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A Class of Mutant CHO Cells Resistant to Cholera Toxin Rapidly Degrades the Catalytic Polypeptide of Cholera Toxin and Exhibits Increased Endoplasmic Reticulum‐Associated Degradation
Author(s) -
Teter Ken,
Jobling Michael G.,
Holmes Randall K.
Publication year - 2003
Publication title -
traffic
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.677
H-Index - 130
eISSN - 1600-0854
pISSN - 1398-9219
DOI - 10.1034/j.1600-0854.2003.00070.x
Subject(s) - endoplasmic reticulum , endoplasmic reticulum associated protein degradation , cholera toxin , biology , kdel , pseudomonas exotoxin , microbiology and biotechnology , exotoxin , ricin , biochemistry , toxin , golgi apparatus , unfolded protein response , recombinant dna , gene
After binding to the eukaryotic cell surface, cholera toxin undergoes retrograde transport to the endoplasmic reticulum. The catalytic A1 polypeptide of cholera toxin (CTA1) then crosses the endoplasmic reticulum membrane and enters the cytosol in a process that may involve the quality control mechanism known as endoplasmic reticulum‐associated degradation. Other toxins such as Pseudomonas exotoxin A and ricin are also thought to exploit endoplasmic reticulum‐associated degradation for entry into the cytosol. To test this model, we mutagenized Chinese hamster ovary cells and selected clones that survived a prolonged coincubation with Pseudomonas exotoxin A and ricin. These lethal endoplasmic reticulum‐translocating toxins bind different surface receptors and target different cytosolic substrates, so resistance to both would likely result from disruption of a shared trafficking or translocation event. Here we characterize two Pseudomonas exotoxin A/ricin‐resistant clones that exhibited increased endoplasmic reticulum‐associated degradation. Both clones acquired the following unselected traits: (i) resistance to cholera toxin; (ii) increased degradation of an endoplasmic reticulum‐localized CTA1 construct; (iii) increased degradation of an established endoplasmic reticulum‐associated degradation substrate, the Z variant of α1‐antitrypsin (α1AT‐Z); and (iv) reduced secretion of both α1AT‐Z and the transport‐competent protein α1AT‐M. Proteosome inhibition partially rescued the α1AT‐M secretion deficiencies. However, the mutant clones did not exhibit increased proteosomal activity against cytosolic proteins, including a second CTA1 construct that was expressed in the cytosol rather than in the endoplasmic reticulum. These results suggested that accelerated endoplasmic reticulum‐associated degradation in the mutant clones produced a cholera toxin/ Pseudomonas exotoxin A/ricin‐resistant phenotype by increasing the coupling efficiency between toxin translocation and toxin degradation .