Clathrin-coated vesicles play an established role in endocytosis from the plasma membrane, but they are also found on internal organelles. We examined the composition of clathrin-coated vesicles on an internal organelle responsible for osmoregulation, the Dictyostelium discoideum contractile vacuole. Clathrin puncta on contractile vacuoles contained multiple accessory proteins typical of plasma membrane-coated pits, including AP2, AP180, and epsin, but not Hip1r. To examine how these clathrin accessory proteins influenced the contractile vacuole, we generated cell lines that carried single and double gene knockouts in the same genetic background. Single or double mutants that lacked AP180 or AP2 exhibited abnormally large contractile vacuoles. The enlarged contractile vacuoles in AP180-null mutants formed because of excessive homotypic fusion among contractile vacuoles. The SNARE protein Vamp7B was mislocalized and enriched on the contractile vacuoles of AP180-null mutants. In vitro assays revealed that AP180 interacted with the cytoplasmic domain of Vamp7B. We propose that AP180 directs Vamp7B into clathrin-coated vesicles on contractile vacuoles, creating an efficient mechanism for regulating the internal distribution of fusion-competent SNARE proteins and limiting homotypic fusions among contractile vacuoles. Dictyostelium contractile vacuoles offer a valuable system to study clathrin-coated vesicles on internal organelles within eukaryotic cells.

AP180, one of many assembly proteins and adaptors for clathrin, stimulates the assembly of clathrin lattices on membranes, but its unique contribution to clathrin function remains elusive. In this study we identified the Dictyostelium discoideum ortholog of the adaptor protein AP180 and characterized a mutant strain carrying a deletion in this gene. Imaging GFP-labeled AP180 showed that it localized to punctae at the plasma membrane, the contractile vacuole, and the cytoplasm and associated with clathrin. AP180 null cells did not display defects characteristic of clathrin mutants and continued to localize clathrin punctae on their plasma membrane and within the cytoplasm. However, like clathrin mutants, AP180 mutants, were osmosensitive. When immersed in water, AP180 null cells formed abnormally large contractile vacuoles. Furthermore, the cycle of expansion and contraction for contractile vacuoles in AP80 null cells was twice as long as that of wild-type cells. Taken together, our results suggest that AP180 plays a unique role as a regulator of contractile vacuole morphology and activity in Dictyostelium. INTRODUCTIONEndocytosis via clathrin-coated vesicles is an essential process for the regulated uptake of nutrients and the trafficking of membrane receptors. Although clathrin is the principal structural player in building a coated vesicle, numerous adaptor and accessory proteins fine tune and regulate endocytosis (Lafer, 2002). Together they assemble clathrin triskelia onto membranes to construct a clathrin-coated pit (ter Haar et al., 2000;Brodsky et al., 2001). Four heterotetrameric adaptor proteins have been identified; each is thought to function at a different cellular location (Kirchhausen, 1999). At the plasma membrane the heterotetrameric protein AP-2 recruits clathrin and initiates the assembly of clathrin triskelia into lattices (Gallusser and Kirchhausen, 1993;Owen et al., 2000). Recently, monomeric assembly proteins have been identified at the plasma membrane that could also regulate clathrin traffic. The monomeric assembly protein, AP180, was first purified from coated vesicles of bovine brain (Ahle and Ungewickell, 1986), although a nonneuronal homologue of AP180, CALM, was identified more recently (Dreyling et al., 1996;Tebar et al., 1999). AP180 binds directly to clathrin and promotes assembly of clathrin triskelia into cages of uniform size (Ahle and Ungewickell, 1986;Prasad and Lippoldt, 1988;Ye and Lafer, 1995b).Analysis of mutants of the AP180 orthologues in Drosophila and Caenorhabditis elegans shows that AP180 regulates synaptic vesicle size as well as the sorting of synaptic proteins such as synaptobrevin (Zhang et al., 1998;Nonet et al., 1999;Bao et al., 2005). In mammalian cells, reduction of AP180 results in irregular clathrin lattices, demonstrating an important role for AP180 in the assembly of clathrin into geometrically precise coated vesicles (Meyerholz et al., 2005). Furthermore, recent studies show that AP180 is involved in the internalization of some receptors like the EGF re...