The yeast Hsp110 Sse1 functionally interacts with the Hsp70 chaperones Ssa and Ssb

There is growing evidence that members of the extended Hsp70 family of molecular chaperones, including the Hsp110 and Grp170 subgroups, collaborate in vivo to carry out essential cellular processes. However, relatively little is known regarding the interactions and cellular functions of Sse1, the yeast Hsp110 homolog. Through co-immunoprecipitation analysis, we found that Sse1 forms heterodimeric complexes with the abundant cytosolic Hsp70s Ssa and Ssb in vivo. Furthermore, these complexes can be efficiently reconstituted in vitro using purified proteins. Binding of Ssa or Ssb to Sse1 was mutually exclusive. The ATPase domain of Sse1 was found to be critical for interaction as inactivating point mutations severely reduced interaction with Ssa and Ssb. Sse1 stimulated Ssa1 ATPase activity synergistically with the co-chaperone Ydj1, and stimulation required complex formation. Ssa1 is required for post-translational translocation of the yeast mating pheromone alpha-factor into the endoplasmic reticulum. Like ssa mutants, we demonstrate that sse1delta cells accumulate prepro-alpha-factor, but not the co-translationally imported protein Kar2, indicating that interaction between Sse1 and Ssa is functionally significant in vivo. These data suggest that the Hsp110 chaperone operates in concert with Hsp70 in yeast and that this collaboration is required for cellular Hsp70 functions.     

Yeast Pdr13p and Zuo1p molecular chaperones are new functional Hsp70 and Hsp40 partners

The deletion of the TOM1 gene encoding a putative ubiquitin ligase causes a temperature sensitive cellular growth in Saccharomyces cerevisiae. The arrested cells exhibit pleiotropic defects in nuclear division, maintenance of nuclear structure and heat stress responses. We previously identified a zuo1 mutation as an extragenic suppressor of the tom1 mutant. ZUO1 encodes a DnaJ-related Hsp40. Here we show that a recessive cold sensitive mutation in PDR13 coding for an Hsp70 suppressed the tom1 mutation. The pdr13 deletion mutant was sensitive to high osmolarity, just like the zuo1 deletion mutant. A zuo1 pdr13 double deletion mutant did not show additive phenotypes. Furthermore, a tagged-Zuo1p was co-immunoprecipitated with a tagged-Pdr13p. Taken together, we propose that Pdr13p and Zuo1p are a new pair of Hsp70:Hsp40 molecular chaperones. In addition, Pdr13p co-sedimented with translating ribosomes and this association was independent of the presence of Zuo1p.     

Roles of cytosolic Hsp70 and Hsp40 molecular chaperones in post-translational translocation of presecretory proteins into the endoplasmic reticulum

Hsp70 molecular chaperones and their co-chaperones work together in various cellular compartments to guide the folding of proteins and to aid the translocation of proteins across membranes. Hsp70s stimulate protein folding by binding exposed hydrophobic sequences thereby preventing irreversible aggregation. Hsp40s stimulate the ATPase activity of Hsp70s and target unfolded proteins to Hsp70s. Genetic and biochemical evidence supports a role for cytosolic Hsp70s and Hsp40s in the post-translational translocation of precursor proteins into endoplasmic reticulum and mitochondria. To gain mechanistic insight, we measured the effects of Saccharomyces cerevisiae Ssa1p (Hsp70) and Ydj1p (Hsp40) on the translocation of histidine-tagged prepro-alpha-factor (ppalphaF6H) into microsomes. Radiolabeled ppalphaF6H was affinity purified from wheat germ translation reactions (or Escherichia coli) to remove endogenous chaperones. We demonstrated that either Ssa1p or Ydj1p stimulates post-translational translocation by preventing ppalphaF6H aggregation. The binding and/or hydrolysis of ATP by Ssa1p were required to maintain the translocation competence of ppalphaF6H. To clarify the contributions of membrane-bound and cytosolic Ydj1p, we compared the efficiency of chaperone-dependent translocation into wild-type and Ydj1p-deficient microsomes. Neither soluble nor membrane-bound Ydj1p was essential for post-translational protein translocation. The ability of Ssa1p, Ydj1p, or both chaperones to restore the translocation competence of aggregated ppalphaF6H was negligible.     

