CHARACTERIZATION OF RAB5-Q79L-STIMULATED ENDOSOME FUSION

Fusion of intracellular membrane bound compartments is a common step i n the transport of macromolecules along the endocytic and secretory pa thways. Previous work has shown that GTP gamma S stimulates endosome f usion in the presence of low concentrations of cytosol. In this study, we have characterized the effect of rab5:Q79L, a mutant with reduced GTPase activity, on endosome fusion in a cell-free assay. rab5:Q79L st imulates in vitro endosome fusion. The stimulatory effects required AT P, were blocked by N-ethylmaleimide (NEM) and anti-NEM-sensitive fusio n (NSF) protein antibody, but could proceed in the absence of cytosol. Stimulation of fusion with rab5:Q79L led to rapid inactivation of the vesicles when tested in a second incubation for fusogenic activity. B y electron microscopy, endosomes connected by tubular structures were frequently observed in the presence of rab5:Q79L. Rab5:Q79L promoted f usion only among early endosomes; when the ligands were chased into mo re mature endocytic compartments, fusion was not observed. Phospholipa se A2 inhibitors blocked rab5:Q791-stimulated fusion. The results indi cate that rab5:Q79L promotes fusion by activating factors already pres ent in the membranes and that NSF and phospholipase A2 activities are required downstream of rab5. (C) 1996 Academic Press, Inc.