More recently, we studied how a membrane lipid might specify trafficking of toxin in the retrograde pathway by using fluorophore-labeled GM 1 and imaging toxin trafficking in live cells (28) . We found that a subset of GM 1 species, those with unsaturated ceramide domains, sorted efficiently from the PM to the trans-Golgi network and the ER. Cross-linking by toxin binding was dispensable for such GM 1 trafficking, but membrane cholesterol and the lipid raft-associated proteins actin and flotillin were required. Our results implicated an endogenous proteindependent mechanism of lipid sorting that is dependent on cer- Maximum signal for the tagged wt holotoxin in panels C through F was adjusted to 1.00, and all data points in the panel were normalized to that value. Data points show the mean response Ϯ SE; n ϭ 2 to 4 for A; n ϭ 3 to 4 for B; n ϭ 2 for C to F; each study was reproduced at least once. amide structure and could explain how the toxin gains access to the ER of host cells to induce disease (28) .