The Unique Dual Function and the Evolutionary Relationship of the giardial epsin-like Protein.

TOUZ MC; VALDEZ-TAUBAS J; MOYANO S; WENDLAND B; FELIZIANI C

La Serena

Workshop; EMBO WORKSHOP Actualizations in Membrane Trafficking in Health and Disease.; 2016

Endocytosis and lysosomal protein trafficking is essential in pathogenic parasites since it is 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. Recently, we have identified a protein containing an ENTH domain that defines monomeric adaptor proteins of the epsin family in Giardia. This domain is present in the epsin or epsin-related (epsinR) adaptor proteins, which are implicated in endocytosis and Golgi-to-endosome protein trafficking, respectively, in other eukaryotic cells. We found that GlENTHp (for Giardia lamblia ENTH protein), like epsin, was associated with alpha AP-2, clathrin and ubiquitin, interacted with PI3,4,5P3, and was involved in receptor-mediated endocytosis. It also bonded gamma AP-1 and PI4P, and was implicated in ER-to-PV trafficking, like epsinR proteins. Our findings showed that GlENTHp participates in the machinery for clathrin-mediated membrane budding acting as a dual epsin-epsinR protein. Nevertheless, because many components of the endocytic machinery are structurally and functionally conserved between eukaryotes, we used the yeast system to explore the similarities as well as the particularities of GlENTHp as a member of the epsin family. The yeast S. cerevisiae expresses two homologues of epsin, Ent1 and Ent2, and two orthologous of epsinR, Ent3 and Ent5. Although the expression of GlENTHp (or its ENTH domain alone) neither complemented growth in ent1Δent2Δ mutant nor restored GFP-CPS1 vacuolar protein trafficking defect in ent3Δent5Δ, it resulted in a dominant non-functional counterpart of Ent3/5p in a wild type strain. This phenotype is linked to a defect in CPS1 localization and α-factor mating pheromone maturation. The finding that GlENTHp acts as an non-functional epsinR in yeast cells, reinforce the phylogenetic data showing that GlENTHp belongs to the epsinR subfamily present in eukaryotes prior to its evolution in different taxa.