Membrane fusion and fission events in intracellular trafficking are controlled by both intraluminal Ca2+ release and phosphoinositide (PIP) signalling.However, the molecular identities of the Ca2+ release channels and the target proteins of PIPs are elusive.By direct patch-clamping of the endolys0somal membrane, we report that PI(3,5)P2, an endolysosome-specific PIP, activates endolysosome-localized mucolipin transient receptor potential (TRPML) channels with potency and binds to the N terminus of TRPML1 with specificity.Both PI(3,5)P2-deficient cells and cells that lack TRPML 1 exhibited enlarged endolysosomes / vacuoles and trafficking defects in the late endocytic pathway.We find that the enlarged vacuole phenotype observed in PI(3,5)P2-deficient mouse fibroblasts is suppressed by overexpression of TRPML1.To further reveal the temporal and spatial dynamics of PI(3,5)P2 signaling, we constructed a "PI(3,5)P2-probe", in which the N terminus of TRPML1 was fused directly with GFP.In both yeast and mammalian cells, this probe exhibited high degree of co-localization with markers of vacuoles/endolysosomes, where the PIKfyve/fabl kinase is localized.We are currently characterizing the probe by pharmacologically and genetically manipulating the level of PI(3,5)P2.We conclude that PI(3,5)P2 is mainly localized in late endosomes and lysosomes and mediates intracellular trafficking by inducing TRPML-dependent Ca2+release from endolysosomes.