A critical role for the proteasome activator PA28 in the Hsp90-dependent protein refolding

The 90-kDa heat shock protein, Hsp90, was previously shown to capture firefly luciferase during thermal inactivation and prevent it from undergoing an irreversible off-pathway aggregation, thereby maintaining it in a folding-competent state. While Hsp90 by itself was not sufficient to refold the denatured luciferase, addition of rabbit reticulocyte lysate remarkably restored the luciferase activity. Here we demonstrate that Hsc70, Hsp40, and the 20 S proteasome activator PA28 are the effective components in reticulocyte lysate. Purified Hsc70, Hsp40, and PA28 were necessary and sufficient to fully reconstitute Hsp90-initiated refolding. Kinetics of substrate binding support the idea that PA28 acts as the molecular link between the Hsp90-dependent capture of unfolded proteins and the Hsc70- and ATP-dependent refolding process.     

Dual Role for Hsc70 in the Biogenesis and Regulation of the Heme-Regulated Kinase of the α Subunit of Eukaryotic Translation Initiation Factor 2

The heme-regulated kinase of the alpha subunit of eukaryotic initiation factor 2 (HRI) is activated in rabbit reticulocyte lysate (RRL) in response to a number of environmental conditions, including heme deficiency, heat shock, and oxidative stress. Activation of HRI causes an arrest of initiation of protein synthesis. Recently, we have demonstrated that the heat shock cognate protein Hsc70 negatively modulates the activation of HRI in RRL in response to these environmental conditions. Hsc70 is also known to be a critical component of the Hsp90 chaperone machinery in RRL, which plays an obligatory role for HRI to acquire and maintain a conformation that is competent to activate. Using de novo-synthesized HRI in synchronized pulse-chase translations, we have examined the role of Hsc70 in the regulation of HRI biogenesis and activation. Like Hsp90, Hsc70 interacted with nascent HRI and HRI that was matured to a state which was competent to undergo stimulus-induced activation (mature-competent HRI). Interaction of HRI with Hsc70 was required for the transformation of HRI, as the Hsc70 antagonist clofibric acid inhibited the folding of HRI into a mature-competent conformation. Unlike Hsp90, Hsc70 also interacted with transformed HRI. Clofibric acid disrupted the interaction of Hsc70 with transformed HRI that had been matured and transformed in the absence of the drug. Disruption of Hsc70 interaction with transformed HRI in heme-deficient RRL resulted in its hyperactivation. Furthermore, activation of HRI in response to heat shock or denatured proteins also resulted in a similar blockage of Hsc70 interaction with transformed HRI. These results indicate that Hsc70 is required for the folding and transformation of HRI into an active kinase but is subsequently required to negatively attenuate the activation of transformed HRI.     

Modulation of the chaperone activities of Hsc70/Hsp40 by Hsp105alpha and Hsp105beta

Hsp105alpha and Hsp105beta are stress proteins found in various mammals including human, mouse, and rat, which belong to the Hsp105/Hsp110 protein family. To elucidate their physiological functions, we examined here the chaperone activity of these stress proteins. Hsp105alpha and Hsp105beta prevented the aggregation of firefly luciferase during thermal denaturation, whereas the thermally denatured luciferase was not reactivated by itself or by rabbit reticulocyte lysate (RRL). On the other hand, Hsp105alpha and Hsp105beta suppressed the reactivation of thermally denatured luciferase by RRL and of chemically denatured luciferase by Hsc70/Hsp40 or RRL. Furthermore, although Hsp105alpha and Hsp105beta did not show ATPase activity, the addition of Hsp105alpha or Hsp105beta to Hsc70/Hsp40 enhanced the amount of hydrolysis of ATP greater than that of the Hsp40-stimulated Hsc70 ATPase activity. These findings suggest that Hsp105alpha and Hsp105beta are not only chaperones that prevent thermal aggregation of proteins, but also regulators of the Hsc70 chaperone system in mammalian cells.     

