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 lysine
75, 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.
