Huntingtin interacting protein 1 (HIP1) is a component of clathrin coats. We previously demonstrated that HIP1 promotes clathrin assembly through its central helical domain, which binds directly to clathrin light chains (CLCs). To better understand the relationship between CLC binding and clathrin assembly we sought to dissect this interaction. Using C-terminal deletion constructs of the HIP1 helical domain, we identified a region between residues 450 and 456 that is required for CLC binding. Within this region, point mutations showed the importance of residues Leu-451, Leu-452, and Arg-453. Mutants that fail to bind CLC are unable to promote clathrin assembly in vitro but still mediate HIP1 homodimerization and heterodimerization with the family member HIP12/HIP1R. Moreover, HIP1 binding to CLC is necessary for HIP1 targeting to clathrincoated pits and clathrin-coated vesicles. Interestingly, HIP1 binds to a highly conserved region of CLC previously demonstrated to regulate clathrin assembly. These results suggest a role for HIP1/CLC interactions in the regulation of clathrin assembly.

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PDZ domains mediate protein interactions primarily through either classical recognition of carboxyl-terminal motifs or PDZ/PDZ domain associations. Several studies have also described internal modes of PDZ recognition, most of which depend on b-finger structures. Here, we describe a novel interaction between the PDZ domain of nNOS and Vac14, the activator of the PtdIns(3)P 5-kinase PIKfyve. Binding assays using various Vac14 deletion constructs revealed a b-finger independent interaction that is based on a novel internal motif. Mutational analyses reveal essential residues within the motif allowing us to define a new type of PDZ domain interaction.