+ Site Statistics
References:
52,654,530
Abstracts:
29,560,856
PMIDs:
28,072,755
+ Search Articles
+ Subscribe to Site Feeds
Most Shared
PDF Full Text
+ PDF Full Text
Request PDF Full Text
+ Follow Us
Follow on Facebook
Follow on Twitter
Follow on LinkedIn
+ Translate
+ Recently Requested

A novel translocation assay demonstrates that entry of the cholera toxin A1 subunit into the cytosol involves the endoplasmic reticulum-associated degradation pathway



A novel translocation assay demonstrates that entry of the cholera toxin A1 subunit into the cytosol involves the endoplasmic reticulum-associated degradation pathway



Abstracts of the General Meeting of the American Society for Microbiology 102: 36-37



The active pool of cholera toxin (CT) moves from the cell surface to the endosomes, passes through the Golgi and travels to the endoplasmic reticulum (ER). The catalytic A1 subunit of CT then crosses the ER membrane and enters the cytosol in a process that has been hypothesized to involve the mechanism of ER-associated degradation (ERAD). In this work we describe a novel assay to detect the movement of CTA1 from the ER to the cytosol and provide supporting evidence for the ERAD model of CT translocation. ER export was monitored by the farnesylation of an enzymatically inactive, CVIM-tagged CTA1 subunit. Farnesylation involves addition of a 15-carbon fatty acid chain to the C-terminal CaaX motif (such as CVIM) of a target protein, occurs exclusively in the cytoplasm, and can be detected by partitioning of the farnesylated protein into the detergent phase of a Triton X-114 solution. Indirect immunoflourescence was initially used to confirm the ER localization of CTA1-CVIM in CHO cells after transient transfection with an expression plasmid that encodes CTA1-CVIM. Pulse-chase experiments then demonstrated that the CTA1-CVIM protein was retained intracellularly and degraded in a proteosome-dependent manner with a half-life of about 60 minutes. Treatment with brefeldin A or chloroquine, agents that block post-ER degradative events, had no effect on the turnover of CTA1-CVIM. Finally, by tracking the appearance of CTA1-CVIM in the detergent phase of cell extracts generated with Triton X-114, we found that translocation of CTA1-CVIM from the ER to the cytoplasm occurred rapidly and that the cytoplasmic pool of farnesylated CTA1-CVIM was also rapidly degraded. Further elucidation of the CT translocation mechanism should be aided by the use of CTA1-CVIM and other related constructs.

(PDF emailed within 1 workday: $29.90)

Accession: 034312571

Download citation: RISBibTeXText


Related references

Transfer of the cholera toxin A1 polypeptide from the endoplasmic reticulum to the cytosol is a rapid process facilitated by the endoplasmic reticulum-associated degradation pathway. Infection and Immunity 70(11): 6166-6171, 2002

Thermal Unfolding of the Pertussis Toxin S1 Subunit Facilitates Toxin Translocation to the Cytosol by the Mechanism of Endoplasmic Reticulum-Associated Degradation. Infection and Immunity 84(12): 3388-3398, 2016

Translocation of cholera toxin from the endoplasmic reticulum to the cytosol. European Journal of Cell Biology 75(SUPPL 48): 98, 1998

Hsp90 is required for transfer of the cholera toxin A1 subunit from the endoplasmic reticulum to the cytosol. Journal of Biological Chemistry 285(41): 31261-7, 2010

N-terminal extension of the cholera toxin A1-chain causes rapid degradation after retrotranslocation from endoplasmic reticulum to cytosol. Journal of Biological Chemistry 285(9): 6145-6152, 2010

The cholera toxin A1(3) subdomain is essential for interaction with ADP-ribosylation factor 6 and full toxic activity but is not required for translocation from the endoplasmic reticulum to the cytosol. Infection and Immunity 74(4): 2259-2267, 2006

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. Traffic 4(4): 232-242, 2003

Cholera toxin: an intracellular journey into the cytosol by way of the endoplasmic reticulum. Toxins 2(3): 310-325, 2010

Toxin instability and its role in toxin translocation from the endoplasmic reticulum to the cytosol. Biomolecules 3(4): 997-1029, 2013

Dependence of ricin toxicity on translocation of the toxin A-chain from the endoplasmic reticulum to the cytosol. Journal of Biological Chemistry 274(48): 34443-34449, Nov 26, 1999

The low lysine content of ricin A chain reduces the risk of proteolytic degradation after translocation from the endoplasmic reticulum to the cytosol. Biochemistry (American Chemical Society) 41(10): 05-13, 2002

Ubiquitination is required for the retro-translocation of a short-lived luminal endoplasmic reticulum glycoprotein to the cytosol for degradation by the proteasome. Journal of Biological Chemistry 273(16): 9734-9743, 1998

Cautionary note on the use of the B subunit of cholera toxin as a ganglioside GM1 probe: detection of cholera toxin A subunit in B subunit preparations by a sensitive adenylate cyclase assay. Journal of Cellular Biochemistry 42(3): 143-152, 1990

The hepatitis B virus precore protein is retrotransported from endoplasmic reticulum (ER) to cytosol through the ER-associated degradation pathway. Journal of Biological Chemistry 283(47): 32352-32360, 2008

Structural and functional interactions between the cholera toxin A1 subunit and ERdj3/HEDJ, a chaperone of the endoplasmic reticulum. Infection and Immunity 79(11): 4739-4747, 2011