Protein disulfide isomerase–like proteins play opposing roles during retrotranslocation

Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
The Journal of Cell Biology (Impact Factor: 9.83). 07/2006; 173(6):853-9. DOI: 10.1083/jcb.200602046
Source: PubMed


Misfolded proteins in the endoplasmic reticulum (ER) are retained in the organelle or retrotranslocated to the cytosol for proteasomal degradation. ER chaperones that guide these opposing processes are largely unknown. We developed a semipermeabilized cell system to study the retrotranslocation of cholera toxin (CT), a toxic agent that crosses the ER membrane to reach the cytosol during intoxication. We found that protein disulfide isomerase (PDI) facilitates CT retrotranslocation, whereas ERp72, a PDI-like protein, mediates its ER retention. In vitro analysis revealed that PDI and ERp72 alter CT's conformation in a manner consistent with their roles in retrotranslocation and ER retention. Moreover, we found that PDI's and ERp72's opposing functions operate on endogenous ER misfolded proteins. Thus, our data identify PDI family proteins that play opposing roles in ER quality control and establish an assay to further delineate the mechanism of CT retrotranslocation.

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Available from: Wayne I Lencer, Oct 09, 2015
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    • "In the current analysis, MG132 treatment led also to the accumulation of a protein disulfide isomerase-like (PDI). PDI facilitates the retrotranslocation of some misfolded proteins from the ER to the proteasome in yeast and animal cells [50], [51]. Immunogold labeling with anti-ubiquitin antibody has provided direct proof of UbP accumulation in ER membranes upon MG132 treatment, leading to significant ER stress [13]. "
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    ABSTRACT: Chemical inhibition of the proteasome has been previously found to effectively impair pollen germination and tube growth in vitro. However, the mediators of these effects at the molecular level are unknown. By performing 2DE proteomic analysis, 24 differentially expressed protein spots, representing 14 unique candidate proteins, were identified in the pollen of kiwifruit (Actinidia deliciosa) germinated in the presence of the MG132 proteasome inhibitor. qPCR analysis revealed that 11 of these proteins are not up-regulated at the mRNA level, but are most likely stabilized by proteasome inhibition. These differentially expressed proteins are predicted to function in various pathways including energy and lipid metabolism, cell wall synthesis, protein synthesis/degradation and stress responses. In line with this evidence, the MG132-induced changes in the proteome were accompanied by an increase in ATP and ROS content and by an alteration in fatty acid composition.
    PLoS ONE 09/2014; 9(9):e108811. DOI:10.1371/journal.pone.0108811 · 3.23 Impact Factor
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    • "As noted above, retrotranslocation of ERAD substrates is preceded by a recognition step. The chaperone BiP, which is known to be involved in identification of non-glycosylated ERAD substrates, and an ER-resident ATPase (Torsin A) promote CTA1 retrotranslocation (Tsai et al., 2001; Winkeler et al., 2003; Forster et al., 2006; Moore et al., 2010). Sel1L and ERdj5, a co-chaperone of BiP, also facilitate CTA1 retrotranslocation, where the J domain of ERdj5 is required (Williams et al., 2013). "
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    ABSTRACT: Endoplasmic reticulum (ER)-associated degradation (ERAD) is a universally important process among eukaryotic cells. ERAD is necessary to preserve cell integrity since the accumulation of defective proteins results in diseases associated with neurological dysfunction, cancer, and infections. This process involves recognition of misfolded or misassembled proteins that have been translated in association with ER membranes. Recognition of ERAD substrates leads to their extraction through the ER membrane (retrotranslocation or dislocation), ubiquitination, and destruction by cytosolic proteasomes. This review focuses on ERAD and its components as well as how viruses use this process to promote their replication and to avoid the immune response.
    Frontiers in Microbiology 07/2014; 5:330. DOI:10.3389/fmicb.2014.00330 · 3.99 Impact Factor
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    • "To additionally test whether in vitro release of CTA1 resembles release in vivo, we examined an uncleavable mutant form of CTA, R192G CTA, that is not released into the cytosol in vivo [21], [29]. The R192 cleavage site is flanked by C187 and C199 that form the lone disulfide bond. "
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    ABSTRACT: Following retrograde trafficking to the endoplasmic reticulum (ER), cholera toxin A1 (CTA1) subunit hijacks ER-associated degradation (ERAD) machinery and retro-translocates into the cytosol to induce toxicity. We previously established a cell-based in vivo assay to identify ER components that regulate this process. However, elucidating cytosolic events that govern CTA1 retro-translocation using this assay is difficult as manipulating cytosolic factors often perturbs toxin retrograde transport to the ER. To circumvent this problem, we developed an in vitro assay in semi-permeabilized cells that directly monitors CTA1 release from the ER into the cytosol. We demonstrate CTA1 is released into the cytosol as a folded molecule in a p97- and proteasome-independent manner. Release nonetheless involves a GTP-dependent reaction. Upon extending this assay to the canonical ERAD substrate T-cell receptor α (TCRα), we found the receptor is unfolded when released into the cytosol and degraded by membrane-associated proteasome. In this reaction, p97 initially extracts TCRα from the ER membrane, followed by TCRα discharge into the cytosol that requires additional energy-dependent cytosolic activities. Our results reveal mechanistic insights into cytosolic events controlling CTA1 and TCRα retro-translocation, and provide a reliable tool to further probe this process.
    PLoS ONE 10/2013; 8(10):e75801. DOI:10.1371/journal.pone.0075801 · 3.23 Impact Factor
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