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Dcr2 targets Ire1 and downregulates the unfolded protein response in Saccharomyces cerevisiae.

ABSTRACT: Accumulation of unfolded proteins in the endoplasmic reticulum triggers the unfolded protein response (UPR). How the UPR is downregulated is not well understood. Inositol requirement 1 (Ire1) is an endoplasmic reticulum transmembrane UPR sensor in Saccharomyces cerevisiae. When the UPR is triggered, Ire1 is autophosphorylated, on Ser 840 and Ser 841, inducing the cytosolic endonuclease activity of Ire1, thereby initiating the splicing and translational de-repression of HAC1 mRNA. Homologous to Atf/Creb1 (Hac1) activates UPR transcription. Here, we report that the dose-dependent cell-cycle regulator 2 (Dcr2) phosphatase functionally and physically interacts with Ire1. We identified genetic interactions between DCR2 and genes, including IRE1, which are involved in secretory processes. Overexpression of DCR2, but not of a catalytically inactive DCR2 allele, significantly delays HAC1 splicing and sensitizes cells to the UPR. Furthermore, Dcr2 physically interacts in vivo with Ire1-S840E,S841E, which mimics phosphorylated Ire1, and Dcr2 de-phosphorylates Ire1 in vitro. Our results are consistent with de-phosphorylation of Ire1 being a mechanism for antagonizing UPR signalling.

SUBMITTER: Guo J

PROVIDER: S-EPMC1679794 | biostudies-literature | 2006 Nov

REPOSITORIES: biostudies-literature

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Dcr2 targets Ire1 and downregulates the unfolded protein response in Saccharomyces cerevisiae.

Guo Jinbai J Polymenis Michael M

EMBO reports 20060922 11

Accumulation of unfolded proteins in the endoplasmic reticulum triggers the unfolded protein response (UPR). How the UPR is downregulated is not well understood. Inositol requirement 1 (Ire1) is an endoplasmic reticulum transmembrane UPR sensor in Saccharomyces cerevisiae. When the UPR is triggered, Ire1 is autophosphorylated, on Ser 840 and Ser 841, inducing the cytosolic endonuclease activity of Ire1, thereby initiating the splicing and translational de-repression of HAC1 mRNA. Homologous to A ...[more]

PMID: 16990850

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Project description:BackgroundThe expression of human virus surface proteins, as well as other mammalian glycoproteins, is much more efficient in cells of higher eukaryotes rather than yeasts. The limitations to high-level expression of active viral surface glycoproteins in yeast are not well understood. To identify possible bottlenecks we performed a detailed study on overexpression of recombinant mumps hemagglutinin-neuraminidase (MuHN) and measles hemagglutinin (MeH) in yeast Saccharomyces cerevisiae, combining the analysis of recombinant proteins with a proteomic approach.ResultsOverexpressed recombinant MuHN and MeH proteins were present in large aggregates, were inactive and totally insoluble under native conditions. Moreover, the majority of recombinant protein was found in immature form of non-glycosylated precursors. Fractionation of yeast lysates revealed that the core of viral surface protein aggregates consists of MuHN or MeH disulfide-linked multimers involving eukaryotic translation elongation factor 1A (eEF1A) and is closely associated with small heat shock proteins (sHsps) that can be removed only under denaturing conditions. Complexes of large Hsps seem to be bound to aggregate core peripherally as they can be easily removed at high salt concentrations. Proteomic analysis revealed that the accumulation of unglycosylated viral protein precursors results in specific cytosolic unfolded protein response (UPR-Cyto) in yeast cells, characterized by different action and regulation of small Hsps versus large chaperones of Hsp70, Hsp90 and Hsp110 families. In contrast to most environmental stresses, in the response to synthesis of recombinant MuHN and MeH, only the large Hsps were upregulated whereas sHsps were not. Interestingly, the amount of eEF1A was also increased during this stress response.ConclusionsInefficient translocation of MuHN and MeH precursors through ER membrane is a bottleneck for high-level expression in yeast. Overexpression of these recombinant proteins induces the UPR's cytosolic counterpart, the UPR-Cyto, which represent a subset of proteins involved in the heat-shock response. The involvement of eEF1A may explain the mechanism by which only large chaperones, but not small Hsps are upregulated during this stress response. Our study highlights important differences between viral surface protein expression in yeast and mammalian cells at the first stage of secretory pathway.

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Dcr2 targets Ire1 and downregulates the unfolded protein response in Saccharomyces cerevisiae.

Publications

Dcr2 targets Ire1 and downregulates the unfolded protein response in Saccharomyces cerevisiae.

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