Soluble lysosomal hydrolases transport in the primitive eukaryote Giardia lamblia

RIVERO MR; MIRAS SL; VRANYCH CV; RÓPOLO AS; TOUZ MC

Villa Carlos Paz. Córdoba.

Congreso; XLIV Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular.; 2008

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Unlike yeast and mammalian cells, the lysosomal sorting pathway is not well defined in Giardia. For instance, in more evolved cells, soluble hydrolases are sorted from the trans-Golgi network to the endosomal/lysosomal system through a receptor-adaptin mediated process. We found that delivery of the soluble acid phosphatase (AcPh) to the lysosome-like peripheral vacuoles (PVs), involved an adaptin-clathrin dependent pathway. These strongly suggest that Giardia possesses a hydrolase receptor for PV delivery. Thus, we performed pull-down assays using a His-tagged AcPh to search for the hydrolase receptor. One of the associated proteins obtained contains a YQII lysosomal-sorting motif necessary for Adaptor Protein 1 (gAP1) binding. By fusion protein expression in transgenic trophozoites together with IFA and confocal microscopy, we found that this putative Giardia Hydrolase Receptor (gHR) colocalizes with AcPh around the nuclei and in the PVs. “In vivo” interactions of gHR with AcPh or gAP1 were confirmed by yeast two-hybrid and verified by IPP assays. In addition, ongoing experiments with PV-residents Cathepsin B, revealed that gHR might be a common hydrolase receptor. These studies will help to determine the minimal machinery necessary for lysosomal trafficking developed throughout eukaryotic evolution or whether it is a consequence of Giardia parasitic life style.