Mitochondrial Hsp70 cannot replace BiP in driving protein translocation into the yeast endoplasmic reticulum

Brodsky, Jeffrey L.; Bäuerle, Marc; Horst, Martin; McClellan, Amie J.

doi:10.1016/s0014-5793(98)01065-5

Cited by 10 publications

(9 citation statements)

References 31 publications

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“…Conversely, GrpE was unable to promote nucleotide release from Ssa1p (reference 37 and data not shown). Such specificity between chaperones and their cofactors has been observed for J protein-Hsp70 interactions (5,11,18,35,41,45,69). Because Hsp70 can be classified in several subclasses depending on the presence or absence of specific structural features within the highly conserved ATPase domain (9), it is reasonable to think that at least in some cases each chaperone and its cofactor(s) have evolved to fit one another.…”

Section: Discussionmentioning

confidence: 97%

Nucleotide Exchange Factor for the Yeast Hsp70 Molecular Chaperone Ssa1p

Kabani

Beckerich

Brodsky

2002

Molecular and Cellular Biology

127

138

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We report on the identification of Fes1p (yBR101cp) as a cytosolic homologue of Sls1p, an endoplasmic reticulum (ER) protein previously shown to act as a nucleotide exchange factor for yeast BiP (M. Kabani, J.-M. Beckerich, and C. Gaillardin, Mol. Cell. Biol. 20:6923-6934, 2000). We found that Fes1p associates preferentially to the ADP-bound form of the cytosolic Hsp70 molecular chaperone Ssa1p and promotes nucleotide release. Fes1p activity was shown to be compartment and species specific since Sls1p and Escherichia coli GrpE could not substitute for Fes1p. Surprisingly, whereas Sls1p stimulated the ATPase activity of BiP in cooperation with luminal J proteins, Fes1p was shown to inhibit the Ydj1p-mediated activation of Ssa1p ATPase activity in steady-state and single-turnover assays. Disruption of FES1 in several wild-type backgrounds conferred a strong thermosensitive phenotype but partially rescued ydj1-151 thermosensitivity. The ⌬fes1 strain was proficient for posttranslational protein translocation, as well as for the ER-associated degradation of two substrates. However, the ⌬fes1 mutant showed increased cycloheximide sensitivity and a general translational defect, suggesting that Fes1p acts during protein translation, a process in which Ssa1p and Ydj1p are known to be involved. In support of this hypothesis, Fes1p was found to be associated with ribosomes.

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Section: Discussionmentioning

confidence: 97%

Nucleotide Exchange Factor for the Yeast Hsp70 Molecular Chaperone Ssa1p

Kabani

Beckerich

Brodsky

2002

Molecular and Cellular Biology

127

138

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“…Functional complementation of kar2 mutants has been achieved with mammalian and plant BiP proteins (Normington et al, 1989;Denecke et al, 1991). However, neither cytosolic nor mitochondrial yeast HSP70s are able to substitute functionally for KAR2 (Brodsky et al, 1993(Brodsky et al, , 1998, likely due to the inability of these proteins to productively interact with proteins such as cochaperones or components of the ER translocation machinery. The partial complementation of kar2-159 mutant cells by Chlamydomonas BiP1 might be due to an ineffective interaction of this protein to substrates or specific partners.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of Protein Synthesis by TOR Inactivation Revealed a Conserved Regulatory Mechanism of the BiP Chaperone in Chlamydomonas

et al. 2011

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The target of rapamycin (TOR) kinase integrates nutritional and stress signals to coordinately control cell growth in all eukaryotes. TOR associates with highly conserved proteins to constitute two distinct signaling complexes termed TORC1 and TORC2. Inactivation of TORC1 by rapamycin negatively regulates protein synthesis in most eukaryotes. Here, we report that down-regulation of TOR signaling by rapamycin in the model green alga Chlamydomonas reinhardtii resulted in pronounced phosphorylation of the endoplasmic reticulum chaperone BiP. Our results indicated that Chlamydomonas TOR regulates BiP phosphorylation through the control of protein synthesis, since rapamycin and cycloheximide have similar effects on BiP modification and protein synthesis inhibition. Modification of BiP by phosphorylation was suppressed under conditions that require the chaperone activity of BiP, such as heat shock stress or tunicamycin treatment, which inhibits N-linked glycosylation of nascent proteins in the endoplasmic reticulum. A phosphopeptide localized in the substrate-binding domain of BiP was identified in Chlamydomonas cells treated with rapamycin. This peptide contains a highly conserved threonine residue that might regulate BiP function, as demonstrated by yeast functional assays. Thus, our study has revealed a regulatory mechanism of BiP in Chlamydomonas by phosphorylation/dephosphorylation events and assigns a role to the TOR pathway in the control of BiP modification.

