A novel monomeric adaptor protein in Giardia lamblia with a dual epsin-like function

FELIZIANI C.; ZAMPONI N.; MIRAS S.L.; ROPOLO A. S.; LANFREDI-RANGEL A.; TOUZ M.C.

Newport

Congreso; GRC- Host parasite interaction; 2012

Salve Regina University

Endocytosis and lysosomal protein trafficking are essential processes in pathogenic parasites since they are directly linked to vital parasite-specific processes, e. g., host cell invasion, nutrition, and cell differentiation into resistant stages, as in the case of Giardia. The epsin N-terminal homology (ENTH) domain is an evolutionarily conserved protein module, which is found primarily in proteins involved in clathrin-mediated trafficking. By searching in the GDB, we found that GlENTHp (for Giardia lamblia ENTH protein) contains an ENTH domain that shares the three dimensional structure with human epsin ENTH domain, displaying an alpha-helical structure composed of 7 alpha-helices. This domain is present in epsin or epsin-related (epsinR) proteins, which are involved in endocytosis and protein trafficking from the Golgi to the lysosomes, respectively. Both epsin and epsinR possess clathrin-binding motifs, but only epsin incorporates an ubiquitin-interaction motif (UIM). Using Giardia´s specific anti-clathrin and anti-ubiquitin antibodies, we determined that GlENTHp interacts with both proteins. Subcellular localization showed that this protein is located mainly in the cytosol and occasionally in the nuclei. Biochemical analysis also showed that GlENTHp binds PI(3,4,5)P3 and PI(4)P, both phosphoinositides linked to anchoring to the plasma membrane and Golgi, respectively. Moreover, protein-protein interaction experiments showed that GlENTHp physically interacts with the alpha subunit of the adaptor protein-2 (AP-2, involved in endocytosis) and with the gamma subunit of AP-1 (implicated in the Golgi-to-lysosome trafficking). Altogether, these results suggest that GlENTHp participates in the machinery for clathrin-mediated membrane budding, functioning as a dual epsin-epsinR protein.