Hsp70 molecular chaperones hydrolyze and re-bind ATP concomitant with the binding and release of aggregation-prone protein substrates. As a result, Hsp70s can enhance protein folding and degradation, the assembly of multi-protein complexes, and the catalytic activity of select enzymes. The ability of Hsp70 to perform these diverse functions is regulated by two other classes of proteins: Hsp40s (also known as J-domain-containing proteins) and Hsp70-specific nucleotide exchange factors (NEFs). Although a NEF for a prokaryotic Hsp70, DnaK has been known and studied for some time, eukaryotic Hsp70s NEFs were discovered more recently. Like their Hsp70 partners, the eukaryotic NEFs also play diverse roles in cellular processes, and recent structural studies have elucidated their mechanism of action.

Introduction

To cope with environmental stresses, such as heat shock, oxidative injury, or glucose-depletion, the expression of a large number of heat shock proteins (Hsps) is induced in all cell types examined. Early work defined these Hsps (some of which are identical to the glucose-responsive proteins, or Grps) by their apparent molecular masses; thus, Hsps with a mass of ˜70 kDa became known as Hsp70s, and ˜40 kDa Hsps are Hsp40s.¹ Subsequent studies indicated that many Hsps also function as molecular chaperones, factors that aid in the maturation, processing, or sub-cellular targeting of other proteins.

Perhaps the best-studied group of molecular chaperones is the Hsp70s.² Hsp70s are found in every organism (with the exception of some archaea³) and in eukaryotes reside in or are associated with each sub-cellular compartment. Hsp70s are highly homologous to one another and are comprised of three domains: A ˜44 kDa amino-terminal ATPase domain, a central ˜18 kDa peptide-binding domain (PBD), and a carboxy-terminal “lid” that closes onto the PBD to capture peptide substrates.⁴ In some Hsp70s, a short carboxy-terminal amino acid motif also mediates the interaction between Hsp70s and co-chaperones containing tetratrico peptide repeat (TPR) domains (see chapters by Daniel et al and Smith and Cox). By virtue of their preferential binding to hydrophobic peptides, Hsp70s retain these aggregation-prone substrates in solution, which in turn permits Hsp70s to enhance: (1) the folding of nascent or temporarily unfolded proteins; (2) the degradation of mis-folded polypeptides; (3) the assembly of multi-protein complexes; and (4) the catalytic activity of enzyme complexes that might require quaternary assembly. It should come as no surprise, then, that Hsp70 over-expression permits the cell to withstand cellular stresses, and that Hsp70s and constitutively expressed Hsp70 homologues, or Hsp70 “cognates” (also known as Hsc70s) play vital roles in cellular physiology.

Hsp70s bind loosely to their peptide substrates when the ATPase domain is occupied by ATP, and tightly when the enzyme is in the ADP-bound state;^5-8 therefore, ADP-ATP exchange is critical for peptide release, and both ATP hydrolysis and nucleotide exchange are accelerated by Hsp70s co-chaperones. Specifically, Hsp40s—which are defined by the presence of an ˜70 amino acid “J” domain—enhance ATP hydrolysis (see the chapter by Rosser and Cyr), whereas ADP release is catalyzed by another group of proteins, known as nucleotide exchange factors (NEFs). In fact, these factors do not “exchange” one nucleotide for another, but because ATP is present at much higher concentrations than ADP in the cell, ATP binding most commonly follows ADP release.

The physiological consequences of eukaryotic Hsp70-Hsp40 interaction are well-characterized.^9-11 In contrast, the contributions of Hsp70 NEFs in eukaryotic cell homeostasis are only now becoming apparent. Therefore, the purpose of this review is to summarize briefly what is known about the best-characterized Hsp70 NEF, the bacterial GrpE protein, and then to discuss in greater detail the more recent discovery of eukaryotic NEFs in the cytoplasm and in the endoplasmic reticulum (ER). Particular emphasis will be placed on the molecular underpinnings by which these NEFs function, and on important but unanswered questions in this field of research.

