TcVps15 as a positive regulator of autophagy in Trypanosoma cruzi

SCHOIJET, ALEJANDRA C.; FLAWIÁ, MIRTHA M.; ALONSO, GUILLERMO D.

Rosario

Congreso; 50th Annual Meeting Argentine Society for Biochemistry and Molecular Biology; 2014

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB).

Autophagy is a tightly controlled degradation process of all eukaryotes. In yeast, the phosphatidylinositol 3-kinase (Vps34) and its membrane targeting unit, the protein kinase Vps15 have been shown to be important regulators of autophagy. However, their role in this process remains elusive in Trypanosoma cruzi. Previously, we have characterized the first class III PI3K in Trypanosoma cruzi, named TcVps34, which has a role in vital processes such as osmoregulation, acidification and endocytosis. Afterward, we biochemically characterized the regulatory kinase TcVps15 in this parasite. In the present work, we aimed to assess the function of TcVps15 in autophagy. Immunofluorescence studies demonstrated that under starvation conditions TcVps15 colocalize with Atg8, an autophagosome marker in T. cruzi. Moreover, assays using acridine orange showed a higher level of acidification in TcVps15 transgenic parasites under nutritional stress. To further study the structure of TcVps15, we performed a mutation in its catalytic domain. Biochemical assays using the recombinant protein showed a six-fold increase in the Km value. We are currently assessing the effects of this mutation in transgenic parasites through immunofluorescence and starvation assays. Taken together, these results provide the first evidence for a function of Vps15 as a positive regulator of autophagy in Trypanosoma cruzi.