Phosphatidylinositol 3-phosphate-interacting domains in PIKfyve. Binding specificity and role in PIKfyve. Endomenbrane localization.

PIKfyve is a phosphatidylinositol (PtdIns) 3-phosphate (P)-metabolizing enzyme, which, in addition to a C-terminally positioned catalytic domain, harbors several evolutionarily conserved domains, including a FYVE finger. The FYVE finger domains are thought to direct the protein localization to intracellular membrane PtdIns 3-P. Recent studies with several FYVE domain proteins challenge this general concept. Here we have examined the binding of PIKfyve's FYVE domain to PtdIns 3-P in vitro and in vivo and a plausible contribution of this binding mechanism for the intracellular localization of the full-length protein. We document now a specific and high affinity interaction of a recombinantly produced PIKfyve FYVE domain peptide fragment with PtdIns 3-P-containing liposomes that requires the presence of the conservative core of basic residues within the FYVE domain. PIKfyve localization to membranes of the late endocytic pathway was found to be absolutely dependent on the presence of an intact FYVE finger. Cell treatment with PI 3-kinase inhibitor wortmannin dissociated endosome-bound PIKfyve, indicating that the protein targeted the membrane PtdIns 3-P. An enzymatically inactive peptide fragment of the PIKfyve catalytic domain was found to also specifically bind to PtdIns 3-P-containing liposomes, with residue Lys-1999 being critical in the interaction. This binding, however, was of relatively low affinity and, in the cellular context, was found ineffective in directing the molecule to PtdIns 3-P-enriched endosomes. Collectively, these results demonstrate that interaction of the FYVE domain with PtdIns 3-P is absolutely necessary for PIKfyve targeting to the membranes of the late endocytic pathway and determine PIKfyve as a downstream effector of PtdIns 3-P.