Dictyostelium Hip1r contributes to spore shape and requires epsin for phosphorylation and localization

Clathrin-coated pits assemble on the plasma membrane to select and sequester proteins within coated vesicles for delivery to intracellular compartments. Although a host of clathrin-associated proteins have been identified, much less is known regarding the interactions between clathrin-associated proteins or how individual proteins influence the function of other proteins. In this study, we present evidence of a functional relationship between two clathrin-associated proteins in Dictyostelium, Hip1r and epsin. Hip1r-null cells form fruiting bodies that yield defective spores that lack the organized fibrils typical of wild-type spores. This spore coat defect leads to formation of round, rather than ovoid, spores in Hip1r-null cells that exhibit decreased viability. Like Hip1r-null cells, epsin-null cells also construct fruiting bodies with round spores, but these spores are more environmentally robust. Double-null cells that harbor deletions in both epsin and Hip1r form fruiting bodies, with spores identical in shape and viability to Hip1r single-null cells. In the growing amoeba, Hip1r is phosphorylated and localizes to puncta on the plasma membrane that also contain epsin. Both the phosphorylation state and localization of Hip1r into membrane puncta require epsin. Moreover, expression of the N-terminal ENTH domain of epsin is sufficient to restore both the phosphorylation and the restricted localization of Hip1r within plasma membrane puncta. The results from this study reveal a novel interaction between two clathrin-associated proteins during cellular events in both growing and developing Dictyostelium cells.