GrpE: The Bacterial Nucleotide Exchange Factor for Hsp70

The replication of the λ bacteriophage genome in E. coli requires DNA helicase activity at the origin of replication (ori). The helicase is initially inhibited by the λP protein, but the protein is displaced by host-encoded Hsp70 and Hsp40 chaperones, which were first named DnaK and DnaJ, respectively, based on the inability of dnaK and dnaJ mutants to support λ replication.¹² Another mutant that prevented λ replication is encoded by the grpE locus.¹³ DnaK-DnaJ-dependent liberation of λP from the ori and replication of the phage genome can be recapitulated in vitro, and it was discovered that decreased amounts of DnaK are required in these assays if GrpE is also present.^14,15 This phenomenon results from the fact that GrpE strips ADP from DnaK, and the combination of DnaJ and GrpE synergistically enhances DnaK's ATPase activity in single-turnover measurements by 50-fold, or even up to 5000-fold, depending on whether GrpE is saturating.^8,16 The DnaK-DnaJ-GrpE “machine” not only regulates multi-protein complex assembly—as observed during phage λ replication—but assists in the folding of newly synthesized and unfolded polypeptides, and homologues of each of these proteins reside in the mitochondria and help drive the import or “translocation” and maturation of nascent polypeptides in this organelle (see chapter by Bursac and Lihgow).^17,18

The Discovery of Hsp70 Nucleotide Exchange Factors in Eukaryotes: Fishing Pays Off

The cytoplasm and ER lumen in eukaryotes contain several Hsp70 and Hsp40 homologues, and it was assumed that GrpE homologues would also reside in these compartments. After many years, the failure to identify them was ascribed either to the fact that GrpE homologues are highly divergent and/or that the Hsp70s in the ER and eukaryotic cytoplasm might have evolved such that GrpE-assisted ADP release is dispensable.¹⁹ Thus, it came as a complete surprise when BAG-1—which was first identified as a cellular partner for Bcl-2, a negative regulator of apoptosis²⁰—was found to catalyze ADP release from mammalian Hsp70.²¹ The binding between BAG-1 and the ATPase domain of Hsp70 is mediated by a ˜50 amino acid “BAG” domain,^22-24 which is present in each of the many isoforms and splice variants of BAG-1 that have been identified. However, it is clear that BAG domain-containing NEFs do not function identically to GrpE, at least in part because their structures are distinct (also see below). For example, GrpE catalyzes the release of both ADP and ATP from DnaK, whereas BAG-1 triggers only ADP release.²⁵ In addition, GrpE augments DnaK-DnaJ-mediated protein folding and assembly, whereas BAG-1 has been found to exert either positive or negative effects on Hsp70-Hsp40-directed protein folding and chaperone activity. These contradictory results stem primarily from the concentrations of BAG-1 employed and the presence or absence of specific co-chaperones.^26,27 Thus, future work is needed to define how BAG domain-containing proteins impact known chaperone activities and how each of the various isoforms function under normal, cellular conditions and at their native concentrations.

For some time it was thought that yeast lacked a BAG domain-containing protein, but the available structure of an Hsp70 ATPase domain in complex with a BAG domain fragment²⁸ brought about the discovery of a highly divergent BAG-1 homologue in the yeast database, Snl1.²⁹ SNL1 was originally identified as a high-copy suppressor of the toxicity produced by the C-terminal fragment of a nuclear pore protein, and one consequence of this fragment is the generation of nuclear membrane “herniations”.³⁰ Therefore, it was proposed that Snl1 modulates nuclear pore complex (NPC) integrity, and consistent with this hypothesis, Snl1 is an integral membrane protein that resides in the nuclear envelope/ER membrane. Proof that Snl1 is a bona fide BAG homologue derived from the fact that Snl1 associates with Hsp70s from yeast and mammals, and that a purified soluble fragment of Snl1 stimulates Hsp40-enhanced ATP hydrolysis by Hsp70 to the same extent as a mammalian BAG domain-containing protein.²⁹

Because the lumen of the ER houses a high concentration of Hsp70 and because of its prominent role in catalyzing the folding of nascent proteins, it was also assumed that a NEF would reside in this compartment. Almost all secreted proteins associate with BiP, the ER lumenal Hsp70, during translocation and folding.³¹ During translocation, BiP is anchored to an integral membrane J-domain-containing protein, but if the subsequent folding of the nascent secreted protein is compromised, BiP interacts instead with soluble Hsp40s to facilitate the “retro-translocation” of the aberrant protein from the ER and into the cytoplasm where it is degraded by the proteasome.³² This process was termed ER associated degradation (ERAD³³) and is conserved amongst all eukaryotes.

