A biochemical method for tracking cholera toxin transport from plasma membrane to Golgi and endoplasmic reticulum.
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 membrane 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 (by 35S-sulfation) and ER (by N-glycosylation). In this chapter, we describe the methods we developed to study this trafficking pathway.
De Luca
Heidi E
HE
Gastrointestinal Cell Biology, Children's Hospital Boston, Harvard Medical School, Boston, MA, USA.
Lencer
Wayne I
WI
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002522:Chlorocebus aethiops; D002771:Cholera / Q000378:metabolism / Q000473:pathology; D002772:Cholera Toxin / Q000378:metabolism* / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism* / Q000473:pathology; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000473:pathology; D006031:Glycosylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D014709:Vero Cells; D021601:trans-Golgi Network / Q000378:metabolism* / Q000473:pathology
D002772:Cholera Toxin