The actin-binding protein Hip1R associates with clathrin during early stages of endocytosis and promotes clathrin assembly in vitro

Huntingtin-interacting protein 1 related (Hip1R) is a novel component of cl athrin-coated pits and vesicles and is a mammalian homologue of Sla2p, an a ctin-binding protein important for both actin organization and endocytosis in yeast. Here, we demonstrate that Hip1R binds via its putative central co iled-coil domain to clathrin, and provide evidence that Hip1R and clathrin are associated in vivo at sites of endocytosis. First, real-time analysis o f Hip1R-YFP and DsRed-clathrin light chain (LC) in live cells revealed that these proteins show almost identical temporal and spatial regulation at th e cell cortex. Second, at the ultrastructure level, immunogold labeling of 'unroofed' cells showed that Hip1R localizes to clathrin-coated pits. Third , overexpression of Hip1R affected the subcellular distribution of clathrin LC. Consistent with a functional role for Hip1R in endocytosis, we also de monstrated that it promotes clathrin cage assembly in vitro. Finally, we sh owed that Hip1R is a rod-shaped apparent dimer with globular heads at eithe r end, and that it can assemble clathrin-coated vesicles and F-actin into h igher order structures. In total, Hip1R's properties suggest an early endoc ytic function at the interface between clathrin, F-actin, and lipids.