To identify BiP partners that might include NEFs and that might facilitate protein translocation, folding, and/or ERAD, genetic selections were performed in different yeasts. First, the SLS1 gene was identified in a synthetic lethal screen in Y. lipolytica strains that lacked a component of the signal recognition particle, which is essential in this organism for protein translocation.³⁴ Later studies established that the Sls1 homologue in S. cerevisiae interacts preferentially with the ADP-bound form of BiP, that Sls1 enhances the Hsp40-mediated stimulation of BiP's ATPase activity, and that Sls1 accelerates the release of ADP and ATP from BiP.³⁵ Second, Stirling and colleagues isolated a gene that at high-copy number suppressed a growth defect in S. cerevisiae lacking an Hsp70-related protein, known as Lhs1, and that were unable to mount an ER stress response.³⁶ The gene, SIL1, is identical to SLS1, and the Sil1 protein was shown to bind selectively to BiP's ATPase domain. Together, these data suggested strongly that Sls1/Sil1 is a BiP NEF. Further support for this hypothesis was provided by the discovery that Sls1/Sil1 is the yeast homologue of BAP, a resident of the mammalian ER that strips nucleotide from BiP and synergistically enhances the J-domain-mediated activation of BiP's ATPase activity.³⁷

Surprisingly, Lhs1, mentioned above as an Hsp70-related protein, also appears to function as a NEF. Lhs1 is a member of the Hsp110/Grp170 family of mammalian molecular chaperones that possess N-terminal ATP binding domains with some homology to the Hsp70 ATPase domain; however, the C-terminal halves are comprised of extended, nonconserved polypeptide binding domains.³⁸ Recent studies from the Stirling laboratory indicate that Lhs1 interacts with BiP in the yeast ER and can strip ADP/ATP from BiP as efficiently as Sls1/Sil1, thus activating BiP's steady-state ATPase activity when combined with a J-domain-containing protein. ³⁹ In turn, BiP activates the ATPase activity of Lhs1, and in both cases the ATP-binding properties of the chaperones are essential for activity. These results indicate that BiP and Lhs1 reciprocally enhance one another's activities, perhaps to coordinate the transfer of polypeptide substrates. Although it is not yet clear whether all members of the Hsp110/Grp170 family are NEFs, another group reported that Hsc70 activates the ATPase activity of a cytosolic, mammalian Hsp110 homologue, Hsp105α, and that Hsp105α inhibits the hydrolysis of ATP-bound Hsc70. These results are consistent with Hsp105α possessing NEF activity.⁴⁰

To identify new cytoplasmic NEFs, we searched the S. cerevisiae genome for Sls1 homologues that lacked an ER-targeting sequence and isolated the FES1 gene.⁴¹ Purified Fes1 catalyzes the release of ADP and ATP from cytoplasmic Hsp70, and the fes1 thermosensitive growth phenotype is rescued by mutations in a cytoplasmic Hsp40. This genetic finding is consistent with the opposing effects of Hsp40s and NEFs on the identity of the Hsp70-bound nucleotide; i.e., Hsp40s drive Hsp70s into the ADP-bound state, whereas NEFs drive Hsp70s into the ATP-bound state. Interestingly, a mammalian Fes1 homolog—known as HspBP1—was identified previously as a Hsp70 interactor in a yeast two-hybrid screen.⁴² Initially, HspBP1 was reported to inhibit nucleotide binding and chaperone activity, but subsequent work by our groups established that HspBP1 also catalyzes nucleotide release from Hsp70.^43,44

Hsp70 NEFs in Eukaryotes Exhibit Diverse Functions

Hsp70s play a prominent role in many cellular processes, and so it was anticipated that the NEFs would also exhibit diverse functions. Thus far, this prediction has been affirmed, but because this field is in its infancy, relatively little is known, and in some cases—as mentioned above for BAG-1—contradictory results have been obtained. In this section we will highlight key findings, direct the reader to the pertinent literature, and speculate on important directions for future studies.

