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Global expression analyses of yeast cells harboring specific members of the cytosolic Hsp70-Ssa family

ABSTRACT: 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.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE32433 | GEO | 2012/12/31

SECONDARY ACCESSION(S): PRJNA147891

REPOSITORIES: GEO

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Global expression analyses of yeast cells harboring specific members of the cytosolic Hsp70-Ssa family

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.

2012-12-31 | E-GEOD-32433 | biostudies-arrayexpress

Regulation of the Hsf1-dependent transcriptome via conserved bipartite contacts with Hsp70 promotes survival in yeast

Project description:Protein homeostasis and cellular fitness in the presence of proteotoxic stress is promoted by heat shock factor 1 (HSF1), which controls basal and stress-induced expression of molecular chaperones and other targets. The major heat shock proteins Hsp70 and Hsp90 in turn participate in a negative feedback loop that ensures appropriate coordination of the heat shock response with environmental conditions. Features of this regulatory circuit in the budding yeast Saccharomyces cerevisiae have been recently defined, most notably regarding direct interaction between Hsf1 and the constitutively expressed Hsp70 Ssa1. We sought to further explore the complex nature of Ssa1/Hsf1 regulation. Ssa1 is found to interact independently with both the previously defined CE2 site in the Hsf1 carboxyl-terminal transcriptional activation domain as well as a novel site that we identify within the amino-terminal activation domain. Consistent with both sites bearing a recognition signature for Hsp70, we demonstrate that Ssa1 contacts Hsf1 using its substrate binding domain and abolishing either regulatory site results in loss of Ssa1 association. Removing Hsp70 regulation of Hsf1 results in global dysregulation of Hsf1 transcriptional activity, with synergistic effects when both sites are disrupted together on both gene expression and cellular fitness. Finally, we find that Hsp70 interacts with both transcriptional activation domains of Hsf1 in the related yeast Lachancea kluyveri, implying a conserved mechanism of regulation to promote cellular proteostasis.

2019-04-16 | GSE129832 | GEO

Dynamic interplay between Ydj1 and Hsp90 for interaction with nucleotide binding domain of Hsp70 regulates client maturation

Project description:Hsp90 constitutes one of the major chaperone machinery in the cell. The Hsp70 assists Hsp90 in its client maturation though the underlying basis of the Hsp70 role remains to be explored. In the present study we identified novel mutations in the nucleotide-binding domain of yeast Ssa1 Hsp70 (Ssa1-T175N and Ssa1-D158N) that adversely affect the maturation of Hsp90 clients v-Src and Ste11. The identified Ssa1 amino acids critical for Hsp90 function were also found to be conserved across species such as in E.coli DnaK and the constitutive Hsp70 isoform (HspA8) in humans. These mutations are distal to the C-terminus of Hsp70 that primarily mediates Hsp90 interaction through the bridge protein Sti1. Intriguingly, we found that the bridge protein Sti1 is critical for cellular viability in cells expressing Ssa1-T175N (A1-T175N) or Ssa1-D158N (A1-D158N) as sole Ssa Hsp70. The growth defect was specific for sti1Δ, as deletion of none of the other Hsp90 co-chaperones showed lethality in A1-T175N or A1-D158N. Mass-spectrometry based whole proteome analysis of A1-T175 cells lacking Sti1 showed an altered abundance of various kinases and transcription factors suggesting compromised Hsp90 activity. Further proteomic analysis showed that pathways involved in signaling, signal transduction, and protein phosphorylation are markedly downregulated in the A1-T175N upon repression of Sti1 under DOX regulated promoter. In contrast to Ssa1, the homologous mutations in Ssa4 (Ssa4-T175N/D158N) supported cell growth even in the absence of Sti1. Overall, our data suggest that Ydj1 competes with Hsp90 for binding to Hsp70, and thus regulates Hsp90 interaction with the nucleotide-binding domain of Hsp70. The study thus provides new insight into the Hsp70-mediated regulation of Hsp90 and broadens our understanding of the intricate complexities of the Hsp70-Hsp90 network. This project was done under Deepak Sharma, CSIR-IMTECH, deepaks@imtech.res.in as a PI of the project.

