Functional analysis of the clathrin assembly protein, AP180, in Dictyostelium discoideum
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AP180, an important coat component of clathrin-coated vesicles, is known to assemble clathrin triskelia into cages of uniform size. To gain insight into the relationship between AP180 and clathrin, the gene that encodes AP180 in Dictyostelium discoideum was cloned and a mutant strain carrying a deletion in this gene was constructed using homologous recombination. Unlike clathrin mutants, AP180 null cells displayed normal pinocytosis, cytokinesis and development into fruiting bodies. However, AP180 mutant cells were osmosensitive, a phenotype also exhibited by clathrin light chain and clathrin heavy chain mutants. The contractile vacuole in AP180 mutant cells became abnormally large in a hypotonic environment and the cycle of expansion and discharge of the vacuole took twice as long compared to that of wild-type cells. Expression of GFP tagged AP180 showed that it localized to punctae at the plasma membrane, cytoplasm and perinuclear area and that it associated extensively with clathrin at these sites. AP180 also localized to the contractile vacuole and in the absence of AP180 more contractile vacuoles were labeled with clathrin. The association of AP180 with the contractile vacuole was affected in the absence of clathrin light chain and the internalization of AP180 into cytoplasmic punctae required the presence of clathrin heavy chain. This work also investigated the dynamics between clathrin, AP180 and AP-2, which is another clathrin assembly protein. A double mutant strain was constructed that had the genes for both AP180 and AP-2 deleted. Of all the clathrin mediated processes examined only osmoregulation was more severe than in the AP180 or AP-2 single mutant cells. The osmosensitivity of the double mutant was an indication that clathrin events at the plasma membrane and the contractile vacuole are linked to some extent. In the absence of both adaptor proteins, the membrane association of clathrin was decreased but not completely abolished whereas the presence of clathrin on the contractile vacuole was markedly decreased. These results present a functional relationship between clathrin, AP180 and AP-2 and suggest that endocytic events mediated by clathrin, AP180 and AP- 2 are important in the normal function of the contractile vacuole.