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Central domain deletions affect the SAXS solution structure and function of yeast Hsp40 proteins Sis1 and Ydj1.

ABSTRACT: Ydj1 and Sis1 are structurally and functionally distinct Hsp40 proteins of the yeast cytosol. Sis1 is an essential gene whereas the ydj1 gene is essential for growth at elevated temperatures and cannot complement sis1 gene deletion. Truncated polypeptides capable of complementing the sis1 gene deletion comprise the J-domain of either Sis1 or Ydj1 connected to the G/F region of Sis1 (but not Ydj1). Sis1 mutants in which the G/F was deleted but G/M maintained were capable of complementing the sis1 gene deletion.To investigate the relevance of central domains on the structure and function of Ydj1 and Sis1 we prepared Sis1 constructs deleting specific domains. The mutants had decreased affinity for heated luciferase but were equally capable of stimulating ATPase activity of Hsp70. Detailed low resolution structures were obtained and the overall flexibility of Hsp40 and its mutants were assessed using SAXS methods. Deletion of either the G/M or the G/M plus CTDI domains had little impact on the quaternary structure of Sis1 analyzed by the SAXS technique. However, deletion of the ZFLR-CTDI changed the relative position of the J-domains in Ydj1 in such a way that they ended up resembling that of Sis1. The results revealed that the G/F and G/M regions are not the only flexible domains. All model structures exhibit a common clamp-like conformation.Our results suggest that the central domains, previously appointed as important features for substrate binding, are also relevant keeping the J-domains in their specific relative positions. The clamp-like architecture observed seems also to be favorable to the interactions of Hsp40 with Hsp70.

SUBMITTER: Silva JC

PROVIDER: S-EPMC3236591 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Central domain deletions affect the SAXS solution structure and function of yeast Hsp40 proteins Sis1 and Ydj1.

Silva Julio C JC Borges Julio C JC Cyr Douglas M DM Ramos Carlos Hi CH Torriani Iris L IL

BMC structural biology 20111019

<h4>Background</h4>Ydj1 and Sis1 are structurally and functionally distinct Hsp40 proteins of the yeast cytosol. Sis1 is an essential gene whereas the ydj1 gene is essential for growth at elevated temperatures and cannot complement sis1 gene deletion. Truncated polypeptides capable of complementing the sis1 gene deletion comprise the J-domain of either Sis1 or Ydj1 connected to the G/F region of Sis1 (but not Ydj1). Sis1 mutants in which the G/F was deleted but G/M maintained were capable of com ...[more]

PMID: 22011374

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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, using S. cerevisiae strain expressing Ssa1 as sole Ssa Hsp70, 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, and proximal to Ydj1 (Hsp40 co-chaperone of Hsp70 family) binding region. 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-T175N 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 repressing Sti1 expression using doxycycline regulatable promoter. In contrast to Ssa1, the homologous mutations in Ssa4 (Ssa4-T175N/D158N), the stress inducible Hsp70 isoform, 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.

Dataset Information

Central domain deletions affect the SAXS solution structure and function of yeast Hsp40 proteins Sis1 and Ydj1.

Publications

Central domain deletions affect the SAXS solution structure and function of yeast Hsp40 proteins Sis1 and Ydj1.

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