Phorbol ester promotes endocytosis by activating a factor involved in endosome fusion

Previous studies indicate that a zinc- and phorbol ester-binding factor is necessary for in vitro endosome fusion and for the effect of RabS on endoso me fusion, Rab5 is a small GTPase that regulates membrane fusion between ea rly endosomes derived from either receptor-mediated endocytosis or fluid-ph ase endocytosis. In its GTP-bound form, Rab5 promotes endocytosis and enhan ces fusion among early endosomes, To determine if PMA stimulates endocytosi s by activating a factor required for endosome fusion, we overexpressed wil d-type RabS, a dominant negative mutant (Rab5:S34N), and a GTPase deficient mutant (Rab5:Q79L) in BHK-21 cells. The phorbol ester PMA stimulates endoc ytosis and increases the number and the size of endocytic vesicles, even in the presence of Rab5:S34N, Zinc depletion with N,N,N',N'-tetrakis-(2-pyrid ylmethyl)ethylenediamine (TPEN) and addition of calphostin C (CPC), an inhi bitor of PKC that interacts with zinc and phorbol ester binding motifs, inh ibited both basal and Rab5-stimulated fluid phase endocytosis. These two re agents also inhibited the size and number of endocytic vesicles promoted by RabS. These results suggest that PMA stimulates endocytosis by regulating the dynamics of the early endosome compartment.