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Development of an assay to discover novel cytosolic factors for cholera toxin retro‐translocation
Author(s) -
Moore Paul Christopher,
Tsai Billy
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.lb107
Subject(s) - cytosol , cholera toxin , endoplasmic reticulum associated protein degradation , endoplasmic reticulum , toxin , microbiology and biotechnology , proteasome , hsp90 , biochemistry , biology , chemistry , heat shock protein , enzyme , gene
Cholera toxin (CT) is transported from the plasma membrane of host cells to the endoplasmic reticulum (ER) where the CTA1 subunit retro‐translocates to the cytosol to induce toxicity. In the ER, the toxin co‐opts machinery that disposes misfolded proteins to the cytosol for ubiquitin‐dependent proteasomal destruction. However, once extracted into the cytosol the toxin evades proteasomal degradation, refolds, and becomes catalytically active. To elucidate this mechanism we developed an in vitro assay that permits release of CTA1 from intact ER membranes upon addition of cytosolic extract. The toxin must be delivered to the ER and processed normally into CTA1 for release. As expected, treatment with the proteasome inhibitor epoxomicin has no affect on toxin release. Furthermore, heat inactivation and energy depletion of the extract indicate that a proteinaceous, energy‐dependent activity exists. Preliminary fractionation of the cytosol indicates that this activity does not co‐fractionate with p97, Hsp90 and Hsp70; primary candidates for the retro‐translocation factor. Concurrent experiments with TCRα show that the assay can be applied to a variety of ERAD substrates. Funding: Burroughs Wellcome & NIH .