Multiple 40-kDa heat-shock protein chaperones function in Tom70-dependent mitochondrial import

Mitochondrial preproteins that are imported via the translocase of the mitochondrial outer membrane (Tom)70 receptor are complexed with cytosolic chaperones before targeting to the mitochondrial outer membrane. The adenine nucleotide transporter (ANT) follows this pathway, and its purified mature form is identical to the preprotein. Purified ANT was reconstituted with chaperones in reticulocyte lysate, and bound proteins were identified by mass spectrometry. In addition to 70-kDa heat-shock cognate protein (Hsc70) and 90-kDa heat-shock protein (Hsp90), a specific subset of cochaperones were found, but no mitochondria-specific targeting factors were found. Interestingly, three different Hsp40-related J-domain proteins were identified: DJA1, DJA2, and DJA4. The DJAs bound preproteins to different extents through their C-terminal regions. DJA dominant-negative mutants lacking the N-terminal J-domains impaired mitochondrial import. The mutants blocked the binding of Hsc70 to preprotein, but with varying efficiency. The DJAs also showed significant differences in activation of the Hsc70 ATPase and Hsc70-dependent protein refolding. In HeLa cells, the DJAs increased new protein folding and mitochondrial import, although to different extents. No single DJA was superior to the others in all aspects, but each had a profile of partial specialization. The Hsp90 cochaperones p23 and Aha1 also regulated Hsp90-preprotein interactions. We suggest that multiple cochaperones with similar yet partially specialized properties cooperate in optimal chaperone-preprotein complexes.     

Neurospora crassa FKBP22 is a novel ER chaperone and functionally cooperates with BiP

FK506 binding proteins (FKBPs) belong to the family of peptidyl prolyl cis-trans isomerases (PPIases) catalyzing the cis/trans isomerisation of Xaa-Pro bonds in oligopeptides and proteins. FKBPs are involved in folding, assembly and trafficking of proteins. However, only limited knowledge is available about the roles of FKBPs in the endoplasmic reticulum (ER) and their interaction with other proteins. Here we show the ER located Neurospora crassa FKBP22 to be a dimeric protein with PPIase and a novel chaperone activity. While the homodimerization of FKBP22 is mediated by its carboxy-terminal domain, the amino-terminal domain is a functional FKBP domain. The chaperone activity is mediated by the FKBP domain but is exhibited only by the full-length protein. We further demonstrate a direct interaction between FKBP22 and BiP, the major Hsp70 chaperone in the ER. The binding to BiP is mediated by the FKBP domain of FKBP22. Interestingly BiP enhances the chaperone activity of FKBP22. Both proteins form a stable complex with an unfolded substrate protein and thereby prevent its aggregation. These results suggest that BiP and FKBP22 form a folding helper complex with a high chaperoning capacity in the ER of Neurospora crassa.     

FKBP38 peptidylprolyl isomerase promotes the folding of cystic fibrosis transmembrane conductance regulator in the endoplasmic reticulum

FK506-binding protein 38 (FKBP38), a membrane-anchored, tetratricopeptide repeat (TPR)-containing immunophilin, associates with nascent plasma membrane ion channels in the endoplasmic reticulum (ER). It promotes the maturation of the human ether-à-go-go-related gene (HERG) potassium channel and maintains the steady state level of the cystic fibrosis transmembrane conductance regulator (CFTR), but the underlying mechanisms remain unclear. Using a combination of steady state and pulse-chase analyses, we show that FKBP38 knockdown increases protein synthesis but inhibits the post-translational folding of CFTR, leading to reduced steady state levels of CFTR in the ER, decreased processing, and impaired cell surface functional expression in Calu-3 human airway epithelial cells. The membrane anchorage of FKBP38 is necessary for the inhibition of protein synthesis but not for CFTR post-translational folding. In contrast, the peptidylprolyl cis/trans isomerase active site is utilized to promote CFTR post-translational folding but is not important for regulation of protein synthesis. Uncoupling FKBP38 from Hsp90 by substituting a conserved lysine in the TPR domain modestly enhances CFTR maturation and further reduces its synthesis. Removing the N-terminal glutamate-rich domain (ERD) slightly enhances CFTR synthesis but reduces its maturation, suggesting that the ERD contributes to FKBP38 biological activities. Our data support a dual role for FKBP38 in regulating CFTR synthesis and post-translational folding. In contrast to earlier prediction but consistent with in vitro enzymological studies, FKBP38 peptidylprolyl cis/trans isomerase plays an important role in membrane protein biogenesis on the cytoplasmic side of the ER membrane, whose activity is negatively regulated by Hsp90 through the TPR domain.     

