Core Protein Machinery for Mammalian Phosphatidylinositol 3,5-Bisphosphate Synthesis and Turnover That Regulates the Progression of Endosomal Transport

作者：Diego Sbrissa, Ognian Christov Ikonomov, Zhiyao Fu, Takeshi Ijuin, Jean Grüenberg, Tadaomi Takenawa, Assia Shisheva · 发表于：Journal of Biological Chemistry · 年份：2007 · DOI：10.1074/jbc.m611678200 · 被引用次数：197 · 研究领域：Cellular transport and secretion、Calcium signaling and nucleotide metabolism、Pancreatic function and diabetes

Perturbations in phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2)-synthesizing enzymes result in enlarged endocytic organelles from yeast to humans, indicating evolutionarily conserved function of PtdIns(3,5)P2 in endosome-related events. This is reinforced by the structural and functional homology of yeast Vac14 and human Vac14 (ArPIKfyve), which activate yeast and mammalian PtdIns(3,5)P2-producing enzymes, Fab1 and PIKfyve, respectively. In yeast, PtdIns(3,5)P2-specific phosphatase, Fig4, in association with Vac14, turns over PtdIns(3,5)P2, 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)P2 in vitro, although the other D5-phosphorylated polyphosphoinositides were also substrates. Ablation of endogenous Sac3 by short interfering RNAs elevated PtdIns(3,5)P2 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)P2 requirement in early endosome dynamics. In vitro reconstitution of carrier vesicle formation from donor early endosomes revea...