Intermolecular Interactions between Hsp90 and Hsp70

Members of the Hsp90 and Hsp70 families of molecular chaperones are imp\ortant for the maintenance of protein homeostasis and cellular recovery following environmental stresses, such as heat and oxidative stress. Moreover, the two chaperones can collaborate in protein remodeling and activation. In higher eukaryotes, Hsp90 and Hsp70 form a functionally active complex with Hop (Hsp90–Hsp70 organizing protein) acting as a bridge between the two chaperones. In bacteria, which do not contain a Hop homolog, Hsp90 and Hsp70, DnaK, directly interact during protein remodeling. Although yeast possesses a Hop-like protein, Sti1, Hsp90, and Hsp70 can directly interact in yeast in the absence of Sti1. Previous studies showed that residues in the middle domain of Escherichia coli Hsp90 are important for interaction with the J-protein binding region of DnaK. The results did not distinguish between the possibility that (i) these sites were involved in direct interaction and (ii) the residues in these sites participate in conformational changes which are transduced to other sites on Hsp90 and DnaK that are involved in the direct interaction. Here we show by crosslinking experiments that the direct interaction is between a site in the middle domain of Hsp90 and the J-protein binding site of Hsp70 in both E. coli and yeast. Moreover, J-protein promotes the Hsp70–Hsp90 interaction in the presence of ATP, likely by converting Hsp70 into the ADP-bound conformation. The identification of the protein–protein interaction site is anticipated to lead to a better understanding of the collaboration between the two chaperones in protein remodeling.     

The Hsp40 J-domain Stimulates Hsp70 When Tethered by the Client to the ATPase Domain

The Escherichia coli Hsp40 DnaJ uses its J-domain (Jd) to couple ATP hydrolysis and client protein capture in Hsp70 DnaK. Fusion of the Jd to peptide p5 (as in Jdp5) dramatically increases the apparent affinity of the p5 moiety for DnaK in the presence of ATP, and Jdp5 stimulates ATP hydrolysis in DnaK by several orders of magnitude. NMR experiments with [¹⁵N]Jdp5 demonstrated that the peptide tethers the Jd to the ATPase domain. Thus, ATP hydrolysis and client protein binding in DnaK are coupled principally through the association of the client with DnaJ. Overexpression of a recombinant Jd was specifically toxic to cells that simultaneously expressed DnaK. No toxicity was observed when overexpressing Jdp5 or mutant Jd or when co-overexpressing the Jd and the nucleotide exchange factor GrpE. The results suggest that the Jd shifts DnaK to a client-bound form by stimulating the DnaK ATPase but only when the Jd is brought to DnaK by a client-Hsp40 complex.     

Interaction of the targeting sequence of chloroplast precursors with Hsp70 molecular chaperones.

We have analyzed the interaction of DnaK and plant Hsp70 proteins with the wild-type ferredoxin-NADP+ reductase precursor (preFNR) and mutants containing amino-acid replacements in the targeting sequence. Using an algorithm already developed [Rüdiger, S., Germeroth, L., Schneider-Mergener, J. & Bukau, B. (1997) EMBO J. 16, 1501-1507] we observed that 75% of the 727 plastid precursor proteins analyzed contained at least one site with high likelihood of DnaK binding in their transit peptides. Statistical analysis showed a decrease of DnaK binding site frequency within the first 15 amino-acid residues of the transit peptides. Using fusion proteins we detected the interaction of DnaK with the transit peptide of the folded preFNR but not with the mature region of the protein. Discharge of DnaK from the presequence was favored by addition of MgATP. When a putative DnaK binding site was artificially added at the N-terminus of the mature protein, we observed formation of complexes with bacterial and plant Hsp70 molecular chaperones. Reducing the likelihood of DnaK binding by directed mutagenesis of the presequence increased the release of bound DnaK. The Hsp70 proteins from plastids and plant cell cytosol also interacted with the preFNR transit peptide. Overall results are discussed in the context of the proposed models to explain the organelle protein import.     