A ribosome-associated peptidyl-prolyl cis/trans isomerase identified as the trigger factor.

Peptidyl-prolyl cis/trans isomerases (PPIases) are enzymes that catalyse protein folding both in vitro and in vivo. We isolated a peptidyl-prolyl cis/trans isomerase (PPIase) which is specifically associated with the 50S subunit of the Escherichia coli ribosome. This association was abolished by adding at least 1.5 M LiCl. Sequencing the N-terminal amino acids in addition to three proteolytic fragments totalling 62 amino acids revealed that this PPIase is identical to the E.coli trigger factor. A comparison of the amino acid sequence of trigger factor with those of other PPIase families shows little similarities, suggesting that trigger factor may represent an additional family of PPIases. Trigger factor was purified to homogeneity on a preparative scale from E.coli and its enzymatic properties were studied. In its activity towards oligopeptide substrates, the trigger factor resembles the FK506-binding proteins (FKBPs). Additionally, the pattern of subsite specificities with respect to the amino acid preceding proline in Suc-Ala-Xaa-Pro-Phe-4-nitroanilides is reminiscent of FKBPs. However, the PPIase activity of the trigger factor was not inhibited by either FK506 or by cyclosporin A at concentrations up to 100 microM. In vitro, the trigger factor catalysed the proline-limited refolding of a variant of RNase T1 much better than all other PPIases that have been examined so far.     

Binding to chaperones allows import of a purified mitochondrial precursor into mitochondria

Refolding of the acid-unfolded precursor to mitochondrial aspartate aminotransferase (pmAAT) is inhibited when cytosolic Hsc70 is included in the refolding reaction (Artigues, A., Iriarte, A., and Martinez-Carrion, M. (1997) J. Biol. Chem. 272, 16852-16861). At low molar excess of Hsc70 pmAAT is recovered in insoluble aggregates containing equal amounts of Hsc70. However, in the presence of a large excess of Hsc70, refolding of pmAAT is still arrested, but the enzyme remains in solution. Similar behavior was observed with two other cytosolic chaperones, bovine Hsp90 and yeast Ydj1. Coimmunoprecipitation of pmAAT using Hsc70 antibodies confirmed the formation of soluble Hsc70-pmAAT complexes at high concentrations of the chaperone. Data from analytical centrifugation, sedimentation in glycerol gradients, and partial purification of the soluble complexes indicate that multiple Hsc70 molecules bind per pmAAT polypeptide chain. The absence of catalytic activity together with the protease susceptibility of pmAAT bound to Hsc70, Hsp90, or Ydj1 suggest that these chaperones bind and maintain pmAAT in a partially unfolded state, analogous to the import-competent conformation of the protein synthesized in cell-free extracts. Remarkably, the purified pmAAT bound to Hsc70 or Ydj1, but not to Hsp90, is imported by isolated mitochondria in a reticulocyte lysate-dependent manner. Thus, both Hsc70 and Ydj1 can trap an import-competent folding intermediate of pmAAT, but productive binding and import into mitochondria require the collaboration of additional cytosolic factors from the lysate.     

Novel inhibitors of heat shock protein Hsp70-mediated luciferase refolding that bind to DnaJ

Inhibitors of both heat shock proteins Hsp90 and Hsp70 have been identified in assays measuring luciferase refolding containing rabbit reticulocyte lysate or purified chaperone components. Here, we report the discovery of a series of phenoxy-N-arylacetamides that disrupt Hsp70-mediated luciferase refolding by binding to DnaJ, the bacterial homolog of human Hsp40. Inhibitor characterization experiments demonstrated negative cooperativity with respect to DnaJ and luciferase concentration, but varying the concentration of ATP had no effect on potency. Thermal shift analysis suggested a direct interaction with DnaJ, but not with Hsp70. These compounds may be useful tools for studying DnaJ/Hsp40 in various cellular processes.     

