THE ROLE OF CDP-DIACYLGLYCEROL SYNTHETASE AND PHOSPHATIDYLINOSITOL SYNTHASE ACTIVITY LEVELS IN THE REGULATION OF CELLULAR PHOSPHATIDYLINOSITOL CONTENT

The regulation of phosphatidylinositol synthesis was examined by cloni ng and expressing in COS-7 cells the human cDNAs encoding the two enzy mes in the biosynthetic pathway, Human CDP-diacylglycerol synthetase ( cds1) and phosphatidylinositol synthase (pis1) clones were identified in the human expressed sequence-tagged (EST) data base, and full-lengt h cDNAs were obtained by library screening, The cds1 cDNA did not poss ess a recognizable mitochondrial import signal, and the activity of th e expressed Cds1 protein was stimulated by nucleoside triphosphates in vitro, indicating that cds1 did not encode the mitochondrial-specific isozyme, There were two mRNA species (3.9 and 5.6 kilobases) detected on Northern blots hybridized with the cds1 probe that were expressed at distinctly different levels in various human tissues, Consistent wi th the presence of the two mRNAs, a cDNA predicted to encode a second human CDP-diacylglycerol synthetase (cds2) was also uncovered in the E ST data base, In contrast to the two cds mRNAs, a single, 2.1-kilobase pis1 mRNA was uniformly expressed in all human tissues examined, Expr ession of the pis1 gene led to the overproduction of both phosphatidyl inositol synthase and phosphatidylinositol:inositol exchange reactions , indicating that the Pis1 polypeptide catalyzed both of these activit ies, Phosphatase treatment of cell extracts abolished the CMP-independ ent phosphatidylinositol:inositol exchange reaction, and exchange acti vity was completely restored by the addition of CMP, Overexpression of cds1 or pis1 alone or in combination did not enhance the rate of phos phatidylinositol biosynthesis, Also, overexpression did not result in a significant proportional increase in the cellular levels of CDP-diac ylglycerol or phosphatidylinositol, These data illustrate that the lev els of Cds1 and Pis1 protein expression are not critical determinants of cellular PtdIns content and argue against a determining role for th e activity of either of these enzymes in the regulation of PtdIns bios ynthesis.