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Georgia Veterinary Scholars Program

GVSP Summer 2009 Scholars

Georgia Veterinary Scholar	Faculty Mentor
Kristen Moles University of Georgia Class of 2012	Dr. Roberto DoCampo Cellular Biology UGA Franklin College of Arts & Sciences

Localization and functional role of Rab32 in Trypanosoma cruzi

* Kristen Moles1,2, Vicente P. Martins1, and Roberto Docampo1
1Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, and  2College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA

Trypanosoma cruzi is a zoonosis and the etiological agent of Chagas disease, an endemic pathology in Latin America with more than 15 million persons infected. Moreover, a substantial increase in seropositive blood donors in USA has been reported lately. T. cruzi is exposed to many osmotic stressors throughout its life cycle and osmoregulation is essential for this parasite. T. cruzi contains a contractile vacuole complex that plays a vital role in cellular responses to osmotic stresses. Volume recovery after hyposmotic stress is mediated by cyclic AMP-dependent translocation of aquaporin to the contractile vacuole. It has been found that the aquaporin is phosphorylated by protein kinase A. Here we focused on studying Rab32, a protein that was identified in the contractile vacuole of T. cruzi by proteomic analysis. As this protein was reported to function as an A-kinase anchoring protein (AKAP), it could be involved in protein kinase A recruitment to the contractile vacuole. Using the PCR technique, the TcRab32 gene was amplified and cloned into TOPO-vector. The recombinant DNA was purified, the gene was confirmed by sequencing and sub-cloned into pET28 for bacterial expression and TcRab32 purification, and into pTEX-N-GFP for expression in T. cruzi. We performed site directed mutagenesis of T24N and Q71L in pTEX-N-GFP::TcRab32 to evaluate the dependence of GTP-binding for membrane association. The prenylation sites C241A/C243A at the C-terminal were also mutated to verify their importance for membrane association. Finally, to determine if TcRab32 binds to PKA we mutated residue L197P that blocks PKA binding.