The Hdj-2/Hsc70 chaperone pair facilitates early steps in CFTR biogenesis

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel constructed from two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBD) and a regulatory (R) domain. The NBDs and R-domain are cytosolic and how they are assembled with the MSDs to achieve the native CFTR structure is not clear. Human DnaJ 2 (Hdj-2) is a co-chaperone of heat shock cognate 70 (Hsc70) which is localized to the cytosolic face of the ER. Whether Hdj-2 directs Hsc70 to facilitate the assembly of cytosolic regions on CFTR was investigated. We report that immature ER forms of CFTR and DeltaF508 CFTR can be isolated in complexes with Hdj-2 and Hsc70. The DeltaF508 mutation is localized in NBD1 and causes the CFTR to misfold. Levels of complex formation between DeltaF508 CFTR and Hdj-2/Hsp70 were approximately 2-fold higher than those with CFTR. The earliest stage at which Hdj-2/Hsc70 could bind CFTR translation intermediates coincided with the expression of NBD1 in the cytosol. Interestingly, complex formation between Hdj-2 and nascent CFTR was greatly reduced after expression of the R-domain. In experiments with purified components, Hdj-2 and Hsc70 acted synergistically to suppress NBD1 aggregation. Collectively, these data suggest that Hdj-2 and Hsc70 facilitate early steps in CFTR assembly. A putative step in the CFTR folding pathway catalyzed by Hdj-2/Hsc70 is the formation of an intramolecular NBD1-R-domain complex. Whether this step is defective in the biogenesis of DeltaF508 CFTR will be discussed.     

Unc45b forms a cytosolic complex with Hsp90 and targets the unfolded myosin motor domain

Myosin folding and assembly in striated muscle is mediated by the general chaperones Hsc70 and Hsp90 and a myosin specific co-chaperone, UNC45. Two UNC45 genes are found in vertebrates, including a striated muscle specific form, Unc45b. We have investigated the role of Unc45b in myosin folding. Epitope tagged murine Unc45b (Unc45b(Flag)) was expressed in muscle and non-muscle cells and bacteria, isolated and characterized. The protein is a soluble monomer in solution with a compact folded rod-shaped structure of approximately 19 nm length by electron microscopy. When over-expressed in striated muscle cells, Unc45b(Flag) fractionates as a cytosolic protein and isolates as a stable complex with Hsp90. Purified Unc45b(Flag) re-binds Hsp90 and forms a stable complex in solution. The endogenous Unc45b in muscle cell lysates is also found associated with Hsp90. The Unc45b(Flag)/Hsp90 complex binds the partially folded myosin motor domain when incubated with myosin subfragments synthesized in a reticulocyte lysate. This binding is independent of the myosin rod or light chains. Unc45b(Flag) does not bind native myosin subfragments consistent with a chaperone function. More importantly, Unc45b(Flag) enhances myosin motor domain folding during de novo motor domain synthesis indicating that it has a direct role in myosin maturation. Thus, mammalian Unc45b is a cytosolic protein that forms a stable complex with Hsp90, selectively binds the unfolded conformation of the myosin motor domain, and promotes motor domain folding.     

Functional divergence between co-chaperones of Hsc70

The ATPase cycle of the chaperone Hsc70 is regulated by co-chaperones; Hsp40/DnaJ-related proteins stimulate ATP hydrolysis by Hsc70 and can bind unfolded polypeptides themselves. Conversely, various nucleotide exchange factors (NEFs) stimulate ADP-ATP exchange by Hsc70. We analyzed the purified Hsp40-related co-chaperones DJA1 (Hdj2) and DJA2 (Hdj3) and found that they had a distinct pattern of binding to a range of polypeptides. DJA2 alone could stimulate Hsc70-mediated refolding of luciferase in the absence of NEF, whereas DJA1 was much less active. The addition of the Bag1 NEF increased refolding by Hsc70 and DJA2, as did the newly characterized NEF Hsp110, but each NEF had a different optimal concentration ratio to Hsc70. Notably, the NEF HspBP1 could not increase refolding by Hsc70 and DJA2 at any concentration, and none of the NEFs improved the refolding activity with DJA1. Instead, DJA1 was inhibitory of refolding with DJA2 and Hsc70. All combinations of DJA1 or DJA2 with the three NEFs stimulated the Hsc70 ATPase rate, although Hsp110 became less effective with increasing concentrations. A chimeric DJA2 having its Hsc70-stimulatory J domain replaced with that of DJA1 was functional for polypeptide binding and ATPase stimulation of Hsc70. However, it could not support efficient Hsc70-mediated refolding and also inhibited refolding with DJA2 and Hsc70. These results suggest a more complex model of Hsc70 mechanism than has been previously thought, with notable functional divergence between Hsc70 co-chaperones.     

