‘TcVps34, a PI 3-kinase involved in osmoregulation and membrane trafficking in Trypanosoma cruzi’

SCHOIJET, A.; MIRANDA, K.; DOCAMPO, R.; TORRES, H.; FLAWIA, M.; ALONSO, G.

Mar del Plata, Buenos Aires, Argentina.

Congreso; XLIII Annual Meeting -Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB).; 2007

SAIB

During its developmental stages in different hosts, T. cruzi faces extreme fluctuations in osmolarity and in the uptake of nutrients that must incorporate through its endocytic system. In this work, we report the identification of TcVps34, a class III phosphatidylinositol 3-kinase of T. cruzi. Recombinant TcVps34 was able to phosphorylate only phosphatidylinositol (PI) to produce phosphatidylinositol 3-phosphate (PI 3-P) and complemented yeast Vps34 knockout strain, indicating that TcVps34 is a functional PI 3-kinase. Microscopy analysis of TcVps34 over-expressing epimastigotes cells revealed functional and large contractile vacuoles as well as some defects in the region near of the citostome and the flagellar pocket. Pre-incubation of wild-type epimastigotes with PI 3-kinase inhibitors reduced the recovery of the parasites to hypo-osmotic stress. Kinetics of proton uptake in TcVps34 over-expressing cells showed higher H+-ATPase and lower H+-PPase activities than in wild-type cells. In addition, endocytosis of FITC-transferrin was reduced in TcVps34 over-expressing cells. Finally, a yeast two-hybrid assay showed interaction of TcVps34 with TcVps15, a ser/thr kinase that interact as a component of a protein complex with Vps34 in yeast. Taken together, these results suggest a potential role for TcVps34 in osmoregulation, vacuolar acidification, and membrane trafficking in T cruzi.