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“…In this regard, yeast mitochondrial Hsp70, loaded into reconstituted endoplasmic reticulum vesicles, could not replace endoplasmic reticulum Hsp70 in an in vitro protein secretion assay (39). The inability of mitochondrial Hsp70 to function in BiP-mediated secretion was attributed to the inability of mitochondrial Hsp70 to associate productively with Sec63p, a BiP-specific DnaJ co-chaperone (39). The possible role of Hsp70 co-chaperones in nuclear transport has not been explored.…”

Section: Discussionmentioning

confidence: 99%

A Nuclear Export Signal Prevents Saccharomyces cerevisiae Hsp70 Ssb1p from Stimulating Nuclear Localization Signal-directed Nuclear Transport

Shul'ga

James

Craig

et al. 1999

Journal of Biological Chemistry

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Hsp70 has been implicated in nuclear localization signal (NLS)-directed nuclear transport. Saccharomyces cerevisiae contains distinct SSA and SSB gene families of cytosolic Hsp70s. The nucleocytoplasmic localization of Ssa1p and Ssb1p was investigated using green fluorescent protein (GFP) fusions. Whereas GFP-Ssa1p localized both to the nucleus and cytoplasm, GFP-Ssb1p appeared only in the cytosol. The C-terminal domain of Ssb1p contains a leucine-rich nuclear export signal (NES) that is necessary and sufficient to direct nuclear export. The accumulation of GFP-Ssb1p in the nuclei of xpo1-1 cells suggests that Ssb1p shuttles across the nuclear envelope. Elevated levels of SSA1 but not SSB1 suppressed the NLS-GFP nuclear localization defects of nup188-⌬ cells. Studies with Ssa1p/Ssb1p chimeras revealed that the Ssb1p NES is sufficient and necessary to inhibit the function of Ssa-or Ssb-type Hsp70s in nuclear transport. Thus, NES-less Ssb1p stimulates nuclear transport in nup188-⌬ cells and NES-containing Ssa1p does not. We conclude that the differential function of Ssa1p and Ssb1p in nuclear transport is due to the NES-directed export of the Ssb1p and not to functional differences in their ATPase or peptide binding domains.The import of proteins into nuclei is mediated by soluble nuclear localization signal (NLS) 1 receptors. SV40 large Tantigen-like NLSs are bound in the cytoplasm by karyopherin ␣ (Kap ␣), which serves as an adapter to link NLS-cargo to karyopherin ␤ (Kap ␤). Kap ␤ mediates docking at the cytoplasmic face of the nuclear pore complex (1). Saccharomyces cerevisiae contains a single Kap ␣ gene encoded by SRP1. Translocation of the NLS-cargo-Kap ␣/␤ ternary complex occurs through the central channel of the nuclear pore complex. Once in the nucleus, the NLS-cargo dissociates and Kap ␣ and ␤ are recycled to the cytoplasm (1, 2). Only a portion of the cellular import traffic is mediated by Kap ␣/␤ heterodimers. Other classes of NLS-cargo, for example shuttling pre-mRNA binding proteins that display M9-type import signals (3), are transported by Kap ␤-like factors that bind directly to cognate NLS-cargo without the aid of Kap ␣ adapters (1, 4). Hsp70/Hsc70s (collectively referred to here as Hsp70s) are conserved molecular chaperones that participate in a variety of cellular functions, including protein folding and transport and the repair of stress-induced damage (5-7). Hsp70s are composed of a 44-kDa N-terminal ATPase domain, an 18-kDa peptide binding domain, and a C-terminal 10-kDa variable domain of unknown function (8, 9). S. cerevisiae contains two families of cytosolic Hsp70 genes, SSA1-4 and SSB1-2 (10, 11). The yeast Ssa-type Hsp70s are similar to the cytosolic Hsp70s found in other organisms including bacteria. To date, Ssb-type Hsp70s have been identified only in fungi. In S. cerevisiae, Ssb1 and Ssb2 are associated with translating ribosomes and can be cross-linked to nascent polypeptides (12, 13).Hsp70s have been proposed to function in NLS-directed nuclear transport by promoting the formati...

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Mitochondrial Hsp70 cannot replace BiP in driving protein translocation into the yeast endoplasmic reticulum

Cited by 10 publications

References 31 publications

Nucleotide Exchange Factor for the Yeast Hsp70 Molecular Chaperone Ssa1p

Nucleotide Exchange Factor for the Yeast Hsp70 Molecular Chaperone Ssa1p

Inhibition of Protein Synthesis by TOR Inactivation Revealed a Conserved Regulatory Mechanism of the BiP Chaperone in Chlamydomonas

A Nuclear Export Signal Prevents Saccharomyces cerevisiae Hsp70 Ssb1p from Stimulating Nuclear Localization Signal-directed Nuclear Transport

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