An actin-binding protein of the Sla2/Huntingtin interacting protein 1 family is a novel component of clathrin-coated pits and vesicles

The actin cytoskeleton has been implicated in endocytosis, yet few molecule s that link these systems have been identified. Here, we have cloned and ch aracterized mHip1R, a protein that is closely related to huntingtin interac ting protein 1 (Hip1). These two proteins are mammalian homologues of Sla2p , an actin-binding protein important for actin organization and endocytosis in yeast. Sequence alignments and secondary structure predictions verified that mHip1R belongs to the Sla2 protein family. Thus, mHip1R contains an N H2-terminal domain homologous to that implicated in Sla2p's endocytic funct ion, three predicted coiled-coils, a leucine zipper, and a talin-like actin -binding domain at the COOH terminus. The talin-like domain of mHip1R binds to F-actin in vitro and colocalizes with F-actin in vivo, indicating that this activity has been conserved from yeast to mammals. mHip1R shows a punc tate immunolocalization and is enriched at the cell cortex and in the perin uclear region. We concluded that the cortical localization represents endoc ytic compartments, because mHip1R colocalizes with clathrin, AP-2, and endo cytosed transferrin, and because mHip1R fractionates biochemically with cla thrin-coated vesicles. Time-lapse video microscopy of mHip1R-green fluoresc ence protein (GFP) revealed a blinking behavior similar to that reported fo r GFP-clathrin, and an actin-dependent inward movement of punctate structur es from the cell periphery. These data show that mHip1R is a component of c lathrin-coated pits and vesicles and suggest that it might link the endocyt ic machinery to the actin cytoskeleton.