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.

Co- and Post-translocation Roles for HSP90 in Cholera Intoxication

“…In vitro reconstitution of the CTA1 translocation event likewise required ATP hydrolysis by Hsp90: the addition of Hsp90/ATP but neither Hsp90 nor Hsp90/ATP␥S to salt-or urea-washed membranes was sufficient for CTA1 export. ATP␥S has been previously reported to block in vitro CTA1 translocation, although the affected host protein was not identified (68). Multiple cytosolic host factors may be active in the CTA1 translocation event, but our in vitro reconstitution assay has shown, for the first time, that Hsp90/ ATP is sufficient for toxin export to the cytosol.…”

“…Western blot analysis from a previous study failed to detect Hsp90 in cytosolic fractions that facilitated in vitro CTA1 translocation, leading some to question the link between Hsp90 and CTA1 translocation (68). However, this interpretation is based upon a negative result from an assay that did not use a salt wash to strip Hsp90 from the membrane fraction.…”

Mouse Mammary Tumor Virus Signal Peptide Uses a Novel p97-Dependent and Derlin-Independent Retrotranslocation Mechanism To Escape Proteasomal Degradation

“…Further, the cytosolic p97 ATPase has been reported to provide the energy for membrane extraction of many ERAD substrates (2, 13), and yet this enzyme is dispensable for CTA1 dislocation to the cytosol (64, 65, 67). More recently, the chaperone Hsp90 has been shown to bind to CTA1 to facilitate its extraction from the ER membrane as well as rapid folding in the cytosol, at least in cell-free systems (71), but others dispute this claim (74). Membrane extraction of most ERAD substrates by the AAA ATPase p97 is followed by E3 ligase-mediated polyubiquitylation (5).…”

Pathogenic Hijacking of ER-Associated Degradation: Is ERAD Flexible?

“…Although the A1 chain has been recognized as a non-ubiquitylated substrate of ERAD (Wernick et al, 2010), a recent study suggests that the ubiquitin ligase activity of HRD1 and/or gp78 is necessary for dislocation of the A1 chain (Bernardi et al, 2010), while it remains unclear whether the A1 chain itself is the target of this poly-ubiquitylation activity. Usage of p97, which typically requires preceding poly-ubiquitylation of the substrate, as a driving force for A1 chain dislocation is very controversial, with evidence both supporting and refuting this (Abujarour et al, 2005;Kothe et al, 2005;Moore et al, 2013). Thus, the presence or absence of poly-ubiquitylation on the A1 chain, the driving force for its dislocation, and the mechanism by which the A1 chain evades ubiquitin-dependent degradation remain controversial.…”