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

Abstract

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.

The ENTH and C-terminal domains ofDictyosteliumepsin cooperate to regulate the dynamic interaction with clathrin-coated pits

“…2D,F). This round spore phenotype was reminiscent of Dictyostelium Hip1r, another clathrin accessory protein (Repass et al, 2007). The restricted phenotype during development supported an essential role for epsin in a specialized pathway that controls the correct morphology of spores.…”

“…At present, little is known about how Dictyostelium spores construct their oblong shape. Interestingly, the clathrin accessory protein Hip1r also forms abnormally round spores in Dictyostelium (Repass et al, 2007). Both epsin-null cells and Hip1r-null cells produce round spores with slightly reduced viability, but Hip1r-null spores are more sensitive to heat and detergent treatment, indicating that the spore phenotype of Hip1r-null cells is more severe (Repass et al, 2007).…”

Molecular basis for coupling the plasma membrane to the actin cytoskeleton during clathrin-mediated endocytosis

“…Interestingly, in Dictyostelium discoideum plasma membrane localization of the Sla2 homolog Hip1r depends on epsin's ENTH domain (36). This dependence strongly suggests that the interaction between the lipid-binding domains of these two proteins is highly conserved.…”

Dynamics of clathrin-mediated endocytosis and its requirement for organelle biogenesis in Dictyostelium

“…Evidence for CME in Dictyostelium is based on the observation that clathrin forms plasma membrane puncta that colocalize with AP2, epsin, Hip1r and AP180 (Stavrou and O'Halloran, 2006;Repass et al, 2007;Brady et al, 2008;Wen et al, 2009;Sosa et al, 2012). In the case of AP2, immunofluorescence studies using an antibody to the a or b1/2 subunits showed that they localize to clathrin puncta (Wen et al, 2009;Sosa et al, 2012).…”