title: Establishment of an in vitro transport assay that reveals mechanistic differences in cytosolic events controlling cholera toxin and T-cell receptor α retro-translocation.
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.
authors.affiliation: Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.
authors.forename: Paul
authors.initials: P
authors.lastname: Moore
authors.forename: Kaiyu
authors.initials: K
authors.lastname: He
authors.forename: Billy
authors.initials: B
authors.lastname: Tsai
mesh_terms: D001681:Biological Assay; D002463:Cell Membrane Permeability; D002772:Cholera Toxin / Q000737:chemistry / Q000378:metabolism*; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism*; D060746:Endoplasmic Reticulum-Associated Degradation / Q000235:genetics; D006160:Guanosine Triphosphate / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D021381:Protein Transport; D059748:Proteolysis; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000737:chemistry / Q000378:metabolism*
chemical_list: D009687:Nuclear Proteins; D016693:Receptors, Antigen, T-Cell, alpha-beta; C094680:nuclear protein import factor p97; D006160:Guanosine Triphosphate; D002772:Cholera Toxin; D046988:Proteasome Endopeptidase Complex