The actin cytoskeleton has been implicated in endocytosis, yet few molecules that link these systems have been identified. Here, we have cloned and characterized mHip1R, a protein that is closely related to huntingtin interacting 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 NH2-terminal domain homologous to that implicated in Sla2p's endocytic function, 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 punctate immunolocalization and is enriched at the cell cortex and in the perinuclear region. We concluded that the cortical localization represents endocytic compartments, because mHip1R colocalizes with clathrin, AP-2, and endocytosed transferrin, and because mHip1R fractionates biochemically with clathrin-coated vesicles. Time-lapse video microscopy of mHip1R–green fluorescence protein (GFP) revealed a blinking behavior similar to that reported for GFP-clathrin, and an actin-dependent inward movement of punctate structures from the cell periphery. These data show that mHip1R is a component of clathrin-coated pits and vesicles and suggest that it might link the endocytic machinery to the actin cytoskeleton.

Polyglutamine expansion in huntingtin is the underlying mutation leading to neurodegeneration in Huntington disease. This mutation influences the interaction of huntingtin with different proteins, including huntingtin-interacting protein 1 (HIP1), in which affinity to bind to mutant huntingtin is profoundly reduced. Here we demonstrate that HIP1 colocalizes with markers of clathrin-mediated endocytosis in neuronal cells and is highly enriched on clathrin-coated vesicles (CCVs) purified from brain homogenates. HIP1 binds to the clathrin adaptor protein 2 (AP2) and the terminal domain of the clathrin heavy chain, predominantly through a small fragment encompassing amino acids 276 -335. This region, which contains consensus clathrin-and AP2-binding sites, functions in conjunction with the coiledcoil domain to target HIP1 to CCVs. Expression of various HIP1 fragments leads to a potent block of clathrinmediated endocytosis. Our findings demonstrate that HIP1 is a novel component of the endocytic machinery.

Huntington disease (HD) is caused by polyglutamine [poly(Q)] expansion in the protein huntingtin (htt). Although the exact mechanism of disease progression remains to be elucidated, altered interactions of mutant htt with its protein partners could contribute to the disease. Using the yeast two-hybrid system, we have isolated a novel htt interacting protein, HIP14. HIP14's interaction with htt is inversely correlated to the poly(Q) length in htt. mRNAs of 9 and 6 bp are transcribed from the HIP14 gene, with the 6 kb transcript being predominantly expressed in the brain. HIP14 protein is enriched in the brain, shows partial co-localization with htt in the striatum, and is found in medium spiny projection neurons, the subset of neurons affected in HD. HIP14 localizes to the Golgi, and to vesicles in the cytoplasm. The HIP14 protein has sequence similarity to Akr1p, a protein essential for endocytosis in Saccharomyces cerevisiae. Expression of human HIP14 results in rescue of the temperature-sensitive lethality in akr1 Delta yeast cells and, furthermore, restores their defect in endocytosis, demonstrating a role for HIP14 in intracellular trafficking. Our findings suggest that decreased interaction between htt and HIP14 could contribute to the neuronal dysfunction in HD by perturbing normal intracellular transport pathways in neurons.

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