Mutations in the Yeast Hsp70, Ssa1, at P417 Alter ATP Cycling, Interdomain Coupling, and Specific Chaperone Functions.
ABSTRACT: The major cytoplasmic Hsp70 chaperones in the yeast Saccharomyces cerevisiae are the Ssa proteins, and much of our understanding of Hsp70 biology has emerged from studying ssa mutant strains. For example, Ssa1 catalyzes multiple cellular functions, including protein transport and degradation, and to this end, the ssa1-45 mutant has proved invaluable. However, the biochemical defects associated with the corresponding Ssa1-45 protein (P417L) are unknown. Consequently, we characterized Ssa1 P417L, as well as a P417S variant, which corresponds to a mutation in the gene encoding the yeast mitochondrial Hsp70. We discovered that the P417L and P417S proteins exhibit accelerated ATPase activity that was similar to the Hsp40-stimulated rate of ATP hydrolysis of wild-type Ssa1. We also found that the mutant proteins were compromised for peptide binding. These data are consistent with defects in peptide-stimulated ATPase activity and with results from limited proteolysis experiments, which indicated that the mutants' substrate binding domains were highly vulnerable to digestion. Defects in the reactivation of heat-denatured luciferase were also evident. Correspondingly, yeast expressing P417L or P417S as the only copy of Ssa were temperature sensitive and exhibited defects in Ssa1-dependent protein translocation and misfolded protein degradation. Together, our studies suggest that the structure of the substrate binding domain is altered and that coupling between this domain and the nucleotide binding domain is disabled when the conserved P417 residue is mutated. Our data also provide new insights into the nature of the many cellular defects associated with the ssa1-45 allele.
Project description:Heat-shock protein 40 (Hsp40) enables Hsp70 to play critical roles in a number of cellular processes, such as protein folding, assembly, degradation and translocation in vivo. Hsp40 recognizes and binds non-native polypeptides and delivers them to Hsp70. Then Hsp40 stimulates the ATPase activity of Hsp70 to fold the polypeptides. By using yeast Hsp40 Sis1 and yeast Hsp70 Ssa1 as our model proteins, we found that the Sis1 peptide-binding fragment interacts directly with the full-length Ssa1 in vitro. Further studies showed that the C-terminal lid domain of Ssa1 could interact with Sis1 peptide-binding domain physically in vitro. The Sis1 peptide-binding fragment forms a stable complex with the Ssa1 C-terminal lid domain in solution. The interactions between these two proteins appear to be charge-charge interactions because high-ionic-strength buffer can dissociate the complex. Further mapping studies showed that the Sis1 peptide-binding fragment binds the extreme C-terminal 15 amino acid residues of Ssa1. A flexible glycine-rich region is followed by these 15 residues in the Ssa1 primary sequence. Atomic force microscopy of the Sis1-Ssa1 complex showed that only one end of the Ssa1 lid domain binds the Sis1 peptide-binding-fragment dimer at the upper level of the huge groove within the Sis1 dimer. Based on the data, we propose an "anchoring and docking" model to illustrate the mechanisms by which Hsp40 interacts with Hsp70 and delivers the non-native polypeptide to Hsp70.
Project description:Heat shock protein (Hsp) 40 facilitates the critical role of Hsp70 in a number of cellular processes such as protein folding, assembly, degradation and translocation in vivo. Hsp40 and Hsp70 stay in close contact to achieve these diverse functions. The conserved C-terminal EEVD motif in Hsp70 has been shown to regulate Hsp40-Hsp70 interaction by an unknown mechanism. Here, we provide a structural basis for this regulation by determining the crystal structure of yeast Hsp40 Sis1 peptide-binding fragment complexed with the Hsp70 Ssa1 C-terminal. The Ssa1 extreme C-terminal eight residues, G634PTVEEVD641, form a beta-strand with the domain I of Sis1 peptide-binding fragment. Surprisingly, the Ssa1 C-terminal binds Sis1 at the site where Sis1 interacts with the non-native polypeptides. The negatively charged residues within the EEVD motif in Ssa1 C-terminal form extensive charge-charge interactions with the positively charged residues in Sis1. The structure-based mutagenesis data support the structural observations.
