A FACTOR WITH A ZINC-BINDING AND PHORBOL ESTER-BINDING DOMAIN IS NECESSARY FOR ENDOSOME FUSION

An inhibitory effect of several zinc chelators on endosome fusion reco nstituted in an in vitro system has been recently reported (A. Aballay et al., 1995, Biochem. J. 312, 919-923). The factor that requires zin c for its activity is still unknown. Since the regulatory domain of pr otein kinase C (PKC) contains cysteine-rich motifs which coordinate zi nc, we suspected that PKC or a PRC-like protein might be that factor. To test this hypothesis, we studied the effect of calphostin C, a spec ific inhibitor of PKC that interacts with the cysteine-rich motif, and PMA (phorbol 12-myristate 13-acetate), an activator of several PRC is oforms that bind to the same region, on endosome fusion. Calphostin C inhibited endosome fusion in a zinc-regulated manner, whereas PMA enha nced endosome fusion. Moreover, fusion was strongly stimulated when bo th PMA and zinc were added together to zinc-depleted fusion reactions. Inhibitors of the catalytic domain of PKC had no effect on the assay suggesting that the kinase activity is not required. In contrast, a gl utathione S-transferase fusion protein containing a cysteine-rich regi on of the regulatory domain of PKC gamma inhibited endosome fusion in a PMA-dependent manner. Western blot analysis demonstrated the presenc e of proteins containing PKC-like cysteine-rich regions that are relea sed horn endosomal fractions by zinc chelators. These results indicate that, the previously proposed zinc-dependent factor required for endo some fusion could be either a PRC isoform or a protein containing the phorbol ester-binding domain of PKC. (C) 1997 Academic Press.