although RF-hsp 70 may not be absolutely required for hsp 70 recycling, our results strongly suggest that efficient recycling of hsp 70 is dependent upon RF-hsp 70 action. The characteristics of RF-hsp 70 suggest that its function may be analogous to activities termed DnaJ and GrpE in bacteria (15) and YDJ1 in yeast (16), which stimulate the ATPase activity of the corresponding hsp 70, increase the rate of release of bound ATP or ADP from hsp 70 (GrpE), and enhance dissociation of hsp 70 from unfolded protein in the presence of ATP (YDJ1). As we will show in the following article, however, the primary structure of RF-hsp 70 is not similar to that of these proteins. Instead, it shows appreciable sequence similarity to the yeast protein STI1 (31,32), a protein that is increased with heat stress but whose function is currently unclear (31). We wish to stress that, although we believe RF-hsp 70 has an important role in promoting the folding or chaperonin activity of hsp 70, we do not believe it is the only activity required for optimal hsp 70 function. For example, although RF-hsp 70 increases the rate of renaturation of heat-denatured luciferase by hsp 70 up to 10-fold in an isolated reaction in the absence of other protein factors, it does not fully reconstitute the activity of the intact reticulocyte lysate (Table I and Refs. 18 and 27). One additional participant in the folding reaction mediated by hsp 70 is hsp 90, which enhances the renaturation of luciferase in the presence of hsp 70 (18) but is not as effective as RF-hsp 70 (Fig. 8). Two other possible candidates are hsp 40, a mammalian homolog to bacterial DnaJ (33,34), and the TCP-1 ring complex that is analogous to bacterial GroEL or hsp 60 (35). Both of these activities have been shown recently to participate with hsp 70 in the folding of newly synthesized luciferase in rabbit reticulocyte lysate (36). In addition, the renaturation of denatured luciferase using fractions from rabbit reticulocyte lysate separated by size appeared to be associated with TCP-1 in a study by Nimmesgern and Hartl (27) but not in the study by Schumacher et al. (18). One possible basis for this difference is that in the former study (27), luciferase was denatured completely with 6 M guanidinium chloride; whereas in the latter (18) and in our experiments above, luciferase was inactivated with mild heat and probably was not denatured completely. It is then possible that folding and activation of fully denatured (27) or newly synthesized (36) luciferase may be dependent upon TCP-1, whereas the reactivation of heat-denatured luciferase (18) may not. Clearly, further investigation will be required to resolve this point as well as to determine more precisely what activities in addition to RF-hsp 70 are required for optimal chaperonin function by hsp 70.
We have also observed partial specificity in the ability of RF-hsp 70 to stimulate the ATPase and chaperonin activities of different hsp 70s. RF-hsp 70 is most effective with its own hsp 70, hsp 70(R). RF-hsp 70 is less effective with hsp 70(B), even less effective with hsp 70(H), and ineffective with hsp 70(bn). These differences presumably reflect differences in the primary structure of the various hsp 70s. Although the complete primary structure of hsp 70(R) is not currently known, we have determined the sequence of its carboxyl-terminal 35 residues 2 and found this to be identical to that of the constitutively expressed rat hsp 70 (19,20) and to differ from that of the constitutively expressed hsp 70(B) (37) at only one site. This has verified that hsp 70(R) is the constitutively expressed form of hsp 70, and it suggests that the partial specificity observed could correlate with how close the primary structure of each hsp 70 is to hsp 70(R). The hsp 70(B) may be most similar, hsp 70(H) is probably more divergent since it is the inducible form of hsp 70 with a very different carboxyl-terminal sequence (38), and hsp 70(bn) is probably the most divergent.