BAG-1 is a positive or negative regulator of chaperone-mediated protein folding, depending on several variables, and to a large extent these contradictory results derive from the use of in vitro assays in which the experimental conditions may vary from the cellular environment and from in vivo expression systems in which super-stoichiometric amounts of wild type or mutant versions of the protein are produced.^26,27,45 Therefore, and as noted above, future studies must employ conditions that more closely mimic those found in the cell. Nevertheless, what is becoming increasingly clear is that BAG-1 can target proteins for proteasome-mediated degradation (see chapter by Höhfeld et al). This attribute results from an embedded ubiquitin-like domain in BAG-1,⁴⁶ which facilitates proteasome interaction. Because BAG-1 also binds Hsp70, it has been proposed that BAG-1 couples Hsp70 to the proteasome to facilitate chaperone-mediated “decisions” during cytoplasmic protein turn-over. In addition to its role in protein degradation, BAG-1 protects cells against apoptosis, consistent with the association between BAG-1 and Bcl-2. BAG-1 is also involved in androgen receptor and transcriptional activation, and associates with and regulates the Raf-1/ERK kinase. Interestingly, some of these activities are independent of the BAG domain, and thus each BAG-1 homologue probably evolved unique functional motifs to diversify its functions. In addition, these data suggest that BAG domain-containing proteins might prove to be targets for pharmacological interventions to treat human diseases.

The discovery of a yeast BAG-1 homologue, Snl1,²⁹ provides researchers with a genetic tool to define better how one member of this protein family functions in the cell. As noted above, Snl1 is thought to stabilize the NPC and perhaps modulate its activity,³⁰ but to date it is not clear how this occurs. Of additional interest is Snl1's localization at the ER membrane, suggesting that the protein might aid Hsp70 and Hsp40 homologues during translocation or ERAD; however, we have found that translocation and ERAD are robust in yeast deleted for SNL1 either alone or when combined with fes1 mutants (J. Bennett, J. Young, and J. L. B., unpublished observations).

In contrast, several lines of evidence suggest that the ER lumenal NEF in yeast, Sls1/Sil1, is involved in ERAD and translocation. First, the mRNA encoding Sls1/Sil1 rises when cells are exposed to stresses that activate the unfolded protein response (UPR).⁴⁷ Other UPR targeted genes include chaperones and enzymes required for protein folding, post-translational modification, and ERAD, and deletion of SLS1/SIL1 in one S. cerevisiae strain background modestly compromises ERAD efficiency.⁴⁷ Second, yeast deleted both for LHS1 (see above) and for SLS1/ SIL1 exhibit strong translocation defects, although more modest translocation defects are evident in lhs1Δ cells.³⁶ Third, Y. lipolytica strains expressing a truncated form of Sls1 that is unable to interact with BiP are translocation-defective.⁴⁸ One explanation for each of these findings is that the NEF simply increases the efficiency at which BiP functions during translocation and ERAD, although this has not been demonstrated directly. It will also be vital in the future to determine whether the mammalian homologue, BAP,³⁷ plays a role in any of these processes.

If BAG-1 and Snl1 are NEFs for cytoplasmic Hsp70s in eukaryotes, why does the cytoplasm harbor the Fes1/HspBP1 proteins? One possibility is that each NEF acts on only a unique Hsp70 or family of Hsp70s. For example, there are seven Hsp70s in the cytoplasm of S. cerevisiae that are grouped into distinct classes: One class (the “Ssas”) facilitates translocation and ERAD, and others (the “Ssbs” and “Ssz”) associate with the ribosome and are involved in translation.^31,49 Although this hypothesis still needs to be examined more thoroughly, we reported that fes1 mutants display phenotypes consistent with defects in translation initiation and that the Fes1 protein is associated with the ribosome, even though Fes1 is a NEF for an Ssa family member.⁴¹ Yeast deleted for FES1 also exhibit defects in the folding of newly synthesized firefly luciferase,^44,50 a process that is similarly dependent on the Ssa chaperones.^51,52 Although preliminary, these data suggest that NEFs might be promiscuous when choosing their Hsp70 partners. Otherwise, little else is known about Fes1 homologues except that the levels of HspBP1 are elevated in tumor cells,⁵³ a result that is consistent with the observation that many tumors contain increased levels of Hsp70.⁵⁴ Clearly, much more work is needed on the roles played by Fes1/HspBP1 family members in the cell, an undertaking that will benefit from the construction of new mutants and assays in which their functions can be better defined.

