A NOVEL ROLE FOR RAB5-GDI IN LIGAND SEQUESTRATION INTO CALTHRIN-COATED PITS

Background: Clathrin-coated pits are formed at the plasma membrane by the assembly of the coat components, namely clathrin and adaptors from the cytosol, Little is known about the regulation and mechanism of th is assembly process. Results: We have used an in vitro assay for clath rin-coated pit assembly to identify a novel component required for the invagination of newly formed coated pits, We have purified this cytos olic component and shown it to be a complex of Rab5 and GDI (guanine-n ucleo tide dissociation inhibitor), that was previously demonstrated t o be involved in downstream processing of endocytic vesicles. Using a combination of quantitative electron microscopy and in vitro endocytos is assays, we have demonstrated that although coat proteins and ATP ar e sufficient to increase the number of new coated pits at the cell sur face in permeabilised cells, the Rab5-GDI complex is required for liga nd sequestration into clathrin-coated pits, Conclusions: We have ident ified Rab5 as a critical cytosolic component required for clathrin-coa ted pit function. Given the well-established role of Rab5 in the fusio n of endocytic, vesicles with endosomes, our results suggest that recr uitment of essential components of the targeting and fusion machinery is coupled to the formation of functional transport vesicles.