Bag-1M accelerates nucleotide release for human Hsc70 and Hsp70 and can act concentration-dependent as positive and negative cofactor.

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 P(i). Surprisingly, under physiological conditions, i.e. low Bag-1M concentrations and presence of P(i), 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.     

Differential inhibition of Hsc70 activities by two Hsc70-binding peptides

The ability of two high-affinity Hsc70-binding peptides [FYQLALT (peptide-Phi) and NIVRKKK (peptide-K)] to differentially inhibit Hsc70-dependent processes in rabbit reticulocyte lysate (RRL) was examined. Both peptide-Phi and peptide-K inhibited chaperone-dependent renaturation of luciferase in RRL. Peptide-Phi, but not peptide-K, blocked Hsp90/Hsc70-dependent transformation of the heme-regulated eIF2 alpha kinase (HRI) into an active, heme-regulatable kinase. In contrast, peptide-K, but not peptide-Phi, inhibited Hsc70-mediated suppression of the activation of mature-transformed HRI. Furthermore, HDJ2 (Human DnaJ homologue 2), but not HDJ1, potentiated the ability of Hsc70 to suppress the activation of HRI in RRL. Mechanistically, peptide-K inhibited, while peptide-Phi enhanced, HDJ2-induced stimulation of Hsc70 ATPase activity in vitro. The data presented support the hypotheses that peptide-Phi acts to inhibit Hsc70 function by binding to the hydrophobic peptide-binding cleft of Hsc70, while peptide-K acts through binding to a site that modulates the interaction of Hsc70 with DnaJ homologues. Overall, the data indicate that peptide-Phi and peptide-K have differential effects on Hsc70 functions under quasi-physiological conditions in RRL, and suggest that therapeutically valuable peptide mimetics can be designed to inhibit specific functions of Hsc70.     

Lipid interaction differentiates the constitutive and stress-induced heat shock proteins Hsc70 and Hsp70

Heat shock proteins play a major role in the process of protein folding, and they have been termed molecular chaperones. Two members of the Hsp70 family, Hsc70 and Hsp70, have a high degree of sequence homology. But they differ in their expression pattern. Hsc70 is constitutively expressed, whereas Hsp70 is stress inducible. These 2 proteins are localized in the cytosol and the nucleus. In addition, they have also been observed in close proximity to cellular membranes. We have recently reported that Hsc70 is capable of interacting with a lipid bilayer forming ion-conductance channels. In the present study, we found that both Hsc70 and Hsp70 interact with lipids and can be differentiated by their characteristic induction of liposome aggregation. These proteins promote the aggregation of phosphatidylserine liposomes in a time- and protein concentration-dependent manner. Although both proteins are active in this process, the level and kinetics of aggregation are different between them. Calcium ions enhance Hsc70 and Hsp70 liposome aggregation, but the effect is more dramatic for Hsc70 than for Hsp70. Addition of adenosine triphosphate blocks liposome aggregation induced by both proteins. Adenosine diphosphate (ADP) also blocks Hsp70-mediated liposome aggregation. Micromolar concentrations of ADP enhance Hsc70-induced liposome aggregation, whereas at millimolar concentrations the nucleotide has an inhibitory effect. These results confirm those of previous studies indicating that the Hsp70 family can interact with lipids directly. It is possible that the interaction of Hsp70s with lipids may play a role in the folding of membrane proteins and the translocation of polypeptides across membranes.     

