A Mammalian Ortholog of Saccharomyces cerevisiae Vac14 That Associates with and Up-Regulates PIKfyve Phosphoinositide 5-Kinase Activity

Abstract

Multivesicular body morphology and size are controlled in part by PtdIns(3,5)P 2 , produced in mammalian cells by PIKfyve-directed phosphorylation of PtdIns(3)P. Here we identify human Vac14 (hVac14), an evolutionarily conserved protein, present in all eukaryotes but studied principally in yeast thus far, as a novel positive regulator of PIKfyve enzymatic activity. In mammalian cells and tissues, Vac14 is a low-abundance 82-kDa protein, but its endogenous levels could be up-regulated upon ectopic expression of hVac14. PIKfyve and hVac14 largely cofractionated, populated similar intracellular locales, and physically associated. A smallinterfering RNA-directed gene-silencing approach to selectively eliminate endogenous hVac14 rendered HEK293 cells susceptible to morphological alterations similar to those observed upon expression of PIKfyve mutants deficient in PtdIns(3,5)P 2 production. Largely decreased in vitro PIKfyve kinase activity and unaltered PIKfyve protein levels were detected under these conditions. Conversely, ectopic expression of hVac14 increased the intrinsic PIKfyve lipid kinase activity. Concordantly, intracellular PtdIns(3)P-to-PtdIns(3,5)P 2 conversion was perturbed by hVac14 depletion and was elevated upon ectopic expression of hVac14. These data demonstrate a major role of the PIKfyve-associated hVac14 protein in activating PIKfyve and thereby regulating PtdIns(3,5)P 2 synthesis and endomembrane homeostasis in mammalian cells.

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

“…The evolutionary conservation of the PtdIns(3,5)P 2 pathway is further substantiated by the recent findings for structural and functional homology between yeast and mammalian Vac14, also known as ArPIKfyve (14,38). They both activate Fab1 and PIKfyve, respectively, and in the case of mammalian cells, this is by a physical association (38 -40).…”

“…Polyclonal anti-HA (R4289), anti-IRAP, anti-GRP94, anti-Rab4, and monoclonal anti-transferrin receptor antibodies were gifts by Drs. Mike Czech, Paul Pilch, Steve Cala, Ira Melmann, and Ian Traubridge and used under previously specified conditions (38,(43)(44)(45)(46).…”

“…Proteins were produced in Escherichia coli BL21(DE3) strain and purified as described (47). Construction of pCMV5-HA-hArPIKfyve, pEGFP-HA-hArPIKfyve, or pEGFP-HA-mPIKfyve WT was detailed elsewhere (10,38,43).…”

“…Polyclonal anti-PIKfyve (R7069, directed against the N terminus) and anti-ArPIKfyve (WS047, directed against the C terminus) were described elsewhere (38,43). The antibodies were used for immunoprecipitation and Western blotting either as crude antisera or after affinity purification on the corresponding GST fusion peptides as described previously (38). Anti-Myc monoclonal antibody was produced by 9E10.2 hybridoma cells (ATCC).…”

“…Expression of Myc-or GFP-tagged Sac3 proteins was confirmed by Western blotting with anti-Myc and anti-GFP antibodies. pRSETb-His 6 -hArPIKfyve and pRSETb-His 6 -mGDI2 were described previously (38,47). Proteins were produced in Escherichia coli BL21(DE3) strain and purified as described (47).…”