Soluble Lysosomal Hidrolases Transport In The Primitive Eucaryote Giardia lamblia

RIVERO M. R.; MIRAS S. L.; VRANYCH C. V.; RÓPOLO A. S; TOUZ M. C.

Congreso; 44th Annual Meeting. Argentine Society for Biochemistry and Molecular Biology Research; 2008

Unlike yeast and mammalian cells, the lysosomal sorting pathwayis not well defined in Giardia. For instance, in more evolved cells,soluble hydrolases are sorted from the trans-Golgi network to theendosomal/lysosomal system through a receptor-adaptin mediatedprocess. We found that delivery of the soluble acid phosphatase(AcPh) to the lysosome-like peripheral vacuoles (PVs), involved anadaptin-clathrin dependent pathway. These strongly suggest thatGiardia possesses a hydrolase receptor for PV delivery. Thus, weperformed pull-down assays using a His-tagged AcPh to search forthe hydrolase receptor. One of the associated proteins obtainedcontains a YQII lysosomal-sorting motif necessary for AdaptorProtein 1 (gAP1) binding. By fusion protein expression intransgenic trophozoites together with IFA and confocalmicroscopy, we found that this putative Giardia HydrolaseReceptor (gHR) colocalizes with AcPh around the nuclei and in thePVs. ?In vivo? interactions of gHR with AcPh or gAP1 wereconfirmed by yeast two-hybrid and verified by IPP assays. Inaddition, ongoing experiments with PV-residents Cathepsin B,revealed that gHR might be a common hydrolase receptor. Thesestudies will help to determine the minimal machinery necessary forlysosomal trafficking developed throughout eukaryotic evolutionor whether it is a consequence of Giardia parasitic life style.