Distinct roles for the Hsp40 and Hsp90 molecular chaperones during cystic fibrosis transmembrane conductance regulator degradation in yeast

Aberrant secreted proteins can be destroyed by ER-associated protein degradation (ERAD), and a prominent, medically relevant ERAD substrate is the cystic fibrosis transmembrane conductance regulator (CFTR). To better define the chaperone requirements during CFTR maturation, the protein was expressed in yeast. Because Hsp70 function impacts CFTR biogenesis in yeast and mammals, we first sought ER-associated Hsp40 cochaperones involved in CFTR maturation. Ydj1p and Hlj1p enhanced Hsp70 ATP hydrolysis but CFTR degradation was slowed only in yeast mutated for both YDJ1 and HLJ1, suggesting functional redundancy. In contrast, CFTR degradation was accelerated in an Hsp90 mutant strain, suggesting that Hsp90 preserves CFTR in a folded state, and consistent with this hypothesis, Hsp90 maintained the solubility of an aggregation-prone domain (NBD1) in CFTR. Soluble ERAD substrate degradation was unaffected in the Hsp90 or the Ydj1p/Hlj1p mutants, and surprisingly CFTR degradation was unaffected in yeast mutated for Hsp90 cochaperones. These results indicate that Hsp90, but not the Hsp90 complex, maintains CFTR structural integrity, whereas Ydj1p/Hlj1p catalyze CFTR degradation.     

Conditional knockdown of proteasomes results in cell-cycle arrest and enhanced expression of molecular chaperones Hsp70 and Hsp40 in chicken DT40 cells

The 26 S proteasome is an evolutionarily conserved ATP-dependent protease complex that degrades poly-ubiquitinated proteins and plays essential roles in a critical part of cellular regulation. In vertebrates, the roles of the proteasome have been widely studied by use of specific inhibitors, but not genetically. Here, we generated a cell line Z(-/-/-)/Z-HA, in which the expression of the catalytic subunit of the proteasome, Z (beta2) could be manipulated. This cell line expresses exogenous Z protein under the control of a tetracycline-repressible promoter in a Z-nullizygous genetic background. Treatment of these cells with doxycycline inhibited Z expression and, hence, the function of the proteasome. The latter resulted in accumulation of poly-ubiquitinated proteins and concomitant induction of molecular chaperones Hsp70 and Hsp40. These results suggest a synergistic role for the proteasome with these molecular chaperones to eliminate misfolded or damaged proteins in vivo. Furthermore, knockdown of the proteasome induced apoptotic cell death following cell-cycle arrest at G(2)/M phase. Our Z(-/-/-)/Z-HA cell line would be useful for evaluating proteolytic processes catalyzed by the proteasome in many biological events in vertebrate cells.     

The ubiquitin-related BAG-1 provides a link between the molecular chaperones Hsc70/Hsp70 and the proteasome

The BAG-1 protein modulates the chaperone activity of Hsc70 and Hsp70 in the mammalian cytosol and nucleus. Remarkably, BAG-1 possesses a ubiquitin-like domain at its amino terminus, suggesting a link to the ubiquitin/proteasome system. Here we show that BAG-1 is indeed associated with the 26 S proteasome in HeLa cells. Binding of the chaperone cofactor to the proteolytic complex is regulated by ATP hydrolysis and is not mediated by Hsc70 and Hsp70. The presented findings reveal a role of BAG-1 as a physical link between the Hsc70/Hsp70 chaperone system and the proteasome. In fact, targeting of BAG-1 to the proteasome promotes an association of the chaperones with the proteolytic complex in vitro and in vivo. A regulatory function of the chaperone cofactor at the interface between protein folding and protein degradation is thus indicated.     

Structural and functional analysis of the Hsp70/Hsp40 chaperone system

As one of the most abundant and highly conserved molecular chaperones, the 70‐kDa heat shock proteins (Hsp70s) play a key role in maintaining cellular protein homeostasis (proteostasis), one of the most fundamental tasks for every living organism. In this role, Hsp70s are inextricably linked to many human diseases, most notably cancers and neurodegenerative diseases, and are increasingly recognized as important drug targets for developing novel therapeutics for these diseases. Hsp40s are a class of essential and universal partners for Hsp70s in almost all aspects of proteostasis. Thus, Hsp70s and Hsp40s together constitute one of the most important chaperone systems across all kingdoms of life. In recent years, we have witnessed significant progress in understanding the molecular mechanism of this chaperone system through structural and functional analysis. This review will focus on this recent progress, mainly from a structural perspective.     

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.     

Cyclophilin 20 is involved in mitochondrial protein folding in cooperation with molecular chaperones Hsp70 and Hsp60.