Project description:Yeast DNA microarray was used to assess and compare the global expression profile of strains harboring different family members of the major cytosolic Hsp70 family. Viability of a yeast strain deleted for all genes encoding members of the Hsp70-Ssa family (Ssa1/2/3/4) was maintained by the presence of a single Ssa family member expressed ectopically from a plasmid vector. The Hsp70-Ssa family constitutes the main source of Hsp70 molecular chaperone activity in the yeast cell. A yeast cell must actively express a member of this family to remain viable. Hsp70-Ssa are highly conserved both within yeast and amongst other species. Ssa1 and 2 are 97% identical at amino acid level and 80% identical to Ssa3 and 4. The aim of this study was to attribute specific functions to single Ssa family members by identifying specific genes or gene families whose expression was altered in the presence (or absence) of Ssa family members. Eight independent RNA samples were pooled to represent a single biological sample for expression analysis. For example, the single sample analyzed for cells harboring only Ssa1 is a pooled sample of eight independent RNA extractions. Hybridization was performed for cells harboring either Ssa1, Ssa2, Ssa3 or Ssa4 as the sole cytosolic Hsp70-Ssa family member. Gene expression profiles of Ssa2/3/4 were all compared to Ssa1.
Project description:Candida albicans Ssa1 and Ssa2 are members of the HSP70 family of heat shock proteins that are expressed on the cell surface and function as receptors for antimicrobial peptides such as histatins. We investigated the role of Ssa1 and Ssa2 in mediating pathogenic host cell interactions and virulence. A C. albicans ssa1?/? mutant had attenuated virulence in murine models of disseminated and oropharyngeal candidiasis, whereas an ssa2?/? mutant did not. In vitro studies revealed that the ssa1?/? mutant caused markedly less damage to endothelial cells and oral epithelial cell lines. Also, the ssa1?/? mutant had defective binding to endothelial cell N-cadherin and epithelial cell E-cadherin, receptors that mediate host cell endocytosis of C. albicans. As a result, this mutant had impaired capacity to induce its own endocytosis by endothelial cells and oral epithelial cells. Latex beads coated with recombinant Ssa1 were avidly endocytosed by both endothelial cells and oral epithelial cells, demonstrating that Ssa1 is sufficient to induce host cell endocytosis. These results indicate that Ssa1 is a novel invasin that binds to host cell cadherins, induces host cell endocytosis, and is critical for C. albicans to cause maximal damage to host cells and induce disseminated and oropharyngeal disease.
Project description:We previously described an Hsp70 mutant (Ssa1-21p), altered in a conserved residue (L483W), that dominantly impairs yeast [PSI(+)] prion propagation without affecting growth. We generated new SSA1 mutations that impaired [PSI(+)] propagation and second-site mutations in SSA1-21 that restored normal propagation. Effects of mutations on growth did not correlate with [PSI(+)] phenotype, revealing differences in Hsp70 function required for growth and [PSI(+)] propagation and suggesting that Hsp70 interacts differently with [PSI(+)] prion aggregates than with other cellular substrates. Complementary suppression of altered activity between forward and suppressing mutations suggests that mutations that impair [PSI(+)] affect a similar Hsp70 function and that suppressing mutations similarly overcome this effect. All new mutations that impaired [PSI(+)] propagation were located in the ATPase domain. Locations and homology of several suppressing substitutions suggest that they weaken Hsp70's substrate-trapping conformation, implying that impairment of [PSI(+)] by forward mutations is due to altered ability of the ATPase domain to regulate substrate binding. Other suppressing mutations are in residues important for interactions with Hsp40 or TPR-containing cochaperones, suggesting that such interactions are necessary for the impairment of [PSI(+)] propagation caused by mutant Ssa1p.
