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 bacteriumVibrio 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 thetrans-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 containsN-glycosylation and tyrosine-sulfation motifs (CT-GS). This provides a biochemical readout for toxin entry into the trans Golgi (by35 S-sulfation) and ER (byN-glycosylation). In this chapter, we describe the methods we developed to study this trafficking pathway.