J-domain Proteins form Binary Complexes with Hsp90 and Ternary Complexes with Hsp90 and Hsp70

Hsp90 and Hsp70 are highly conserved molecular chaperones that help maintain proteostasis by participating in protein folding, unfolding, remodeling and activation of proteins. Both chaperones are also important for cellular recovery following environmental stresses. Hsp90 and Hsp70 function collaboratively for the remodeling and activation of some client proteins. Previous studies using E. coli and S. cerevisiae showed that residues in the Hsp90 middle domain directly interact with a region in the Hsp70 nucleotide binding domain, in the same region known to bind J-domain proteins. Importantly, J-domain proteins facilitate and stabilize the interaction between Hsp90 and Hsp70 both in E. coli and S. cerevisiae. To further explore the role of J-domain proteins in protein reactivation, we tested the hypothesis that J-domain proteins participate in the collaboration between Hsp90 and Hsp70 by simultaneously interacting with Hsp90 and Hsp70. Using E. coli Hsp90, Hsp70 (DnaK), and a J-domain protein (CbpA), we detected a ternary complex containing all three proteins. The interaction involved the J-domain of CbpA, the DnaK binding region of E. coli Hsp90, and the J-domain protein binding region of DnaK where Hsp90 also binds. Additionally, results show that E. coli Hsp90 interacts with E. coli J-domain proteins, DnaJ and CbpA, and that yeast Hsp90, Hsp82, interacts with a yeast J-domain protein, Ydj1. Together these results suggest that the complexes may be transient intermediates in the pathway of collaborative protein remodeling by Hsp90 and Hsp70.     

Structure-function analysis of HscC,the Escherichia coli member of a novel subfamily of specialized Hsp70 chaperones

Hsp70 chaperones assist protein folding processes through nucleotide-controlled cycles of substrate binding and release. In our effort to understand the structure-function relationship within the Hsp70 family of proteins, we characterized the Escherichia coli member of a novel Hsp70 subfamily, HscC, and identified considerable differences to the well studied E. coli homologue, DnaK, which together suggest that HscC is a specialized chaperone. The basal ATPase cycle of HscC had k(cat) and K(m) values that were 8- and 10,000-fold higher than for DnaK. The HscC ATPase was not affected by the nucleotide exchange factor of DnaK GrpE and stimulated 8-fold by DjlC, a DnaJ protein with a putative transmembrane domain, but not by other DnaJ proteins tested. Substrate binding dynamics and substrate specificity differed significantly between HscC and DnaK. These differences are explicable by distinct structural variations. HscC does not have general chaperone activity because it did not assist refolding of a denatured model substrate. In vivo, HscC failed to complement temperature sensitivity of DeltadnaK cells. Deletion of hscC caused a slow growth phenotype that was suppressed after several generations. Triple knock-outs of all E. coli genes encoding Hsp70 proteins (DeltadnaK DeltahscA DeltahscC) were viable, indicating that Hsp70 proteins are not strictly essential for viability. An extensive search for DeltahscC phenotypes revealed a hypersensitivity to Cd(2+) ions and UV irradiation, suggesting roles of HscC in the cellular response to these stress treatments. Together our data show that the Hsp70 structure exhibits an astonishing degree of adaptive variations to accommodate requirements of a specialized function.     

Diurnal regulation of Hsp70s in leaf tissue

Steady-state mRNA levels for three Hsp70s were found to be regulated by a distinctive light/dark mechanism in spinach leaves. Messenger RNAs for the chloroplast stromal and two cytosolic forms displayed a diurnal expression pattern under isothermal conditions that appeared to be independent of circadian control. While protein blot data showed relatively constant Hsp70 protein levels, the higher Hsp70 mRNA levels in the light paralleled the diurnal cycle of total cell protein synthesis. Fractionation studies showed that the major cytosolic Hsp70 cognate group was associated with polysomes. Therefore, the variation of Hsp70 mRNAs is consistent with the diurnal metabolic activity of plant photosynthetic cells in which the demand of protein biogenesis for chaperone function and tissue temperature are highest during the day.     

