Elucidating the functions of protein disulfide isomerase family proteins during quality control in the endoplasmic reticulum

• Main Author: ster, Michele L
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2009
• Subjects: Cellular biology
• ISBN: 9781109114836; 1109114834

The endoplasmic reticulum (ER) quality control system retains nascent polypeptides in the organelle until they are folded properly and mediates the ER-to-cytosol transport (i.e., retrotranslocation) of misfolded proteins for proteasomal degradation. In mammalian cells, the retrotranslocation machinery is exploited by the A1 subunit of cholera toxin (CTA1) for its transport into the cytosol where it induces toxicity. Using a semipermeabilized cell system characterized here, we found the ER factor protein disulfide isomerase (PDI) facilitates CTA1 retrotranslocation consistent with its previously demonstrated ability to unfold the toxin in vitro. In contrast, the PDI family protein ERp72 retains CTA1 in the ER and stabilizes a folded conformation of the toxin. PDI and ERp72's opposing functions also operate on proteins misfolded in the ER. Thus, we identified PDI family proteins that play opposing roles during ER quality control and established an assay to study CTA1 retrotranslocation. Because PDI's unfoldase activity correlates with retrotranslocation, we investigated the mechanism of this activity. PDI comprises two redox active (a and a') and two redox inactive ( b and b') thioredoxin-like domains, a linker ( x), and a C-terminal domain (c) arranged abb'xa'c. Using recombinant PDI fragments, we show that binding of CTA1 by PDI's bb'xa' domains is necessary and sufficient to trigger unfolding. The linear arrangement of bb'xa' and type of a domain (a' versus a) C-terminal to bb'x are additional determinants of activity. These data suggest a general mechanism for PDI's unfoldase activity: the concurrent and specific binding of bb'xa' to particular regions along the CTA1 molecule triggers its unfolding. Furthermore, we show that PDI's bb' domains are indispensable to the unfolding reaction, whereas the function of its a' domain can be substituted partially by the a' domain from ERp57 (abb'xa'c) or ERp72 (ca°abb'xa'), PDI family proteins that do not unfold CTA1 normally. However, PDI's bb' domains were insufficient to convert full-length ERp57 into an unfoldase because ERp57's a domain inhibited toxin binding. Thus, we propose that generating an unfoldase from thioredoxin-like domains requires PDI's bb'(x) domains followed by an a' domain but not preceded by an a domain that can inhibit binding.