Project description:Heat shock proteins of 70 kDa (Hsp70s) and their J domain-containing Hsp40 cofactors are highly conserved chaperone pairs that facilitate a large number of cellular processes. The observation that each Hsp70 partners with many J domain-containing proteins (JDPs) has led to the hypothesis that Hsp70 function is dictated by cognate JDPs. If this is true, one might expect highly divergent Hsp70-JDP pairs to be unable to function in vivo. However, we discovered that, when a yeast cytosolic JDP, Ydj1, was targeted to the mammalian endoplasmic reticulum (ER), it interacted with the ER-lumenal Hsp70, BiP, and bound to BiP substrates. Conversely, when a mammalian ER-lumenal JDP, ERdj3, was directed to the yeast cytosol, it rescued the temperature-sensitive growth phenotype of yeast-containing mutant alleles in two cytosolic JDPs, HLJ1 and YDJ1, and activated the ATP hydrolysis rate of Ssa1, the yeast cytosolic Hsp70 that partners with Hlj1 and Ydj1. Surprisingly, ERdj3 mutants that were compromised for substrate binding were unable to rescue the hlj1ydj1 growth defect even though they stimulated the ATPase activity of Ssa1. Yet, J domain mutants of ERdj3 that were defective for interaction with Ssa1 restored the growth of hlj1ydj1 yeast. Taken together, these data suggest that the substrate binding properties of certain JDPs, not simply the formation of unique Hsp70-JDP pairs, are critical to specify in vivo function.
Project description:Genetic screens using Saccharomyces cerevisiae have identified an array of cytosolic Hsp70 mutants that are impaired in the ability to propagate the yeast [PSI(+)] prion. The best characterized of these mutants is the Ssa1 L483W mutant (so-called SSA1-21), which is located in the substrate-binding domain of the protein. However, biochemical analysis of some of these Hsp70 mutants has so far failed to provide major insight into the specific functional changes in Hsp70 that cause prion impairment. In order to gain a better understanding of the mechanism of Hsp70 impairment of prions we have taken an in silico approach and focused on the Escherichia coli Hsp70 ortholog DnaK. Using steered molecular dynamics simulations (SMD) we demonstrate that DnaK variant L484W (analogous to SSA1-21) is predicted to bind substrate more avidly than wild-type DnaK due to an increase in numbers of hydrogen bonds and hydrophobic interactions between chaperone and peptide. Additionally the presence of the larger tryptophan side chain is predicted to cause a conformational change in the peptide-binding domain that physically impairs substrate dissociation. The DnaK L484W variant in combination with some SSA1-21 phenotypic second-site suppressor mutations exhibits chaperone-substrate interactions that are similar to wild-type protein and this provides a rationale for the phenotypic suppression that is observed. Our computational analysis fits well with previous yeast genetics studies regarding the functionality of the Ssa1-21 protein and provides further evidence suggesting that manipulation of the Hsp70 ATPase cycle to favor the ADP/substrate-bound form impairs prion propagation. Furthermore, we demonstrate how SMD can be used as a computational tool for predicting Hsp70 peptide-binding domain mutants that impair prion propagation.
