title: Identification of contractile vacuole proteins in Trypanosoma cruzi.
abstract: Contractile vacuole complexes are critical components of cell volume regulation and have been shown to have other functional roles in several free-living protists. However, very little is known about the functions of the contractile vacuole complex of the parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, other than a role in osmoregulation. Identification of the protein composition of these organelles is important for understanding their physiological roles. We applied a combined proteomic and bioinfomatic approach to identify proteins localized to the contractile vacuole. Proteomic analysis of a T. cruzi fraction enriched for contractile vacuoles and analyzed by one-dimensional gel electrophoresis and LC-MS/MS resulted in the addition of 109 newly detected proteins to the group of expressed proteins of epimastigotes. We also identified different peptides that map to at least 39 members of the dispersed gene family 1 (DGF-1) providing evidence that many members of this family are simultaneously expressed in epimastigotes. Of the proteins present in the fraction we selected several homologues with known localizations in contractile vacuoles of other organisms and others that we expected to be present in these vacuoles on the basis of their potential roles. We determined the localization of each by expression as GFP-fusion proteins or with specific antibodies. Six of these putative proteins (Rab11, Rab32, AP180, ATPase subunit B, VAMP1, and phosphate transporter) predominantly localized to the vacuole bladder. TcSNARE2.1, TcSNARE2.2, and calmodulin localized to the spongiome. Calmodulin was also cytosolic. Our results demonstrate the utility of combining subcellular fractionation, proteomic analysis, and bioinformatic approaches for localization of organellar proteins that are difficult to detect with whole cell methodologies. The CV localization of the proteins investigated revealed potential novel roles of these organelles in phosphate metabolism and provided information on the potential participation of adaptor protein complexes in their biogenesis.
authors: Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, Georgia, United States of America.
authors: Paul N
authors: PN
authors: Ulrich
authors: Veronica
authors: V
authors: Jimenez
authors: Miyoung
authors: M
authors: Park
authors: Vicente P
authors: VP
authors: Martins
authors: James
authors: J
authors: Atwood
authors: Kristen
authors: K
authors: Moles
authors: Dalis
authors: D
authors: Collins
authors: Peter
authors: P
authors: Rohloff
authors: Rick
authors: R
authors: Tarleton
authors: Silvia N J
authors: SN
authors: Moreno
authors: Ron
authors: R
authors: Orlando
authors: Roberto
authors: R
authors: Docampo
mesh_terms: D015153:Blotting, Western; D003285:Contractile Proteins / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D058977:Molecular Sequence Annotation; D021381:Protein Transport; D020543:Proteome / Q000378:metabolism; D015800:Protozoan Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D014349:Trypanosoma cruzi / Q000166:cytology / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D003285:Contractile Proteins; D020543:Proteome; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D025262:Vacuolar Proton-Translocating ATPases