Role of p97 AAA-ATPase in the Retrotranslocation of the Cholera Toxin A1 Chain, a Non-ubiquitinated Substrate

Wayne I. Lencer,Heidi E. De Luca,Eli Kern,Jessica S. Wagner,Michael Kothe,Tom A. Rapoport,Yihong Ye

doi:10.1074/jbc.m503138200

Abstract

The enzymatic A1 chain of cholera toxin retrotranslocates across the endoplasmic reticulum membrane into the cytosol, where it induces toxicity. Almost all other retrotranslocation substrates are modified by the attachment of polyubiquitin chains and moved into the cytosol by the ubiquitin-interacting p97 ATPase complex. The cholera toxin A1 chain, however, can induce toxicity in the absence of ubiquitination, and the motive force that drives retrotranslocation is not known. Here, we use adenovirus expressing dominant-negative mutants of p97 to test whether p97 is required for toxin action. We find that cholera toxin still functions with only a small decrease in potency in cells that cannot retrotranslocate other substrates at all. These results suggest that p97 does not provide the primary driving force for extracting the A1 chain from the endoplasmic reticulum, a finding that is consistent with a requirement for polyubiquitination in p97 function.

Highlights

Cholera toxin (CT)1 enters host cells by co-opting two fundamental aspects of cell function that reverse membrane and protein transport in the secretory pathway
These results suggest that p97 does not provide the primary driving force for extracting the A1 chain from the endoplasmic reticulum, a finding that is consistent with a requirement for polyubiquitination in p97 function
We showed that the CT A1 chain still reaches the cytosol in US11 cells that cannot retrotranslocate the MHC Class I heavy chain at all

Summary

Introduction

Cholera toxin (CT)1 enters host cells by co-opting two fundamental aspects of cell function that reverse membrane and protein transport in the secretory pathway. Expression of KA and QQ mutant p97 in US11 cells induces an increase in high molecular weight polyubiquitinated proteins as detected by immunoblot of total cell lysates using antibodies against ubiquitin (Fig. 1B, lower panel, compare lanes 5 and 7 with 1 and 2).

Results

Conclusion