ATTENUATED ENDOCYTOSIS AND TOXICITY OF A MUTANT CHOLERA TOXIN WITH DECREASED ABILITY TO CLUSTER GM1

GI Cell Biology, the Department of Pediatrics, Children's Hospital and Harvard Medical School, and the Harvard Digestive Diseases Center, Boston, Massachusetts, 02115; Department of Microbiology, University of Colorado Denver School of Medicine, Aurora, Colorado, 80045; Departments of Molecular Physiology & Biophysics and Cell & Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232

^* To whom correspondence should be addressed. Email: wayne.lencer{at}childrens.harvard.edu.

	Abstract

Cholera toxin (CT) moves from the plasma membrane (PM) of host cells to the endoplasmic reticulum (ER) by binding to the lipid raft ganglioside GM1 (GM1). The homopentomeric B-subunit of the toxin can bind up to five GM1 molecules at once. Here, we examine the role of polyvalent binding of GM1 in CT action by producing chimeric cholera toxins that have B-subunits with only one or two normal binding pockets for GM1. The chimeric toxins have attenuated affinity for binding to host cell plasma membranes as expected. Nevertheless, like wild type (wt) CT, the CT chimeras induce toxicity, fractionate with detergent-resistant membranes extracted from toxin-treated cells, display restricted diffusion in the plane of the PM in intact cells, and remain bound to GM1 when they are immunoprecipitated. Thus, binding normally to two or perhaps only one GM1 molecule is sufficient for association with lipid rafts in the PM and toxin action. The chimeric toxins, however, are much less potent than wt toxin and they enter the cell by endocytosis more slowly, suggesting that clustering GM1 by the B-subunit enhances the efficiency of toxin uptake and perhaps also trafficking to the ER.