AP180-mediated trafficking of Vamp7B limits homotypic fusion of Dictyostelium contractile vacuoles.
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.
Wen
Yujia
Y
Department of Molecular Cell and Developmental Biology, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA.
Stavrou
Irene
I
Bersuker
Kirill
K
Brady
Rebecca J
RJ
De Lozanne
Arturo
A
O'Halloran
Theresa J
TJ
D033942:Adaptor Proteins, Vesicular Transport / Q000032:analysis / Q000235:genetics / Q000502:physiology; D000818:Animals; D022163:Clathrin-Coated Vesicles / Q000502:physiology*; D004023:Dictyostelium / Q000166:cytology* / Q000235:genetics / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D017930:Genes, Reporter; D007091:Image Processing, Computer-Assisted; D008561:Membrane Fusion / Q000502:physiology*; D034041:Monomeric Clathrin Assembly Proteins / Q000502:physiology*; D025941:Protein Interaction Mapping; D021381:Protein Transport / Q000502:physiology*; D015800:Protozoan Proteins / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D050600:SNARE Proteins / Q000235:genetics / Q000502:physiology*; D014617:Vacuoles / Q000502:physiology* / Q000648:ultrastructure
D033942:Adaptor Proteins, Vesicular Transport; D034041:Monomeric Clathrin Assembly Proteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; C439023:clathrin assembly protein AP180
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