Project description:BACKGROUND: Cytosolic Hsp70 is a ubiquitous molecular chaperone that is involved in responding to a variety of cellular stresses. A major function of Hsp70 is to prevent the aggregation of denatured proteins by binding to exposed hydrophobic regions and preventing the accumulation of amorphous aggregates. To gain further insight into the functional redundancy and specialisation of the highly homologous yeast Hsp70-Ssa family we expressed each of the individual Ssa proteins as the sole source of Hsp70 in the cell and assessed phenotypic differences in prion propagation and stress resistance. Additionally we also analysed the global gene expression patterns in yeast strains expressing individual Ssa proteins, using microarray and RT-qPCR analysis. RESULTS: We confirm and extend previous studies demonstrating that cells expressing different Hsp70-Ssa isoforms vary in their ability to propagate the yeast [PSI+] prion, with Ssa3 being the most proficient. Of the four Ssa family members the heat inducible isoforms are more proficient in acquiring thermotolerance and we show a greater requirement than was previously thought, for cellular processes in addition to the traditional Hsp104 protein disaggregase machinery, in acquiring such thermotolerance. Cells expressing different Hsp70-Ssa isoforms also display differences in phenotypic response to exposure to cell wall damaging and oxidative stress agents, again with the heat inducible isoforms providing better protection than constitutive isoforms. We assessed global transcriptome profiles for cells expressing individual Hsp70-Ssa isoforms as the sole source of cytosolic Hsp70, and identified a significant difference in cellular gene expression between these strains. Differences in gene expression profiles provide a rationale for some phenotypic differences we observed in this study. We also demonstrate a high degree of correlation between microarray data and RT-qPCR analysis for a selection of genes. CONCLUSIONS: The Hsp70-Ssa family provide both redundant and variant-specific functions within the yeast cell. Yeast cells expressing individual members of the Hsp70-Ssa family as the sole source of Ssa protein display differences in global gene expression profiles. These changes in global gene expression may contribute significantly to the phenotypic differences observed between the Hsp70-Ssa family members.
Project description:Plus-strand RNA virus replication occurs via the assembly of viral replicase complexes involving multiple viral and host proteins. To identify host proteins present in the cucumber necrosis tombusvirus (CNV) replicase, we affinity purified functional viral replicase complexes from yeast. Mass spectrometry analysis of proteins resolved by two-dimensional gel electrophoresis revealed the presence of CNV p33 and p92 replicase proteins as well as four major host proteins in the CNV replicase. The host proteins included the Ssa1/2p molecular chaperones (yeast homologues of Hsp70 proteins), Tdh2/3p (glyceraldehyde-3-phosphate dehydrogenase, an RNA-binding protein), Pdc1p (pyruvate decarboxylase), and an unknown approximately 35-kDa acidic protein. Copurification experiments demonstrated that Ssa1p bound to p33 replication protein in vivo, and surface plasmon resonance measurements with purified recombinant proteins confirmed this interaction in vitro. The double mutant strain (ssa1 ssa2) showed 75% reduction in viral RNA accumulation, whereas overexpression of either Ssa1p or Ssa2p stimulated viral RNA replication by approximately threefold. The activity of the purified CNV replicase correlated with viral RNA replication in the above-mentioned ssa1 ssa2 mutant and in the Ssa overexpression strains, suggesting that Ssa1/2p likely plays an important role in the assembly of the CNV replicase.
Project description:Several cellular chaperones have been shown to affect the propagation of the yeast prions [PSI(+)], [PIN(+)] and [URE3]. Ssa1 and Ssa2 are Hsp70 family chaperones that generally cause pro-[PSI(+)] effects, since dominant-negative mutants of Ssa1 or Ssa2 cure [PSI(+)], and overexpression of Ssa1 enhances de novo [PSI(+)] appearance and prevents curing by excess Hsp104. In contrast, Ssa1 was shown to have anti-[URE3] effects, since overexpression of Ssa1 cures [URE3]. Here we show that excess Ssa1 or Ssa2 can also cure [PSI(+)]. This curing is enhanced in the presence of [PIN(+)]. During curing, Sup35-GFP fluorescent aggregates get bigger and fewer in number, which leads to their being diluted out during cell division, a phenotype that was also observed during the curing of [PSI(+)] by certain variants of [PIN(+)]. The sizes of the detergent-resistant [PSI(+)] prion oligomers increase during [PSI(+)] curing by excess Ssa1. Excess Ssa1 likewise leads to an increase in oligomer sizes of low, medium and very high [PIN(+)] variants. While these phenotypes are also caused by inhibition of Hsp104 or Sis1, the overexpression of Ssa1 did not cause any change in Hsp104 or Sis1 levels.