Successive and synergistic action of the Hsp70 and Hsp100 chaperones in protein disaggregation

Proteins belonging to the B-subtype of the Hsp100/Clp chaperone family execute a crucial role in cellular thermotolerance. They cooperate with the Hsp70 chaperones in reactivation of thermally aggregated protein substrates. We investigated the initial events of the disaggregation reaction in real time using denatured, aggregated green fluorescent protein (GFP) as a substrate. Bacterial Hsp70 (DnaK), its co-chaperones (DnaJ and GrpE), and Hsp100 (ClpB) were incubated with aggregated GFP, and the increase in GFP fluorescence was monitored. Incubation of aggregated GFP with DnaK/DnaJ/GrpE but not with ClpB resulted in the rapid initiation of the disaggregation reaction. Under the same conditions a complex between DnaK, DnaJ, and GFP, but not ClpB, was formed as demonstrated by sedimentation analysis and light scattering experiments. Chaperone-dependent disaggregation of chemically denatured aggregated luciferase showed that, similar to GFP disaggregation, incubation with Hsp70 results in the rapid start of the reactivation reaction. For both aggregated GFP and luciferase, incubation with Hsp70 chaperones changes the initial rate but not the overall efficiency or rate of the refolding reaction. Our results clearly demonstrate that the interaction of DnaK and its co-chaperones with aggregated substrate is the rate-limiting reaction at the initial steps of disaggregation.     

Hsc62, Hsc56, and GrpE,the third Hsp70 chaperone system of Escherichia coli

Hsc62 is the third Hsp70 homolog of Escherichia coli, which we found previously. Hsc62 is structurally and biochemically similar to DnaK, but hscC gene encoding Hsc62 did not compensate for the defects in the dnaK-null mutant of E. coli MC4100 strain. We cloned the ybeV gene and purified the gene product named Hsc56, a 55,687-Da protein with a J-domain like sequence. Hsc56 stimulated the ATPase activity of only Hsc62 but not those of the other Hsp70 homologs, DnaK and Hsc66. Hsc56 contains the -His-Pro-Glu- sequence corresponding to the His-Pro-Asp motif in DnaJ, which is indispensable for DnaJ to interact with DnaK. Conversion of -His-Pro-Glu- to -Ala-Ala-Ala- abolished the ability of Hsc56 to stimulate the ATPase activity of Hsc62. GrpE, a nucleotide exchange factor for DnaK, also stimulated the ATPase activity of Hsc62 in the presence of Hsc56. Hsc62-Hsc56-GrpE is probably a new Hsp70 chaperone system of E. coli.     

Multistep mechanism of substrate binding determines chaperone activity of Hsp70

The 70 kDa heat shock proteins (the Hsp70 family) assist refolding of their substrates through ATP-controlled binding. We have analyzed mutants of DnaK, an Hsp70 homolog, altered in key residues of its substrate binding domain. Substrate binding occurs by a dynamic mechanism involving: a hydrophobic pocket for a single residue that is crucial for affinity, a two-layered closing device involving independent action of an alpha-helical lid and an arch, and a superimposed allosteric mechanism of ATP-controlled opening of the substrate binding cavity that operates largely through a beta-structured subdomain. Correlative evidence from mutational analysis suggests that the ADP and ATP states of DnaK differ in the frequency of the conformational changes in the alpha-helical lid and beta-domain that cause opening of the substrate binding cavity. The affinity for substrates, as defined by this mechanism, determines the efficiency of DnaJ-mediated and ATP hydrolysis mediated locking-in of substrates and chaperone activity of DnaK.     

DnaJ dramatically stimulates ATP hydrolysis by DnaK: insight into targeting of Hsp70 proteins to polypeptide substrates

Most, if not all, of the cellular functions of Hsp70 proteins require the assistance of a DnaJ homologue, which accelerates the weak intrinsic ATPase activity of Hsp70 and serves as a specificity factor by binding and targeting specific polypeptide substrates for Hsp70 action. We have used pre-steady-state kinetics to investigate the interaction of the Escherichia coli DnaJ and DnaK proteins, and the effects of DnaJ on the ATPase reaction of DnaK. DnaJ accelerates hydrolysis of ATP by DnaK to such an extent that ATP binding by DnaK becomes rate-limiting for hydrolysis. At high concentrations of DnaK under single-turnover conditions, the rate-limiting step is a first-order process, apparently a change of DnaK conformation, that accompanies ATP binding and proceeds at 12-15 min-1 at 25 degrees C and 1-1.5 min-1 at 5 degrees C. By prebinding ATP to DnaK and subsequently adding DnaJ, the effects of this slow step may be bypassed, and the maximal rate-enhancement of DnaJ on the hydrolysis step is approximately 15 000-fold at 5 degrees C. The interaction of DnaJ with DnaK.ATP is likely a rapid equilibrium relative to ATP hydrolysis, and is relatively weak, with a KD of approximately 20 microM at 5 degrees C, and weaker still at 25 degrees C. In the presence of saturating DnaJ, the maximal rate of ATP hydrolysis by DnaK is similar to previously reported rates for peptide release from DnaK.ATP. This suggests that when DnaK encounters a DnaJ-bound polypeptide or protein complex, a significant fraction of such events result in ATP hydrolysis by DnaK and concomitant capture of the polypeptide substrate in a tight complex with DnaK.ADP. Furthermore, a broadly applicable kinetic mechanism for DnaJ-mediated specificity of Hsp70 action arises from these observations, in which the specificity arises largely from the acceleration of the hydrolysis step itself, rather than by DnaJ-dependent modulation of the affinity of Hsp70 for substrate polypeptides.     