We studied the role of mitochondrial cyclophilin 20 (CyP20), a peptidyl-prolyl cis-trans isomerase, in preprotein translocation across the mitochondrial membranes and protein folding inside the organelle. The inhibitory drug cyclosporin A did not impair membrane translocation of preproteins, but it delayed the folding of an imported protein in wild-type mitochondria. Similarly, Neurospora crassa mitochondria lacking CyP20 efficiently imported preproteins into the matrix, but folding of an imported protein was significantly delayed, indicating that CyP20 is involved in protein folding in the matrix. The slow folding in the mutant mitochondria was not inhibited by cyclosporin A. Folding intermediates of precursor molecules reversibly accumulated at the molecular chaperones Hsp70 and Hsp60 in the matrix. We conclude that CyP20 is a component of the mitochondrial protein folding machinery and that it cooperates with Hsp70 and Hsp60. It is speculated that peptidyl-prolyl cis-trans isomerases in other cellular compartments may similarly promote protein folding in cooperation with chaperone proteins.     

Plant Hsp70 molecular chaperones: Protein structure, gene family, expression and function

The Hsp70 molecular chaperones of plants are encoded by a multi-gene family whose members are developmentally regulated and differentially expressed in response to temperature stress and other conditions that interrupt normal protein folding or favor protein denaturation. Under non-stressful conditions, Hsp70 cognates function in concert with a variety of co-chaperones to facilitate folding of de novo synthesized proteins, assist in transport of precursor proteins into organelles and to help target damaged proteins for degradation. Stress-induced Hsp70s function to mitigate aggregation of stress-denatured proteins and to refold non-native proteins restoring their biological function through iterative cycles of adenine nucleotide hydrolysis-dependent peptide binding and release. Much of what is known about how plant Hsp70s function comes from the study of Hsp70s from other types of organisms. Owing to their unique biology, much remains to be learned about the many functions Hsp70s play in plants.     

Recruitment of Hsp70 chaperones: a crucial part of viral survival strategies

Virus proliferation depends on the successful recruitment of host cellular components for their own replication, protein synthesis, and virion assembly. In the course of virus particle production a large number of proteins are synthesized in a relatively short time, whereby protein folding can become a limiting step. Most viruses therefore need cellular chaperones during their life cycle. In addition to their own protein folding problems viruses need to interfere with cellular processes such as signal transduction, cell cycle regulation and induction of apoptosis in order to create a favorable environment for their proliferation and to avoid premature cell death. Chaperones are involved in the control of these cellular processes and some viruses reprogram their host cell by interacting with them. Hsp70 chaperones, as central components of the cellular chaperone network, are frequently recruited by viruses. This review focuses on the function of Hsp70 chaperones at the different stages of the viral life cycle emphasizing mechanistic aspects.     

The mammalian disaggregase machinery: Hsp110 synergizes with Hsp70 and Hsp40 to catalyze protein disaggregation and reactivation in a cell-free system

Bacteria, fungi, protozoa, chromista and plants all harbor homologues of Hsp104, a AAA+ ATPase that collaborates with Hsp70 and Hsp40 to promote protein disaggregation and reactivation. Curiously, however, metazoa do not possess an Hsp104 homologue. Thus, whether animal cells renature large protein aggregates has long remained unclear. Here, it is established that mammalian cytosol prepared from different sources possesses a potent, ATP-dependent protein disaggregase and reactivation activity, which can be accelerated and stimulated by Hsp104. This activity did not require the AAA+ ATPase, p97. Rather, mammalian Hsp110 (Apg-2), Hsp70 (Hsc70 or Hsp70) and Hsp40 (Hdj1) were necessary and sufficient to slowly dissolve large disordered aggregates and recover natively folded protein. This slow disaggregase activity was conserved to yeast Hsp110 (Sse1), Hsp70 (Ssa1) and Hsp40 (Sis1 or Ydj1). Hsp110 must engage substrate, engage Hsp70, promote nucleotide exchange on Hsp70, and hydrolyze ATP to promote disaggregation of disordered aggregates. Similarly, Hsp70 must engage substrate and Hsp110, and hydrolyze ATP for protein disaggregation. Hsp40 must harbor a functional J domain to promote protein disaggregation, but the J domain alone is insufficient. Optimal disaggregase activity is achieved when the Hsp40 can stimulate the ATPase activity of Hsp110 and Hsp70. Finally, Hsp110, Hsp70 and Hsp40 fail to rapidly remodel amyloid forms of the yeast prion protein, Sup35, or the Parkinson's disease protein, alpha-synuclein. However, Hsp110, Hsp70 and Hsp40 enhanced the activity of Hsp104 against these amyloid substrates. Taken together, these findings suggest that Hsp110 fulfils a subset of Hsp104 activities in mammals. Moreover, they suggest that Hsp104 can collaborate with the mammalian disaggregase machinery to rapidly remodel amyloid conformers.     

