STUDY OF MOLECULES INVOLVED IN THE PROTEIN TRAFFICKING TO LYSOSOME-LIKE VACUOLES IN GIARDIA LAMBLIA

FELIZIANI C; TOUZ MC; ROPOLO AS

Ascochinga

Congreso; . XXIV Reunión Científica Anual. Sociedad de Protozoología.; 2010

STUDY OF MOLECULES INVOLVED IN THE PROTEIN TRAFFICKING TO LYSOSOME-LIKE VACUOLES IN GIARDIA LAMBLIA Feliziani C, Touz MC and Rópolo A Instituto M y M Ferreyra. Córdoba. E-mail: cfeliziani@immf.uncor.edu We focus our effort to elucidate the roles of Rab11 (Rab GTPase family, which has been identified as a regulator of endosomal recycling), ENTH protein (a clathrin-associated protein involved in transport of lysosomal enzymes to late endosomes), and EEA1 (Early Endosome Antigen 1 protein that binds vesicles containing phosphatidylinositol 3-phosphate, required for endosomal transport). Homologue sequences were obtained by searching the Giardia Genome Database. The analysis of the participation of these proteins in the trafficking towards lysosome-like peripheral vacuoles (PVs) was initiated by cloning and expression of HA-fusion proteins in Giardia trophozoites. Using IFA and confocal microscopy we determined their subcellular localization. Protein-protein interaction will be studied by IPP, YTH and in vivo techniques. Further analysis utilizing proteomic strategies will allow us to characterize each protein in the context of their role in lysosome trafficking and function. Additionally, in this work, we developed monoclonal antibodies (mAbs) directed against purified PVs. After cell-fractionation, the PV-containing fraction was isolated and used as immunogen to subcutaneously immunize BALB/c mice. The mice producing PV-positive polyclonal Abs were further euthanized and the spleen cells fused to the NSO myeloma cell line. Antibody-secreting hybridomas were screened by indirect immunofluorescence and dot-blotting using non-encysting trophozoites. The mAbs generated provide a unique set of tools to understand the assembly and function of these particular organelles and whether G. lamblia has many of the endomembrane protein transport elements and sorting functions of higher cells. Giardia appears as a great system to study not only the evolution of the secretory pathway in eukaryotes but also "why" and "how" an organism may have lost some essential subcellular organelles when becoming a parasite, while maintaining the minimal machinery necessary for fundamental cellular function