RAB5, AN EARLY ACTING ENDOSOMAL GTPASE, SUPPORTS IN-VITRO ENDOSOME FUSION WITHOUT GTP HYDROLYSIS

Endocytosis is regulated by several GTPases including Rab5 and one or more heterotrimeric G proteins. We show here that RabS, in the GTP gam ma S (guanosine 5'-O-(thiotriphosphate))-bound form, fully supports in vitro endosome fusion, indicating that GTP hydrolysis is not required , whereas Rab5:S34N and Rab5:N133I, mutants unable to bind GTP, are po tent inhibitors of endosome fusion. Double mutants (Rab5:S34N/Delta C4 and Rab5: N133I/Delta C4) lacking the C-terminal prenylation site wer e inactive, indicating that prenylation is required. Endosomes became resistant to the inhibitory effects of Rab5:S34N by preincubating the vesicles with cytosol prior to the addition of the inhibitor. The acqu isition of resistance to Rab5:S34N was more rapid than to N-ethylmalei mide, indicating that Rab5 mutants are early acting. G(beta gamma) sub units of heterotrimeric G proteins block endosome fusion. However the effect of G(beta gamma) Was abrogated by Rab5.GTP gamma S, indicating that a heterotrimeric G protein may operate upstream of Rab5.