Endocytic mechanisms responsible for uptake of GPI-linked diphtheria toxinreceptor

We have here used diphtheria toxin as a tool to investigate the type of end ocytosis used by a glycosylphosphatidylinositol-linked molecule, a glycosyl phosphatidylinositol-linked version of the diphtheria toxin receptor that i s able to mediate intoxication. The receptor is expressed in HeLa cells whe re clathrin-dependent endocytosis can be blocked by overexpression of mutan t dynamin, Diphtheria toxin intoxicates cells by first binding to cell-surf ace receptors, then the toxin is endocytosed, and upon exposure to low endo somal pH, the toxin enters the cytosol where it inhibits protein synthesis. Inhibition of protein synthesis by the toxin can therefore be used to prob e the entry of the glycosylphosphatidylinositol-linked receptor into an aci dic compartment. Furthermore, degradation of the toxin can be used as an in dicator of entry into the endosomal/lysosomal compartment. The data show th at although expression of mutant dynamin inhibits intoxication mediated via the wild-type receptors, mutant dynamin does not affect intoxication or en docytosis and degradation of diphtheria toxin bound to the glycosglphosphat idylinositol-linked receptor. Confocal microscopy demonstrated that diphthe ria toxin is transported to vesicles containing EEA1, a marker for early en dosomes, Biochemical and ultrastructural studies of the HeLa cells used rev eal that they have very low levels of caveolin-1 and that they contain very few if any caveolae at the cell surface. Furthermore, the endocytic uptake of diphtheria toxin bound to the glycosylphosphatidylinositol-linked recep tor was not reduced by methyl-beta-cyclodextrin or by nystatin which both d isrupt caveolar structure and functions. Thus, uptake of a glycosylphosphat idylinositol-linked protein, in this case the diphtheria toxin receptor, in to the endosomal/lysosomal system can occur independently of both caveolae and clathrin-coated vesicles.