Perturbations in PtdIns(3,5)P(2) synthesizing enzymes result in enlarged endocytic organelles from yeast to humans, indicating evolutionary conserved function of PtdIns(3,5)P(2) in endosome-related events. This is reinforced by the structural and functional homology of yeast Vac14 and hVac14 (ArPIKfyve), which activate yeast and mammalian PtdIns(3,5)P(2)-producing enzymes, Fab1 and PIKfyve, respectively. In yeast, PtdIns(3,5)P(2)-specific phosphatase, Fig4, in association with Vac14, turns over PtdIns(3,5)P(2) but whether such a mechanism operates in mammalian cells and what the identity of mammalian Fig4 may be, are unknown. Here we have identified and characterized Sac3, a Sac domain phosphatase, as the Fig4 mammalian counterpart. Endogenous Sac3, a widespread 97-kDa protein, formed a stable ternary complex with ArPIKfyve and PIKfyve. Concordantly, Sac3 cofractionated and colocalized with ArPIKfyve and PIKfyve. The intrinsic Sac3(WT) phosphatase activity preferably hydrolyzed PtdIns(3,5)P(2) in vitro although the other D-5-phosphorylated polyphosphoinositides were also substrates. Ablation of endogenous Sac3 by siRNAs elevated PtdIns(3,5)P(2) in (32)P-labeled HEK293 cells. Ectopically expressed Sac3(WT) in COS cells colocalized with, and dilated EEA1-positive endosomes, consistent with the PtdIns(3,5)P(2) requirement in early endosome dynamics. In vitro reconstitution of carrier vesicle formation from donor early endosomes revealed a gain of function upon Sac3 loss, whereas PIKfyve or ArPIKfyve protein depletion produced a loss of function. These data demonstrate a coupling between the machinery for PtdIns(3,5)P(2) synthesis and turnover achieved through a physical assembly of PIKfyve, ArPIKfyve and Sac3. We suggest that the tight regulation in PtdIns(3,5)P(2) homeostasis is mechanistically linked to early endosome dynamics in the course of cargo transport.
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|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|