Abstract
Asiatic cholera is a rapidly progressing disease resulting in extreme diarrhea and even death. The causative agent, cholera toxin, is an AB5 -subunit enterotoxin produced by the bacterium Vibrio cholera. The toxin must enter the intestinal cell to cause disease. Entry is achieved by the B-subunit binding to a membrane lipid that carries the toxin all the way from the plasma mem-brane through the trans-Golgi to the endoplasmic reticulum (ER). Once in the ER, a portion of the A-subunit, the A1 chain, unfolds and separates from the B-subunit to retro-translocate to the cytosol. The A1 chain then activates adenylyl cyclase to cause disease. To study this pathway in intact cells, we used a mutant toxin with C-terminal extension of the B-subunit that contains N-glycosylation and tyrosine-sulfation motifs (CT-GS). This provides a biochemical readout for toxin entry into the trans Golgi (by35 S-sulfation) and ER (by N-glycosylation). In this chapter, we describe the methods we developed to study this trafficking pathway.
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De Luca, H.E., Lencer, W.I. (2006). A Biochemical Method for Tracking Cholera Toxin Transport From Plasma Membrane to Golgi and Endoplasmic Reticulum. In: Colgan, S.P. (eds) Cell-Cell Interactions. Methods in Molecular Biology™, vol 341. Humana Press. https://doi.org/10.1385/1-59745-113-4:127
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DOI: https://doi.org/10.1385/1-59745-113-4:127
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