A plasma membrane pool of phosphatidylinositol 4-phosphate is generated by phosphatidylinositol 4-kinase type-III alpha: studies with the PH domains of the oxysterol binding protein and FAPP1.
The PH domains of OSBP and FAPP1 fused to GFP were used to monitor PI(4)P distribution in COS-7 cells during manipulations of PI 4-kinase (PI4K) activities. Both domains were associated with the Golgi and small cytoplasmic vesicles, and a small fraction of OSBP-PH was found at the plasma membrane (PM). Inhibition of type-III PI4Ks with 10 microM wortmannin (Wm) significantly reduced but did not abolish Golgi localization of either PH domains. Downregulation of PI4KIIalpha or PI4KIIIbeta by siRNA reduced the localization of the PH domains to the Golgi and in the former case any remaining Golgi localization was eliminated by Wm treatment. PLC activation by Ca2+ ionophores dissociated the domains from all membranes, but after Ca2+ chelation, they rapidly reassociated with the Golgi, the intracellular vesicles and with the PM. PM association of the domains was significantly higher after the Ca2+ transient and was abolished by Wm pretreatment. PM relocalization was not affected by down-regulation of PI4KIIIbeta or -IIalpha, but was inhibited by down-regulation of PI4KIIIalpha, or by 10 microM PAO, which also inhibits PI4KIIIalpha. Our data suggest that these PH domains detect PI(4)P formation in extra-Golgi compartments under dynamic conditions and that various PI4Ks regulate PI(4)P synthesis in distinct cellular compartments.
Balla
Andras
A
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Tuymetova
Galina
G
Tsiomenko
Arnold
A
Várnai
Péter
P
Balla
Tamas
T
D019870:1-Phosphatidylinositol 4-Kinase / Q000737:chemistry* / Q000378:metabolism; D000111:Acetylcysteine / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002118:Calcium / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D002352:Carrier Proteins / Q000737:chemistry*; D002462:Cell Membrane / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D015536:Down-Regulation; D004533:Egtazic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores; D050356:Lipid Metabolism; D018613:Microscopy, Confocal; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D011987:Receptors, Steroid / Q000737:chemistry*; D013347:Subcellular Fractions; D013997:Time Factors; D014162:Transfection; D000077191:Wortmannin; D000098825:Oxysterol Binding Proteins
D019870:1-Phosphatidylinositol 4-Kinase; D000111:Acetylcysteine; D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes; D002118:Calcium; D002352:Carrier Proteins; D002614:Chelating Agents; D004247:DNA; D004533:Egtazic Acid; D004791:Enzyme Inhibitors; D049452:Green Fluorescent Proteins; D015759:Ionomycin; D007476:Ionophores; D018129:Phosphatidylinositol Phosphates; D017853:Phosphotransferases (Alcohol Group Acceptor); D020033:Protein Isoforms; D034741:RNA, Small Interfering; D011987:Receptors, Steroid; D000077191:Wortmannin; D000098825:Oxysterol Binding Proteins; C487037:PLEKHA3 protein, human; C037178:phosphatidylinositol 4-phosphate; C581087:PI4KB protein, human