A GDP GTP EXCHANGE-STIMULATORY ACTIVITY FOR THE RAB5-RABGDI COMPLEX ON CLATHRIN-COATED VESICLES FROM BOVINE BRAIN/

Small GTPases of the Rab family are key regulators of intracellular tr ansport, They are associated with the cytoplasmic surface of distinct exocytic and endocytic organelles and with transport vesicles connecti ng these compartments, Rab proteins are also present in the cytosol in the GDP-bound conformation complexed to Rab GDP dissociation inhibito r (RabGDI), Upon membrane association, RabGDI is released, and the Rab protein is converted into the GTP-bound form, In this paper we have i nvestigated whether Rab5, which regulates the clathrin-coated vesicle- mediated pathway of endocytosis, can directly associate with the membr ane of clathrin-coated vesicles (CCV) purified from bovine brain in vi tro. We found that RabGDI can specifically deliver Rab5 but not Rab7, which is localized to late endosomes, to CCV, Furthermore, CCV contain a heat- and trypsin-sensitive activity that stimulates the dissociati on of GDP from Rab5, but not from Rab7, and the subsequent binding of GTP, The activity was found to be associated with the CCV membrane but not with the coat components. CCV weakly stimulated GDP release from either post-translationally modified or unmodified Rab5 alone, However , maximal GDP dissociation stimulation required the presence of RabGDI , suggesting that the factor(s) responsible for the membrane associati on and GDP/GTP exchange of Rab5 recognize the protein complexed to Rab GDI, These data demonstrate that CCV are competent for acquiring Rab5 and for converting the molecule into the GTP-bound active form.