title: 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.
abstract: 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 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)P2 synthesis and turnover achieved through a physical assembly of PIKfyve, ArPIKfyve, and Sac3. We suggest that the tight regulation in PtdIns(3,5)P2 homeostasis is mechanistically linked to early endosome dynamics in the course of cargo transport.
authors.affiliation: Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
authors.forename: Diego
authors.initials: D
authors.lastname: Sbrissa
authors.forename: Ognian C
authors.initials: OC
authors.lastname: Ikonomov
authors.forename: Zhiyao
authors.initials: Z
authors.lastname: Fu
authors.forename: Takeshi
authors.initials: T
authors.lastname: Ijuin
authors.forename: Jean
authors.initials: J
authors.lastname: Gruenberg
authors.forename: Tadaomi
authors.initials: T
authors.lastname: Takenawa
authors.forename: Assia
authors.initials: A
authors.lastname: Shisheva
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism*; D006706:Homeostasis; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000096:biosynthesis* / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport
chemical_list: D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D046912:Multiprotein Complexes; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates; D017346:Protein Serine-Threonine Kinases; C494631:VAC14 protein, human; C106336:phosphatidylinositol 3,5-diphosphate; C120564:Sac3 protein kinase; C492139:PIKFYVE protein, human