The giardial epsin-like protein possesses a dual epsin-epsinR role in clathrin-mediated trafficking

FELIZIANI C; ZAMPONI N; GOTTIG N; ROPOLO AS; LANFREDI-RANGEL; TOUZ MC

Mar del Plata

Congreso; SAP; 2014

BACKGROUND: Giardia lacks a defined endosomal/lysosomal system but possesses peripheral vacuoles (PVs) that play roles in endocytosis, degradation, recycling, and secretion of proteins during growth and differentiation. Analysis of the role of heterotetrameric clathrin adaptor proteins in protein trafficking to the PVs, suggested that this organism preserved a highly reduced set of proteins involved in endosomal/lysosomal trafficking. In this work, we identified the protein GlENTHp (for Giardia lamblia ENTH protein) containing an ENTH domain present in the monomeric adaptors epsin or epsin-related (epsinR) proteins, which are implicated in the endocytosis and Golgi-to-endosomes protein trafficking, respectively. METHODS: Immunofluorescence, immunoblotting, immunoprecipitation and yeast-two hybrid assays were carried out in stable clones expressing HA-tagged GlENTHp or GlENTHp mutants. Lipid binding was tested by performing protein-lipid blot overlay assays. The structure of the PVs in wild-type and transgenic parasites were examined by electron microscopy. RESULTS: We found that GlENTHp binds clathrin, ubiquitin, and interacts with the alpha subunit of AP-2 and the gamma subunit of AP-1, with the events of endocytosis and lysosomal hydrolase trafficking being equally affected by GlENTHp downregulation. GlENTHp targeting to membrane required a conserved lysine75, which allowed its binding to PI3,4,5P3 and PI4P, polyphosphoinositides linked in other cells to anchoring to the plasma membrane and Golgi, respectively. Either downregulation of GlENTHp or overexpression of a nonfunctional GlENTHp showed an unusual accumulation of dense material in the PVs and severely affected trophozoite growth. CONCLUSIONS: Our findings suggest that the dual epsin-epsinR role of GlENTHp is implicated in the maintenance of the PV homeostasis providing new avenues for therapeutic intervention against Giardia and related parasites.