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PLoS One. 2013 Oct 11;8(10):e75801. doi: 10.1371/journal.pone.0075801. eCollection 2013.

Establishment of an in vitro transport assay that reveals mechanistic differences in cytosolic events controlling cholera toxin and T-cell receptor α retro-translocation.

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Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.


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.

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