title: Vacuolar protein sorting protein 13A, TtVPS13A, localizes to the tetrahymena thermophila phagosome membrane and is required for efficient phagocytosis.
abstract: Vacuolar protein sorting 13 (VPS13) proteins have been studied in a number of organisms, and mutations in VPS13 genes have been implicated in two human genetic disorders, but the function of these proteins is poorly understood. The TtVPS13A protein was previously identified in a mass spectrometry analysis of the Tetrahymena thermophila phagosome proteome (M. E. Jacobs et al., Eukaryot. Cell 5:1990-2000, 2006), suggesting that it is involved in phagocytosis. In this study, we analyzed the structure of the macronuclear TtVPS13A gene, which was found to be composed of 17 exons spanning 12.5 kb and was predicted to encode a protein of 3,475 amino acids (aa). A strain expressing a TtVPS13A-green fluorescent protein (GFP) fusion protein was constructed, and the protein was found to associate with the phagosome membrane during the entire cycle of phagocytosis. In addition, Tetrahymena cells with a TtVPS13A knockout mutation displayed impaired phagocytosis. Specifically, they grew slowly under conditions where phagocytosis is essential, they formed few phagosomes, and the digestion of phagosomal contents was delayed compared to wild-type cells. Overall, these results provide evidence that the TtVPS13A protein is required for efficient phagocytosis.
authors.affiliation: Department of MMS Biology, University of Connecticut Health Center, Farmington, CT 06032, USA.
authors.forename: Haresha S
authors.initials: HS
authors.lastname: Samaranayake
authors.forename: Ann E
authors.initials: AE
authors.lastname: Cowan
authors.forename: Lawrence A
authors.initials: LA
authors.lastname: Klobutcher
mesh_terms: D001483:Base Sequence; D017353:Gene Deletion; D006801:Humans; D013058:Mass Spectrometry; D010587:Phagocytosis / Q000502:physiology*; D010588:Phagosomes / Q000737:chemistry / Q000378:metabolism*; D020543:Proteome / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D016808:Tetrahymena thermophila / Q000235:genetics* / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D020543:Proteome; D011993:Recombinant Fusion Proteins; D033921:Vesicular Transport Proteins; D012313:RNA