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Sbrissa D, et al.  (2007) Core protein machinery for mammalian phosphatidylinositol 3,5-bisphosphate synthesis and turnover that regulates the progression of endosomal transport. Novel Sac phosphatase joins the ArPIKfyve-PIKfyve complex. J Biol Chem 282(33):23878-91

Abstract: 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.

Status: Published

Type: Journal Article

PubMed ID: 17556371

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Sbrissa D Ikonomov OC Fu Z Ijuin T Gruenberg J Takenawa T Shisheva A Papers in SGD Colleagues in SGD PubMed Google Scholar

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