Regulation of phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae by CDP-diacylglycerol.
Regulation of the 45- and 55-kDa forms of Saccharomyces cerevisiae membrane-associated phosphatidylinositol (PI) 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase) by phospholipids was examined using Triton X-100/phospholipid-mixed micelles. CDP-diacylglycerol and phosphatidylglycerol inhibited 45-kDa PI 4-kinase activity in a dose-dependent manner. Kinetic analyses of the 45-kDa PI 4-kinase showed that phosphatidylglycerol was a competitive inhibitor with respect to PI (Ki = 2 mol %), and CDP-diacylglycerol was a mixed type of inhibitor with respect to PI (Ki = 4 mol %) and MgATP (Ki = 5 mol %). 55-kDa PI 4-kinase activity was not significantly affected by phospholipids. The physiological relevance of CDP-diacylglycerol inhibition of 45-kDa PI 4-kinase activity was examined using plasma membranes from inositol auxotrophic (ino1) cells. Immunoblot analysis showed that 45-kDa PI 4-kinase expression in plasma membranes was not affected by inositol starvation of ino1 cells. However, both 45-kDa PI 4-kinase activity and its product PI 4-phosphate were reduced in plasma membranes from inositol-starved ino1 cells. The CDP-diacylglycerol concentration (9.6 mol %) in plasma membranes of inositol-starved ino1 cells was 12-fold higher than its concentration (0.8 mol %) in plasma membranes of inositol-supplemented cells. Plasma membranes of inositol-starved ino1 cells also had increased levels of phosphatidate, phosphatidylserine, phosphatidylethanolamine, and cardiolipin. However, these phospholipids did not affect pure 45-kDa PI 4-kinase activity. The concentration of CDP-diacylglycerol in plasma membranes of inositol-starved ino1 cells was in the range of the inhibitor constants determined for CDP-diacylglycerol by kinetic analyses using pure 45-kDa PI 4-kinase. These results raised the suggestion that 45-kDa PI 4-kinase activity may be regulated in vivo by CDP-diacylglycerol.
Nickels
J T
JT
Department of Food Science, Cook College, Rutgers University, New Brunswick, New Jersey 08903.
Buxeda
R J
RJ
Carman
G M
GM
D019870:1-Phosphatidylinositol 4-Kinase; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D003567:Cytidine Diphosphate Diglycerides / Q000494:pharmacology*; D002798:Diacylglycerol Cholinephosphotransferase / Q000378:metabolism*; D007294:Inositol / Q000378:metabolism; D007700:Kinetics; D008563:Membrane Lipids / Q000302:isolation & purification / Q000378:metabolism; D010715:Phosphatidylglycerols / Q000494:pharmacology; D010743:Phospholipids / Q000302:isolation & purification / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
D003567:Cytidine Diphosphate Diglycerides; D008563:Membrane Lipids; D010715:Phosphatidylglycerols; D010743:Phospholipids; D029701:Saccharomyces cerevisiae Proteins; D007294:Inositol; D017853:Phosphotransferases (Alcohol Group Acceptor); D019870:1-Phosphatidylinositol 4-Kinase; C441760:PIK1 protein, S cerevisiae; D002798:Diacylglycerol Cholinephosphotransferase