The Mechanism of Action of Hsp70 Nucleotide Exchange Factors: Results from Structural and Biochemical Studies

The first Hsp70 NEF structure determined was the bacterial GrpE in complex with the ATPase domain of its associated Hsp70, DnaK of E. coli (Fig. 1A).⁵⁵ In the crystal structure and in solution, GrpE forms tight dimers that asymmetrically contact only one ATPase domain.^56,57 GrpE has a bipartite structure composed of an alpha-helical N-terminal part and a small beta-sheet domain at the C-terminus. The alpha-helical fragment forming the dimer interface extends far beyond the measures of the ATPase domain and might contact the substrate-binding region of DnaK. Indeed, whereas full-length GrpE interferes with substrate binding, GrpE missing 33 residues at the N-terminus does not. The interaction with the ATPase domain of DnaK is mediated primarily by the beta-sheet region of one GrpE molecule inserting into the cleft between subdomains IB and IIB of the ATPase domain. The highly conserved ATPase domain of Hsp70/Hsc70/DnaK has a bilobal structure that is conventionally divided further into four subdomains, IA and IB forming lobe I, and IIA and IIB lobe II, respectively.⁵⁸ The ATP binding site is located at the bottom of a cleft between subdomains IB and IIB close to the center of the domain. In the structure of the ADP complex of the ATPase domain of mammalian Hsp70, residues from all four subdomains contact the nucleotide. Comparison of the GrpE-DnaK complex with this structure indicated that binding of GrpE induces a 14º rotation of subdomain IIB, resulting in an opening of the nucleotide binding cleft incompatible with nucleotide binding.

Figure 1

Comparison of the Hsp70 nucleotide exchange factor structures. Panels A-C depict the crystal structures of the complexes of GrpE-DnaK, BAG-Hsc70 and HspBP1-Hsp70, respectively.^,, The peptide backbones are shown in ribbon representation with the nucleotide (more...)

The BAG domain of BAG-1 assumes a structure completely unrelated to GrpE, forming a ˜60 Å long three-helix bundle, both in solution and in complex with the ATPase domain of Hsc70 (Fig. 1B).^59,60 In the complex, highly conserved polar residues in helices 2 and 3 contact subdomains IB and IIB of the ATPase domain. The majority of interactions are, however, formed with subdomain IIB.⁶¹ The binding of BAG locks the ATPase domain of Hsc70 in a conformation very similar to DnaK in complex with GrpE, with subdomain IIB rotated outward by 14º. These data suggest convergent evolution of the NEFs and are analogous to the structurally divergent nucleotide exchange factors of small G-proteins, all of which employ a common structural switch.⁶² Although the ATPase sequences are highly conserved in the Hsp70 family, BAG-1 and GrpE do accelerate nucleotide exchange exclusively on their respective binding partners Hsc70/Hsp70 and DnaK, and it is important to note that the sequences of the ATPase domains of the inducible Hsp70 and the constitutive Hsc70 are virtually identical.⁶³

