Phosphoinositide-binding proteins mark, shape and functionally modulate highly-diverged endocytic compartments in the parasitic protist Giardia lamblia.

Phosphorylated derivatives of phosphatidylinositol (PIPs), are key membrane lipid residues involved in clathrin-mediated endocytosis (CME). CME relies on PI(4,5)P-2 to mark endocytic sites at the plasma membrane (PM) associated to clathrin-coated vesicle (CCV) formation. The highly diverged parasitic protist Giardia lamblia presents disordered and static clathrin assemblies at PM invaginations, contacting specialized endocytic organelles called peripheral vacuoles (PVs). The role for clathrin assemblies in fluid phase uptake and their link to internal membranes via PIP-binding adaptors is unknown. Here we provide evidence for a robust link between clathrin assemblies and fluid-phase uptake in G. lamblia mediated by proteins carrying predicted PX, FYVE and NECAP1 PIP-binding modules. We show that chemical and genetic perturbation of PIP-residue binding and turnover elicits novel uptake and organelle-morphology phenotypes. A combination of co-immunoprecipitation and in silico analysis techniques expands the initial PIP-binding network with addition of new members. Our data indicate that, despite the partial conservation of lipid markers and protein cohorts known to play important roles in dynamic endocytic events in well-characterized model systems, the Giardia lineage presents a strikingly divergent clathrin-centered network. This includes several PIP-binding modules, often associated to domains of currently unknown function that shape and modulate fluid-phase uptake at PVs. Author summary In well-characterized model eukaryotes, clathrin-mediated endocytosis is a key process for uptake of extracellular material and is regulated by more than 50 known proteins. A large number of these proteins carry phosphoinositide (PIP)-binding domains and play a central role in the regulation of endocytosis. Here, we report on the detailed functional characterization of PIP-binding proteins in the intestinal parasitic protist Giardia lamblia. We show evidence that proteins carrying specific PIP-binding domains are directly involved in fluid-phase uptake. Furthermore, using co-immunoprecipitation assays, we confirm these proteins' association to G. lamblia's clathrin assemblies. In addition, using state-of-the-art imaging strategies, we demonstrate a previously unappreciated level of complexity involving PIPs and their partner proteins in marking and shaping G. lamblia's unique endocytic compartments. Our data contribute substantially to an updated working model for G. lamblia's host-parasite interface, demonstrating how uptake in this parasite is directly regulated by a variety of PIP residues and PIP-binding modules, which have been re-routed from conserved pathways, likely as a result of host-parasite co-evolution.