2022-10-20 | PXD034983 | Pride

Saccharomyces cerevisiae

Project description:Global expression analyses of yeast cells harboring specific members of the cytosolic Hsp70-Ssa family

| PRJNA147891 | ENA

The Quantitative Changes in the Yeast Hsp70 and Hsp90 Interactomes upon DNA Damage

Project description:The molecular chaperones Hsp70 and Hsp90 participate in many important cellular processes, including how cells respond to DNA damage. In this study, we applied quantitative affinity-purification mass spectrometry (AP-MS) proteomics to understand the protein network through which Hsp70 and Hsp90 exert their effects on the DNA damage response (DDR). We characterized the interactomes of the yeast Hsp70 isoform Ssa1 and Hsp90 isoform Hsp82 before and after exposure to methyl methanesulfonate. We identified 256 chaperone interactors, 146 of which are novel. Although the majority of chaperone interaction remained constant under DNA damage, 5 proteins (Coq5, Ast1, Cys3, Ydr210c and Rnr4) increased in interaction with Ssa1 and/or Hsp82. This data is related to article “Quantitative proteomics of the yeast Hsp70/Hsp90 interactomes during DNA damage reveals chaperone-dependent regulation of ribonucleotide reductase”.

2014-12-08 | PXD001284 | Pride

Rapid acetylation of yeast Hsp70 mediates the cellular response to heat stress

Project description:Hsp70 is a highly conserved molecular chaperone critical for the folding of new and denatured proteins. While traditional models state that cells respond to stress by upregulating inducible HSPs, this response is relatively slow and is limited by transcriptional and translational machinery. Recent studies have identified a number of post-translational modifications (PTMs) on Hsp70 that act to fine-tune its function. We utilized mass spectrometry to determine whether yeast Hsp70 (Ssa1) is differentially modified upon heat shock. We uncovered four lysine residues on Ssa1, K86, K185, K354 and K562 that are deacetylated in response to heat shock. Mutation of these sites cause a substantial remodeling of the Hsp70 interaction network of co-chaperone partners and client proteins while preserving essential chaperone function. Acetylation/deacetylation at these residues alter expression of other heat-shock induced chaperones as well as directly influencing Hsf1 activity. Taken together our data suggest that cells may have the ability to respond to heat stress quickly though Hsp70 deacetylation, followed by a slower, more traditional transcriptional response.

2020-02-24 | PXD015185 | Pride

HSP70 binds RNA and regulates transcription by RNA Polymerase III [RNA-seq]

Project description:We examined the RNA binding ability of the 70-kDa heat shock protein (HSP70) family member HSPA1A and identified the RNA species bound to HSP70 in vivo. We used a UV-crosslinkiung and immunoprecipitation based approach named FLASH-seq (Aktaş et al., 2017) using 3xFLAG-Histidine-Biotin tagged ectopically expressed protein in HEK293T cells.

2024-01-17 | GSE191244 | GEO

HSP70 binds RNA and regulates transcription by RNA Polymerase III [ChIP-seq]

2024-01-17 | GSE191243 | GEO

HSP70 binds RNA and regulates transcription by RNA Polymerase III [FLASH-seq]

Project description:We examined the RNA binding ability of the 70-kDa heat shock protein (HSP70) family member HSPA1A and HSP8 and identified the RNA species bound to HSP70 or HSP8 in vivo. We used a UV-crosslinkiung and immunoprecipitation based approach named FLASH-seq (Akta? et al., 2017) using 3xFLAG-Histidine-Biotin tagged ectopically expressed protein in HEK293T cells.

2024-01-17 | GSE191242 | GEO

Genetic analysis of Hsp70 phosphorylation sitesreveals a role in Candida albicanscell and colony morphogenesis

Project description:Heat shock proteins are best known for their role as chaperonins involved in general proteostasis, but they can also participate in specific cellular regulatory pathways, e.g. via their post-translational modification. Hsp70/Ssa1 is a central cytoplasmic chaperonin in eukaryotes, which also participates in cell cycle regulation via its phosphorylation at a specific residue. Here we analyze the role of Ssa1 phosphorylation in the morphogenesis of the fungus Candida albicans, a common human opportunistic pathogen. C. albicans can assume alternative yeast and hyphal (mold) morphologies, an ability that contributes to its virulence. We identified 11 phosphorylation sites on C. albicans Ssa1, of which 8were only detected in the hyphalcells. Genetic analysis of these sites revealedallele-specific effects on growth at high temperature, cell and colony morphology, and resistance to cell wall-active drugs.This analysis could help direct screens for Ssa1-specific drugs to combat C. albicans virulence.The pleiotropic effects of many Ssa1 mutations are consistent with the large number of Ssa1 client proteins, whereas the lack of concordance between the phenotypes of the different alleles suggests that different sites on Ssa1 can affect interaction with specific classes of client protein, and that modification of these sites can play cellular regulatory roles, consistent with the “chaperone code” hypothesis.

2018-11-21 | PXD010827 | Pride

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