Effect of evolution of the C-terminal region on chaperone activity of Hsp70

Dynamic interdomain interactions within the Hsp70 protein is the basis for the allosteric and functional properties of Hsp70s. While Hsp70s are generally conserved in terms of structure, allosteric behavior, and some overlapping functions, Hsp70s also contain variable sequence regions which are related to distinct functions. In the Hsp70 sequence, the part with the greatest sequence variation is the C-terminal α-helical lid subdomain of substrate-binding domain (SBDα) together with the intrinsically disordered region. Dynamic interactions between the SBDα and β-sandwich substrate-binding subdomain (SBDβ) contribute to the chaperone functions of Hsp70s by tuning kinetics of substrate binding. To investigate how the C-terminal region of Hsp70 has evolved from prokaryotic to eukaryotic organisms, we tested whether this region can be exchanged among different Hsp70 members to support basic chaperone functions. We found that this region from eukaryotic Hsp70 members cannot substitute for the same region in Escherichia coli DnaK to facilitate normal chaperone activity of DnaK. In contrast, this region from E. coli DnaK and Saccharomyces cerevisiae Hsp70 (Ssa1 and Ssa4) can partially support some roles of human stress inducible Hsp70 (hHsp70) and human cognate Hsp70 (hHsc70). Our results indicate that the C-terminal region from eukaryotic Hsp70 members cannot properly support SBDα–SBDβ interactions in DnaK, but this region from DnaK/Ssa1/Ssa4 can still form some SBDα–SBDβ interactions in hHsp70 or hHsc70, which suggests that the mode for SBDα–SBDβ interactions is different in prokaryotic and eukaryotic Hsp70 members. This study provides new insight in the divergency among different Hsp70 homologs and the evolution of Hsp70s.     

Bacterial Hsp70 (DnaK) and mammalian Hsp70 interact differently with lipid membranes

The cellular response to stress is orchestrated by the expression of a family of proteins termed heat shock proteins (hsp) that are involved in the stabilization of basic cellular processes to preserve cell viability and homeostasis. The bulk of hsp function occurs within the cytosol and subcellular compartments. However, some hsp have also been found outside cells released by an active mechanism independent of cell death. Extracellular hsp act as signaling molecules directed at activating a systemic response to stress. The export of hsp requires the translocation from the cytosol into the extracellular milieu across the plasma membrane. We have proposed that membrane insertion is the initial step in this export process. We investigated the interaction of the major inducible hsp from mammalian (Hsp70) and bacterial (DnaK) species with liposomes. We found that mammalian Hsp70 displayed a high specificity for negatively charged phospholipids, such as phosphatidyl serine, whereas DnaK interacted with all lipids tested regardless of the charge. Both proteins were inserted into the lipid bilayer as demonstrated by resistance to acid or basic washes that was confirmed by partial protection from proteolytic cleavage. Several regions of mammalian Hsp70 were inserted into the membrane with a small portion of the N-terminus end exposed to the outer phase of the liposome. In contrast, the N-terminus end of DnaK was inserted into the membrane, exposing the C-terminus end outside the liposome. Mammalian Hsp70 was found to make high oligomeric complexes upon insertion into the membranes whereas DnaK only formed dimers within the lipid bilayer. These observations suggest that both Hsp70s interact with lipids, but mammalian Hsp70 displays a high degree of specificity and structure as compared with the bacterial form.     