HspBP1, a member of the third class of Hsp70 nucleotide exchange factors, is again structurally unrelated to both GrpE and the BAG domain (Fig. 1C). The core domain, which is sufficient for Hsp70 binding, is composed entirely of alpha-helical repeats containing four regular Armadillo repeats in the central region.⁴⁴ Armadillo repeats comprise three helices arranged in an open triangle and are found in many functionally unrelated eukaryotic proteins as a versatile structural building block. In the crystal structure of the complex with lobe II of the Hsp70 ATPase domain, the slightly curved core domain of HspBP1 embraces subdomain IIB.⁴⁴ By comparison with BAG-1, HspBP1 binds sideways onto subdomain IIB, which would generate a steric conflict of its N-terminus with subdomain IB if the ATPase domain adopts a similar conformation as in the complexes with ADP or BAG-1. Indeed, probing the conformation of the entire ATPase domain in complex with HspBP1 by limited proteolysis and fluorescence spectroscopy suggests a less compact conformation for the ATPase domain as compared to the complex with BAG or in the absence of NEFs. It is thus likely that HspBP1 and its homologs trigger nucleotide exchange by a mechanism distinct from BAG domain proteins and GrpE. The distortion of the Hsp70 ATPase domain might be sufficient to dissociate bound ADP, however rotation of subdomain IIB as observed in the BAG-Hsc70 complex might also occur. The different conformations imposed on the ATPase domain of Hsp70/Hsc70 by HspBP1 and BAG-1 may also differentially affect crosstalk between the substrate binding domain and downstream effectors, like CHIP.^64-66 Because each Hsp70 NEF class is conserved to varying extents (Fig. 2), it is likely that NEF homologs of those for which structures have been determined will function similarly.

Figure 2

Amino acid sequence alignments of select nucleotide exchange factors. A) Alignment of the structured portion of E. coli GrpE with mitochondrial GrpE from S. cerevisiae and with chloroplast type-I GrpE from N. tabacum. B) Alignment of the BAG domains of (more...)

Conclusions

As outlined above, initial characterizations and structural studies of eukaryotic Hsp70 NEFs have proceeded rapidly since the relatively recent identification of this family of co-chaperones. What has been more difficult to discern, however, is the spectrum of cellular activities engineered by these proteins. Other Hsp70 co-chaperones appear to augment a sub-set of cellular activities that are normally carried-out by the chaperone (see other chapters in this volume), and we predict that the same rule will apply to Hsp70 NEFs. To some extent this prediction has been borne-out, since unique NEFs facilitate protein folding, translocation, and translation. However, only a relatively small number of Hsp70-catalyzed activities have been examined in these initial studies, and in some cases heterologous reporters (e.g., firefly luciferase) were employed. It is thus imperative that novel cellular assays using endogenous substrates are developed in which the effects of depleting or mutating specific NEFs can be investigated. To this end, the described structural analyses will surely improve our ability to mutate Hsp70-interacting residues on distinct NEFs, and the resulting mutated proteins can then be examined in both genetic (i.e., yeast) and mammalian systems.

Another feature of eukaryotic NEFs that remains mysterious but that will likely become an active area of research is whether these proteins contain built-in stress sensors. Previous work established that the paired, N-terminal helices in E. coli GrpE dimers undergo a reversible transition at ˜48°C, and that the transition reduces nucleotide exchange activity or association with DnaK. As a result, the steady-state population of DnaK becomes predominantly associated with ADP and bound tightly to peptide substrates at elevated temperatures.⁶⁷ More recent data indicate that this “thermosensor” is important for the DnaK-DnaJ-GrpE-mediated prevention of protein aggregation and protection of enzyme activity after heat shock.⁶⁸ Therefore, GrpE function is one component of the cellular “thermometer” that controls protein folding in the cell. Although the eukaryotic NEFs discussed in this review lack homology to GrpE, it will be interesting to examine whether eukaryotic NEF activities are similarly regulated by temperature or other stresses in vivo.

Finally, given the importance of the Hsp70 chaperone system in human physiology and medicine, we predict that Hsp70 NEFs will emerge as important players in maintaining cellular homeostasis. In turn, we anticipate that defects in the activities of select NEFs will be implicated in disease. Recent data support this supposition: Mice have been found that contain a spontaneous, recessive mutation in the gene encoding a Sil1 (Sls1) homologue.⁶⁹ These “woozy” (wz) mice accumulate protein inclusions in the ER and nucleus of Purkinje cells and thus become ataxic. Consistent with a role for the murine NEF in protein quality control, the UPR is induced in Purkinje cells from wz mutants. The discovery of the wz mutation likely represents only the first of many examples in which loss of a NEF homologue in mammals leads to a specific disease or disease-like phenomenon. Therefore, we also predict that the “hunt” will be on for other mutations in mammalian Hsp70 NEFs that impact cellular homeostasis.

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