Browsing by Subject "AP2"
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Item APβ1/2 and Hip1r : insights into early and late stage clathrin adaptors in Dictyostelium discoideum(2012-05) Sosa, Ramiro Thomas; O'Halloran, Theresa; Gross, Jeffrey M.; Johnson, Arlen W.; Mehdy, Mona; Morgan, Jennifer R.Clathrin-mediated endocytosis is the process whereby specific cargoes are internalized into coated vesicles from the plasma membrane. Numerous clathrin adaptors facilitate this process by linking the coat protein clathrin to the plasma membrane by associating with PI(4,5)P2 and binding to membrane-bound cargo. Here, I investigated the role of two clathrin adaptors, APβ1/2 and Hip1r, in clathrin-mediated endocytosis. I found that Dictyostelium APβ1/2 functions in both the AP1 and AP2 complexes, unlike vertebrates, which have distinct β subunits for each AP complex. I found that APβ1/2 function is required for several clathrin-dependent processes, including cytokinesis, development and osmoregulation. I also uncovered a role for APβ1/2 in the stability other subunits of the AP1 and AP2 complexes. Finally, phenotypic comparisons of APβ1/2 mutant cells with cells missing subunits that are specific to the AP1 or AP2 complex allowed me to distinguish between endocytic defects and endosomal trafficking defects in clathrin mutants. My investigation of Hip1r centered on the known requirement for Hip1r in actin dynamics during endocytosis and a possible role for Hip1r phosphorylation in regulating actin. To determine how phosphorylation contributes to Hip1r function, I identified a specific serine residue that serves as a Hip1r phosphorylation site. I also identified a novel role for the kinase PKB in Hip1r phosphorylation. I determined that phosphorylation is not required for Hip1r localization to the plasma membrane. Similar to Hip1r, PKB is required for proper actin dynamics during endocytosis. My results support a model in which epsin recruits Hip1r to the plasma membrane during formation of clathrin-coated vesicles. Here, Hip1r functions as both a clathrin adaptor and a negative regulator of actin polymerization. I propose that phosphorylation of Hip1r by PKB triggers a reduction in the affinity of Hip1r for clathrin, which may stimulate actin polymerization and tethering of clathrin-coated vesicles with the actin cytoskeleton.Item Contribution of AP2 and AP180 to clathrin function in Dictyostelium discoideum(2009-05) Wen, Yujia, 1975-; O'Halloran, TheresaAP2 complex protein is an essential clathrin adaptor protein during clathrin mediated endocytosis. However, this view has been challenged in simple organisms. To gain insight into this conflict, the role of AP2 in clathrin localization and other clathrin related processes were assessed in Dictyostelium discoideum. In Dictyostelium, deleting function AP2 caused mild phenotypes in clathrin membrane localization, cytokinesis, osmoregulation and cell development. This supported the idea that AP2 have significant roles in multicellular organisms but not in unicellular system. Clathrin mediated processes carries important function not only on the plasma membrane but also on some internal organelles. But clathrin coated vesicles on internal organelles are not as well studied as on the plasma membrane. To understand more of the clathrin coated vesicles on internal organelles, the clathrin coated vesicles on Dictyostelium discoideum contractile vacuole were studied. Contractile vacuole associated clathrin coated vesicles contained clathrin adaptor proteins AP2, AP180, and epsin but not Hip1r. The absence of AP180 or AP2 produced abnormal large vacuoles, but the absence of epsin did not cause any detectable contractile vacuole abnormality. The enlarged contractile vacuoles in AP180 minus cells were caused by excessive homotypic fusion among contractile vacuoles. Using both GST-pull down and immunostaining AP180 was identified as the possible adaptor protein for a contractile vacuole-associated SNARE protein, Vamp7B. Therefore recycling Vamp7B from contractile vacuole by AP180 through clathrin coated vesicles could be an efficient way to prevent excessive homotypic fusions among contractile vacuoles. Dictyostelium contractile vacuoles offer a valuable system to study clathrin coated vesicles on cell internal organelles.