Genomic analysis of the Hsp70 superfamily in Arabidopsis thaliana

The Arabidopsis genome contains at least 18 genes encoding members of the 70-kilodalton heat shock protein (Hsp70) family, 14 in the DnaK subfamily and 4 in the Hsp110/SSE subfamily. While the Hsp70s are highly conserved, a phylogenetic analysis including all members of this family in Arabidopsis and in yeast indicates the homology of Hsp70s in the subgroups, such as those predicted to localize in the same subcellular compartment and those similar to the mammalian Hsp110 and Grp170. Gene structure and genome organization suggest duplication in the origin of some genes. The Arabidopsis hsp70s exhibit distinct expression profiles; representative genes of the subgroups are expressed at relatively high levels during specific developmental stages and under thermal stress.     

Structural features required for the interaction of the Hsp70 molecular chaperone DnaK with its cochaperone DnaJ.

Hsp70 family members together with their Hsp40 cochaperones function as molecular chaperones, using an ATP-controlled cycle of polypeptide binding and release to mediate protein folding. Hsp40 plays a key role in the chaperone reaction by stimulating the ATPase activity and activating the substrate binding of Hsp70. We have explored the interaction between the Escherichia coli Hsp70 family member, DnaK, and its cochaperone partner DnaJ. Our data show that the binding of ATP, subsequent conformational changes in DnaK, and DnaJ-stimulated ATP hydrolysis are all required for the formation of a DnaK-DnaJ complex as monitored by Biacore analysis. In addition, our data imply that the interaction of the J-domain with DnaK depends on the substrate binding state of DnaK.     

DnaK as Antibiotic Target: Hot Spot Residues Analysis for Differential Inhibition of the Bacterial Protein in Comparison with the Human HSP70

DnaK, the bacterial homolog of human Hsp70, plays an important role in pathogens survival under stress conditions, like antibiotic therapies. This chaperone sequesters protein aggregates accumulated in bacteria during antibiotic treatment reducing the effect of the cure. Although different classes of DnaK inhibitors have been already designed, they present low specificity. DnaK is highly conserved in prokaryotes (identity 50–70%), which encourages the development of a unique inhibitor for many different bacterial strains. We used the DnaK of Acinetobacter baumannii as representative for our analysis, since it is one of the most important opportunistic human pathogens, exhibits a significant drug resistance and it has the ability to survive in hospital environments. The E.coli DnaK was also included in the analysis as reference structure due to its wide diffusion. Unfortunately, bacterial DnaK and human Hsp70 have an elevated sequence similarity. Therefore, we performed a differential analysis of DnaK and Hsp70 residues to identify hot spots in bacterial proteins that are not present in the human homolog, with the aim of characterizing the key pharmacological features necessary to design selective inhibitors for DnaK. Different conformations of DnaK and Hsp70 bound to known inhibitor-peptides for DnaK, and ineffective for Hsp70, have been analysed by molecular dynamics simulations to identify residues displaying stable and selective interactions with these peptides. Results achieved in this work show that there are some residues that can be used to build selective inhibitors for DnaK, which should be ineffective for the human Hsp70.     

Hsp70 chaperone machine remodels protein aggregates at the initial step of Hsp70-Hsp100-dependent disaggregation

Exposure to temperatures over a certain limit leads to massive protein aggregation in the cell. Disaggregation of such aggregates is largely dependent on the Hsp100 and Hsp70 chaperones. The exact role of the Hsp70 chaperone machine (composed of DnaK, DnaJ, and GrpE) in the Hsp100-dependent process remains unknown. In this study we focused on the Hsp70 role at the initial step of the disaggregation process. Two different aggregated model substrates, green fluorescent protein (GFP) and firefly luciferase, were incubated with the Hsp70 machine resulting in efficient fragmentation of large aggregates into smaller ones. Our data suggest that the observed fragmentation is achieved first by extraction of polypeptides from aggregates in Hsp70 chaperone machine-dependent manner and not by direct fragmentation of large aggregates. In the absence of Hsp100 (ClpB) these "extracted" polypeptides were not able to fold properly and promptly reassociated into new aggregates. The extracted GFP molecules were efficiently recognized and sequestered by a molecular trap, the mutant GroEL D87K, which binds stably to unfolded but not to native polypeptides. The binding of extracted GFP molecules to the GroEL trap prevented their reaggregation. We propose that the Hsp70 machine disentangles polypeptides from protein aggregates prior to Hsp100 action.     

