A Giardial ENTH protein regulates vacuolar homeostasis and reveals a bifunctional role in clathrin-mediated trafficking

MARÍA C TOUZ; ROPOLO ANDREA; CONSTANZA FELIZIANI

Mexico DF

Congreso; 13th International Congress of Parasitology 2014; 2014

BACKGROUND: Giardia lacks a defined endosomal/lysosomal system but possessesperipheral vacuoles (PVs) that play roles in endocytosis, degradation, recycling, andsecretion of proteins during growth and differentiation. Analysis of the role ofheterotetrameric clathrin adaptor proteins in protein trafficking to the PVs, suggestedthat this organism preserved a highly reduced set of proteins involved inendosomal/lysosomal trafficking. In this work, we identified the protein GlENTHp (forGiardia lamblia ENTH protein) containing an ENTH domain present in the monomericadaptors epsin or epsin-related (epsinR) proteins, which are implicated in theendocytosis and Golgi-to-endosomes protein trafficking, respectively.METHODS: Immunofluorescence, immunoblotting, immunoprecipitation and yeast-twohybrid assays were carried out in stable clones expressing HA-tagged GlENTHp orGlENTHp mutants. Lipid binding was tested by performing protein-lipid blot overlayassays. The structure of the PVs in wild-type and transgenic parasites were examinedby electron microscopy.RESULTS: We found that GlENTHp binds clathrin, ubiquitin, and interacts with thealpha subunit of AP-2 and the gamma subunit of AP-1, with the events of endocytosisand lysosomal hydrolase trafficking being equally affected by GlENTHpdownregulation. GlENTHp targeting to membrane required a conserved lysine75, whichallowed its binding to PI3,4,5P3 and PI4P, polyphosphoinositides linked in other cells toanchoring to the plasma membrane and Golgi, respectively. Either downregulation ofGlENTHp or overexpression of a nonfunctional GlENTHp showed an unusualaccumulation of dense material in the PVs and severely affected trophozoite growth.CONCLUSIONS: Our findings suggest that the dual epsin-epsinR role of GlENTHp isimplicated in the maintenance of the PV homeostasis providing new avenues fortherapeutic intervention against Giardia and related parasites.