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.

Acidocalcisomes of Trypanosoma brucei have an inositol 1,4,5-trisphosphate receptor that is required for growth and infectivity

“…Interestingly, apart from one study that described a functional IP 3 receptor in Paramecium tetraurelia (18), biochemical evidence that the other genes identified encode for functional IP 3 receptors is still lacking. Proteomic data from enriched contractile vacuole fractions of T. cruzi (20) included spectra from the putative IP 3 Rs, suggesting that these proteins are expressed in subcellular fractions (20).In this work, we report the characterization of the IP 3 R from T. brucei, which localizes to their acidocalcisomes. IP 3 was able to release Ca 2+ from permeabilized cells and isolated acidocalcisomes, and UV light photolysis of caged IP 3 produced a significant increase in intracellular Ca 2+ in live trypanosomes.…”

Trypanosoma brucei Vacuolar Transporter Chaperone 4 (TbVtc4) Is an Acidocalcisome Polyphosphate Kinase Required for in Vivo Infection

“…This resistance to osmotic stress is essential for digenetic trypanosomatids as they encounter drastic osmotic changes in both the insect vectors and vertebrate hosts (23,25,26). Regulation of cell volume is, in addition, a homeostatic process needed at all times by all cells.…”

The Contractile Vacuole as a Key Regulator of Cellular Water Flow in Chlamydomonas reinhardtii

“…The same proteins/ cellular processes have been implicated in CV function in Amoeba, Dictyostelium, Paramecium, Trypanosoma, and green algae (e.g., proton pumps and aquaporins) (3,4,6). However, we still do not know anything about what other factors (ion transport systems) are needed to build up the necessary osmotic gradient in any system.…”