Modulation of chaperone activities of Hsp70 and Hsp70-2 by a mammalian DnaJ/Hsp40 homolog, DjA4

Type I DnaJs comprise one type of Hsp70 cochaperones. Previously, we showed that two type I DnaJ cochaperones, DjA1 (HSDJ/Hdj-2/Rdj-1/dj2) and DjA2 (cpr3/DNAJ3/Rdj-2/dj3), are important for mitochondrial protein import and luciferase refolding. Another type I DnaJ homolog, DjA4 (mmDjA4/dj4), is highly expressed in heart and testis, and the coexpression of Hsp70 and DjA4 protects against heat stress-induced cell death. Here, we have studied the chaperone functions of DjA4 by assaying the refolding of chemically or thermally denatured luciferase, suppression of luciferase aggregation, and the ATPase of Hsp70s, and compared these activities with those of DjA2. DjA4 stimulates the hydrolysis of ATP by Hsp70. DjA2, but not DjA4, together with Hsp70 caused denatured luciferase to refold efficiently. Together with Hsp70, both DjA2 and DjA4 are efficient in suppressing luciferase aggregation. bag-1 further stimulates ATP hydrolysis and protein refolding by Hsp70 plus DjA2 but not by Hsp70 plus DjA4. Hsp70-2, a testis-specific Hsp70 family member, behaves very similarly to Hsp70 in all these assays. Thus, Hsp70 and Hsp70-2 have similar activities in vitro, and DjA2 and DjA4 can function as partner cochaperones of Hsp70 and Hsp70-2. However, DjA4 is not functionally equivalent in modulating Hsp70s.     

Domain requirements of DnaJ-like (Hsp40) molecular chaperones in the activation of a steroid hormone receptor

DnaJ-like proteins function in association with Hsp70 molecular chaperones to facilitate protein folding. We previously demonstrated that a yeast DnaJ-like protein, Ydj1p, was important for activation of heterologously expressed steroid hormone receptors (Caplan, A. J., Langley, E., Wilson, E. M., and Vidal, J. (1995) J. Biol. Chem. 270, 5251-5257). In the present study, we analyzed Ydj1p function by assaying hormone binding to the human androgen receptor (AR) heterologously expressed in yeast. We analyzed hormone binding in strains that were wild type or deleted for the YDJ1 gene. In the deletion mutant, the AR did not bind hormone to the same extent as the wild type. Introduction of mutant forms of Ydj1p to the deletion strain revealed that the J-domain is necessary but not sufficient for Ydj1p action, and that other domains of the protein are also functionally important. Of three human DnaJ-like proteins introduced into the deletion mutant, only Hdj2, which displays full domain conservation with Ydj1p, suppressed the hormone binding defect of the deletion mutant. By comparison of the domains shared by these three human proteins, and with mutants of Ydj1p that were functional, it was deduced that the cysteine-rich zinc binding domain is important for Hdj2/Ydj1p action in hormone receptor function. A model for the mechanism of DnaJ-like protein action is discussed.     

Allosteric drugs: the interaction of antitumor compound MKT-077 with human Hsp70 chaperones

Hsp70 (heat shock protein 70 kDa) chaperones are key to cellular protein homeostasis. However, they also have the ability to inhibit tumor apoptosis and contribute to aberrant accumulation of hyperphosphorylated tau in neuronal cells affected by tauopathies, including Alzheimer's disease. Hence, Hsp70 chaperones are increasingly becoming identified as targets for therapeutic intervention in these widely abundant diseases. Hsp70 proteins are allosteric machines and offer, besides classical active-site targets, also opportunities to target the mechanism of allostery. In this work, it is demonstrated that the action of the potent anticancer compound MKT-077 (1-ethyl-2-[[3-ethyl-5-(3-methylbenzothiazolin-2-yliden)]-4-oxothiazolidin-2-ylidenemethyl] pyridinium chloride) occurs through a differential interaction with Hsp70 allosteric states. MKT-077 is therefore an “allosteric drug.” Using NMR spectroscopy, we identify the compound's binding site on human HSPA8 (Hsc70). The binding pose is obtained from NMR-restrained docking calculations, subsequently scored by molecular-dynamics-based energy and solvation computations. Suggestions for the improvement of the compound's properties are made on the basis of the binding location and pose.     

