A novel role for Rab5-GDI in ligand sequestration into clathrin-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 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.
McLauchlan
H
H
Department of Biochemistry, University of Dundee, Dundee, DD1 4HN, UK.
Newell
J
J
Morrice
N
N
Osborne
A
A
West
M
M
Smythe
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002417:Cattle; D002478:Cells, Cultured; D002966:Clathrin / Q000502:physiology*; D003034:Coated Pits, Cell-Membrane / Q000502:physiology*; D004705:Endocytosis; D020558:GTP Phosphohydrolases / Q000378:metabolism / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D020730:Guanine Nucleotide Dissociation Inhibitors; D008024:Ligands; D046911:Macromolecular Substances; D008954:Models, Biological; D008969:Molecular Sequence Data; D011485:Protein Binding; D012756:Sheep; D014168:Transferrin / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins
D002966:Clathrin; C086796:GDP dissociation inhibitor 1; D020730:Guanine Nucleotide Dissociation Inhibitors; D008024:Ligands; D046911:Macromolecular Substances; D014168:Transferrin; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins