In vivo bipartite interaction between the Hsp40 Sis1 and Hsp70 in Saccharomyces cerevisiae.
The essential Hsp40, Sis1, is a J-protein cochaperone for the Ssa class of Hsp70's of Saccharomyces cerevisiae. Sis1 is required for the maintenance of the prion [RNQ(+)], as Sis1 lacking its 55-amino-acid glycine-rich region (G/F) does not maintain [RNQ(+)]. We report that overexpression of Sis1DeltaG/F in an otherwise wild-type strain ... had a negative effect on both cell growth and [RNQ(+)] maintenance, while overexpression of wild-type Sis1 did not. Overexpression of the related Hsp40 Ydj1 lacking its G/F region did not cause inhibition of growth, indicating that this dominant effect of Sis1DeltaG/F is not a characteristic shared by all Hsp40's. Analysis of small deletions within the SIS1 G/F region indicated that the observed dominant effects were caused by the absence of sequences known to be important for Sis1's unique cellular functions. These inhibitory effects of Sis1DeltaG/F were obviated by alterations in the N-terminal J-domain of Sis1 that affect interaction with Ssa's ATPase domain. In addition, a genetic screen designed to isolate additional mutations that relieved these inhibitory effects identified two residues in Sis1's carboxy-terminal domain. These alterations disrupted the interaction of Sis1 with the 10-kD carboxy-terminal regulatory domain of Ssa1, indicating that Sis1 has a bipartite interaction with Ssa in vivo.
Mesh Terms:
Adenosine Triphosphatases, Amino Acid Sequence, Binding, Competitive, Cell Proliferation, Dose-Response Relationship, Drug, Enzyme-Linked Immunosorbent Assay, Fungal Proteins, Gene Deletion, Gene Expression Regulation, Fungal, Gene Library, Genes, Fungal, Genetic Techniques, Green Fluorescent Proteins, HSP40 Heat-Shock Proteins, HSP70 Heat-Shock Proteins, Heat-Shock Proteins, Immunoblotting, Luciferases, Molecular Sequence Data, Mutation, Plasmids, Prions, Protein Binding, Protein Conformation, Protein Structure, Secondary, Protein Structure, Tertiary, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Substrate Specificity, Temperature, Time Factors
Adenosine Triphosphatases, Amino Acid Sequence, Binding, Competitive, Cell Proliferation, Dose-Response Relationship, Drug, Enzyme-Linked Immunosorbent Assay, Fungal Proteins, Gene Deletion, Gene Expression Regulation, Fungal, Gene Library, Genes, Fungal, Genetic Techniques, Green Fluorescent Proteins, HSP40 Heat-Shock Proteins, HSP70 Heat-Shock Proteins, Heat-Shock Proteins, Immunoblotting, Luciferases, Molecular Sequence Data, Mutation, Plasmids, Prions, Protein Binding, Protein Conformation, Protein Structure, Secondary, Protein Structure, Tertiary, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Substrate Specificity, Temperature, Time Factors
Genetics
Date: Apr. 01, 2005
PubMed ID: 15687271
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