Cooperation of molecular chaperones with the ubiquitin/proteasome system

Molecular chaperones and energy-dependent proteases have long been viewed as opposing forces that control protein biogenesis. Molecular chaperones are specialized in protein folding, whereas energy-dependent proteases such as the proteasome mediate efficient protein degradation. Recent data, however, suggest that molecular chaperones directly cooperate with the ubiquitin/proteasome system during protein quality control in eukaryotic cells. Modulating the intracellular balance of protein folding and protein degradation may open new strategies for the treatment of human diseases that involve chaperone pathways such as cancer and diverse amyloid diseases.     

Hsp70 and Hsp40 Functionally Interact with Soluble Mutant Huntingtin Oligomers in a Classic ATP-dependent Reaction Cycle

Inclusion bodies of aggregated mutant huntingtin (htt) fragments are a neuropathological hallmark of Huntington disease (HD). The molecular chaperones Hsp70 and Hsp40 colocalize to inclusion bodies and are neuroprotective in HD animal models. How these chaperones suppress mutant htt toxicity is unclear but might involve direct effects on mutant htt misfolding and aggregation. Using size exclusion chromatography and atomic force microscopy, we found that mutant htt fragments assemble into soluble oligomeric species with a broad size distribution, some of which reacted with the conformation-specific antibody A11. Hsp70 associated with A11-reactive oligomers in an Hsp40- and ATP-dependent manner and inhibited their formation coincident with suppression of caspase 3 activity in PC12 cells. Thus, Hsp70 and Hsp40 (DNAJB1) dynamically target specific subsets of soluble oligomers in a classic ATP-dependent reaction cycle, supporting a pathogenic role for these structures in HD.     

S100A1 is a novel molecular chaperone and a member of the Hsp70/Hsp90 multichaperone complex

Although calmodulin is known to be a component of the Hsp70/Hsp90 multichaperone complex, the functional role of the protein remains uncertain. In this study, we have identified S100A1, but not calmodulin or other S100 proteins, as a potent molecular chaperone and a new member of the multichaperone complex. Glutathione S-transferase pull-down assays and co-immunoprecipitation experiments indicated the formation of stable complexes between S100A1 and Hsp90, Hsp70, FKBP52, and CyP40 both in vitro and in mammalian cells. S100A1 potently protected citrate synthase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, and rhodanese from heat-induced aggregation and suppressed the aggregation of chemically denatured rhodanese and citrate synthase during the refolding pathway. In addition, S100A1 suppressed the heat-induced inactivation of citrate synthase activity, similar to that for Hsp90 and p23. The chaperone activity of S100A1 was antagonized by calmodulin antagonists, such as fluphenazine and prenylamine, that is, indeed an intrinsic function of the protein. The overexpression of S100A1 in COS-7 cells protected transiently expressed firefly luciferase and Escherichia coli beta-galactosidase from inactivation during heat shock. The results demonstrate a novel physiological function for S100A1 and bring us closer to a comprehensive understanding of the molecular mechanisms of the Hsp70/Hsp90 multichaperone complex.     

Structural basis of functional cooperation of Tim15/Zim17 with yeast mitochondrial Hsp70

Mitochondrial heat-shock protein 70 (mtHsp70) and its partner proteins drive protein import into the matrix. Tim15/Zim17/Hep1 is a mtHsp70 partner protein on the matrix side of the inner mitochondrial membrane. We determined the nuclear magnetic resonance (NMR) structure of the core domain of Tim15. On the basis of the NMR structure, we created Tim15 mutants and tested their ability to complement the functional defects of Tim15 depletion and to suppress self-aggregation of mtHsp70 in vivo. A pair of basic residues, Arg 106 and His 107, conserved Asp 111 and flexible loop 133-137, and were important (Arg 106-His 107 pair and Asp 111) or partly important (the loop 133-137) for yeast cell growth, mitochondrial protein import and the suppression of mtHsp70 aggregation. Therefore, the function of Tim15 in yeast cell growth is well correlated with its ability to suppress mtHsp70 aggregation, although it is still unknown whether inhibition of mtHsp70 aggregation is the primary function of Tim15.