EVIDENCE FOR A SYMMETRICAL REQUIREMENT FOR RAB5-GTP IN IN-VITRO ENDOSOME-ENDOSOME FUSION

Early endosome fusion, which has been extensively characterized using an in vitro reconstitution assay, is Rab5-dependent. To examine the re quirement for Rab5 on both fusion partners, we prepared cytosol and en dosomes depleted of Rab5, Unlike control cytosol, Rab5-depleted cytoso l was only marginally active in the in vitro endosome fusion. However, fusion could be restored by the addition of wild-type Rab5 or Rab5 D1 36N, a mutant whose nucleotide specificity favors xanthine over guanin e. The addition of Rab5 D136N restored fusion only in the presence of XTP. In the absence of XTP or in the presence of XDP, Rab5 D136N faile d to restore fusion, When fusion was carried out with endosomal vesicl es depleted of Rab GTPases (by preincubation of vesicles with GDP diss ociation inhibitor), together with cytosol immunodepleted of Rab5, fus ion was virtually absent. We then used immunodepleted cytosol and GDP dissociation inhibitor-treated vesicles to determine whether Rab5 is r equired by both fusion partners. Using separate sets of endosomal vesi cles, we found that priming both sets of Rab5-depleted vesicles with R ab5 Q79L, a GTPase defective mutant, substantially stimulated endosome fusion. Priming one set of vesicles with Rab5 Q79L and a second set o f vesicles with Rab5 S34N failed to activate fusion. When both sets of Rab5-depleted vesicles mere primed with Rab5 D136N supplemented with XTP, endosome fusion was stimulated, similar to that observed with Rab 5 Q79L. However, when one set of vesicles was preincubated with Rab5 D 136N plus XTP and the second set with Rab5 D136N and XDP, no stimulati on of fusion was observed. We conclude that Rab5-GTP is required on bo th fusion partners for docking and fusion of early endosomes. To confi rm the fusion of Rab5-GTP-positive vesicles in vivo, we expressed GFP- Rab5 Q79L in fibroblasts and observed fusion of Rab5-positive vesicles . We failed to record fusion of Rab5-positive vesicles with Rab5-negat ive vesicles. We conclude that Rab5-GTP is required on both sets of en dosomes for fusion in vitro and in living cells.