A 14-mer Hsp70 peptide stimulates natural killer (NK) cell activity

Compared with normal cells, tumor cell lines exhibit an unusual plasma membrane localization of heat shock protein 70 (Hsp70). This tumor-selective Hsp70 membrane expression has been found to correlate with an increased sensitivity to lysis mediated by human natural killer (NK) cells that transiently adhere to plastic following cytokine stimulation. A human Hsp70-specific monoclonal antibody (mAb) detects membrane-bound Hsp70 on viable tumor cells and blocks the immune response of NK cells against Hsp70-expressing tumor cells. By peptide scanning (pep-scan) analysis, the epitope of this mAb was mapped as the C-terminal-localized 8-mer NLLGRFEL (NLL, amino acids [aa] 454-461). Most interestingly, similar to full-length Hsp70 protein, the N-terminal-extended 14-mer peptide TKDNNLLGRFELSG (TKD, aa 450-463) was able to stimulate the cytolytic and proliferative activity of NK cells at concentrations equivalent to full-length Hsp70 protein. Blocking studies revealed that an excess of the 14-mer peptide TKDNNLLGRFELSG inhibits the cytolytic activity of NK cells similar to that of Hsp70 protein. In comparison, other TKD-related peptides, including the 8-mer antibody epitope NLLGRFEL (aa 454-461), the 12-mer TKDNNLLGRFEL (aa 450-461), the 13-mer C-terminal-extended peptide NLLGRFELSGIPP (aa 454-466), the 14-mer TKD-equivalent sequences of Hsp70hom TKDNNLLGRFELTG (aa 450-463), Hsc70 TKDNNLLGKFELTG (aa 450-463), and DnaK AADNKSLGQFNLDG (aa 447-460) failed to activate NK activity.     

The J-domain of Hsp40 couples ATP hydrolysis to substrate capture in Hsp70

The Escherichia coli Hsp40 DnaJ uses its J-domain to target substrate polypeptides for binding to the Hsp70 DnaK, but the mechanism of J-domain function has been obscured by a substrate-like interaction between DnaJ and DnaK. ATP hydrolysis in DnaK is associated with a conformational change that captures the substrate, and both DnaJ and substrate can stimulate ATP hydrolysis. However, substrates cannot trigger capture by DnaK in the presence of ATP, and substrates stimulate a DnaK conformational change that is uncoupled from ATP hydrolysis. The role of the J-domain was examined using the fluorescent derivative of a fusion protein composed of the J-domain and a DnaK-binding peptide. In the absence of ATP, DnaK-binding affinity of the fusion protein is similar to that of the unfused peptide. However, in the presence of ATP, the affinity of the fusion protein is dramatically increased, which is opposite to the decrease in DnaK affinity typically exhibited by peptides. Binding of a fusion protein that contains a defective J-domain is insensitive to ATP. According to results from isothermal titration calorimetry, the J-domain binds to the DnaK ATPase domain with weak affinity (K(D) = 23 microM at 20 degrees C). The interaction is characterized by a positive enthalpy, small heat capacity change (DeltaC(p)= -33 kcal mol(-1)), and increasing binding affinity for increasing temperatures in the physiological range. In conditions that support binding of the J-domain to the ATPase domain, the J-domain accelerates ATP hydrolysis and a simultaneous conformational change in DnaK that is associated with peptide capture. The defective J-domain is inactive, despite the fact that it binds to the DnaK ATPase domain with higher than wild-type affinity. The results are most consistent with an allosteric mechanism of J-domain action in which the J-domain couples ATP hydrolysis to peptide capture by accelerating ATP hydrolysis and delaying DnaK closure until ATP is hydrolyzed.     

Regulation of Hsp70 function by a eukaryotic DnaJ homolog.

We report that a purified cytoplasmic Hsp70 homolog from Saccharomyces cerevisiae, Hsp70SSA1, exhibits a weak ATPase activity, which is stimulated by a purified eukaryotic dnaJp homolog (YDJ1p). Stable complex formation between Hsp70SSA1 and the permanently unfolded protein carboxymethylated alpha-lactalbumin (CMLA) was assayed by native gel electrophoresis. The affinity of Hsp70SSA1 for CMLA appeared to be regulated by YDJ1p. Significant reduction in both CMLA-Hsp70SSA1 complex formation and the release of CMLA pre-bound to Hsp70SSA1 was observed only in the presence of both YDJ1p and ATP. Thus, Hsp70SSA1 and YDJ1p interact functionally in the execution of Hsp70SSA1 chaperone activities in the eukaryotic cell.