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.
Highlights
To cause disease, cholera toxin (CT) binds to ganglioside GM1 receptor on the plasma membrane of host cells, becomes endocytosed within vesicles, and traffics through the Golgi apparatus en route to the endoplasmic reticulum (ER)
When cells were intoxicated with wildtype (WT) CT and subjected to the fractionation procedure, we found catalytic A subunit (CTA), cholera toxin A1 (CTA1) and the receptor binding subunit of CT, CTB, in P1 and S1 (Figure 1B, top panel, lane 2 and 4)
In this study we developed an in vitro retro-translocation assay performed using semi-permeabilized cells to clarify cytosolic events controlling CTA1 release from the ER membrane into the cytosol, a critical CT intoxication step
Summary
Cholera toxin (CT) binds to ganglioside GM1 receptor on the plasma membrane of host cells, becomes endocytosed within vesicles, and traffics through the Golgi apparatus en route to the endoplasmic reticulum (ER). A host cell protease proteolytically cleaves the catalytic A subunit (CTA) into the CTA1 and CTA2 peptides before the toxin reaches the ER [7]. CTA remains as a single polypeptide chain after cleavage because of a disulfide bond that links CTA1 and CTA2. In the ER, subsequent reduction of this disulfide bond by oxidoreductases generates free CTA1 peptide. It activates a signal transduction cascade that leads to chloride ion and water secretion across the plasma membrane, resulting in massive diarrhea that typifies the cholera disease
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