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 this assembly process. RESULTS: We have used an in vitro assay for clathrin-coated pit assembly to identify a novel component required for the invagination of newly formed coated pits. We have purified this cytosolic component and shown it to be a complex of Rab5 and GDI (guanine-nucleotide dissociation inhibitor), that was previously demonstrated to be involved in downstream processing of endocytic vesicles. Using a combination of quantitative electron microscopy and in vitro endocytosis assays, we have demonstrated that although coat proteins and ATP are sufficient to increase the number of new coated pits at the cell surface in permeabilised cells, the Rab5-GDI complex is required for ligand sequestration into clathrin-coated pits. CONCLUSIONS: We have identified Rab5 as a critical cytosolic component required for clathrin-coated pit function. Given the well-established role of Rab5 in the fusion of endocytic vesicles with endosomes, our results suggest that recruitment of essential components of the targeting and fusion machinery is coupled to the formation of functional transport vesicles.
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 this assembly process. RESULTS: We have used an in vitro assay for clathrin-coated pit assembly to identify a novel component required for the invagination of newly formed coated pits. We have purified this cytosolic component and shown it to be a complex of Rab5 and GDI (guanine-nucleotide dissociation inhibitor), that was previously demonstrated to be involved in downstream processing of endocytic vesicles. Using a combination of quantitative electron microscopy and in vitro endocytosis assays, we have demonstrated that although coat proteins and ATP are sufficient to increase the number of new coated pits at the cell surface in permeabilised cells, the Rab5-GDI complex is required for ligand sequestration into clathrin-coated pits. CONCLUSIONS: We have identified Rab5 as a critical cytosolic component required for clathrin-coated pit function. Given the well-established role of Rab5 in the fusion of endocytic vesicles with endosomes, our results suggest that recruitment of essential components of the targeting and fusion machinery is coupled to the formation of functional transport vesicles.