FK506-binding protein 51 interacts with Clostridium botulinum C2 toxin and FK506 inhibits membrane translocation of the toxin in mammalian cells

The binary Clostridium botulinum C2 toxin consists of the binding/translocation component C2IIa and the separate enzyme component C2I. C2IIa delivers C2I into the cytosol of eukaryotic target cells where C2I ADP-ribosylates actin. After receptor-mediated endocytosis of the C2IIa/C2I complex, C2IIa forms pores in membranes of acidified early endosomes and unfolded C2I translocates through the pores into the cytosol. Membrane translocation of C2I is facilitated by the activities of host cell chaperone Hsp90 and the peptidyl-prolyl cis/trans isomerase (PPIase) cyclophilin A. Here, we demonstrated that Hsp90 co-precipitates with C2I from lysates of C2 toxin-treated cells and identified the FK506-binding protein (FKBP) 51 as a novel interaction partner of C2I in vitro and in intact mammalian cells. Prompted by this finding, we used the specific pharmacological inhibitor FK506 to investigate whether the PPIase activity of FKBPs plays a role during membrane translocation of C2 toxin. Treatment of cells with FK506 protected cultured cells from intoxication with C2 toxin. Moreover, FK506 inhibited the pH-dependent translocation of C2I across membranes into the cytosol but did not interfere with the enzyme activity of C2I or binding of C2 toxin to cells. Furthermore, FK506 treatment delayed intoxication with the related binary actin ADP-ribosylating toxins from Clostridium perfringens (iota toxin) and Clostridium difficile (CDT) but not with the Rho-glucosylating Clostridium difficile toxin A (TcdA). In conclusion, our results support the hypothesis that clostridial binary actin-ADP-ribosylating toxins share a specific FKBP-dependent translocation mechanism during their uptake into mammalian cells.     

A precursor-specific role for Hsp40/Hsc70 during tail-anchored protein integration at the endoplasmic reticulum

Tail-anchored (TA) protein synthesis at the endoplasmic reticulum (ER) represents a distinct and novel process that provides a paradigm for understanding post-translational membrane insertion in eukaryotes. The major route for delivering TA proteins to the ER requires both ATP and one or more cytosolic factors that facilitate efficient membrane insertion. Until recently, the identity of these cytosolic components was elusive, but two candidates have now been suggested to promote ATP-dependent TA protein integration. The first is the cytosolic chaperone complex of Hsp40/Hsc70, and the second is a novel ATPase denoted Asna-1 or TRC40. In this study we focus on the role of the Hsp40/Hsc70 complex in promoting TA protein biogenesis at the ER. We show that the membrane integration of most TA proteins is stimulated by Hsp40/Hsc70 when using purified components and a reconstituted system. In contrast, when both Hsp40/Hsc70 and Asna-1/TRC40 are provided as a complete system, small molecule inhibition of Hsp40/Hsc70 indicates that only a subset of TA proteins are obligatory clients for this chaperone-mediated delivery route. We show that the hydrophobicity of the TA region dictates whether a precursor is delivered to the ER via the Hsp40/Hsc70 or Asna-1/TRC40-dependent route, and we conclude that these distinct cytosolic ATPases are responsible for two different ATP-dependent pathways of TA protein biogenesis.     

Distinct isoforms of the cofactor BAG-1 differentially affect Hsc70 chaperone function

In the mammalian cytosol and nucleus the activity of the molecular chaperone Hsc70 is regulated by chaperone cofactors that modulate ATP binding and hydrolysis by Hsc70. Among such cofactors is the anti-apoptotic protein BAG-1. Remarkably, BAG-1 is expressed as multiple isoforms, which are distinguished by their amino termini. We investigated whether distinct isoforms differ with respect to their Hsc70-regulating activity. By comparing the mainly cytosolic isoforms BAG-1M and BAG-1S, opposite effects of the two isoforms were observed in chaperone-assisted folding reactions. Whereas BAG-1M was found to inhibit the Hsc70-mediated refolding of nonnative polypeptide substrates, the BAG-1S isoform stimulated Hsc70 chaperone activity. The opposite effects are not due to differences in the regulation of the ATPase activity of Hsc70 by the two isoforms. Both isoforms stimulated ATP hydrolysis by Hsc70 in an Hsp40-dependent manner through an acceleration of ADP-ATP exchange. Our results reveal that the different amino termini of the distinct BAG-1 isoforms determine the outcome of an Hsc70-mediated folding event, most likely by transiently interacting with the polypeptide substrate. Employing isoforms of a cofactor with different substrate binding properties appears to provide the means to influence the chaperone function of Hsc70 in addition to modulating its ATPase cycle.