SacIp dysfunction results in bypass of the requirement for phosphatidylinositol transfer protein (Sec14p) function in yeast Golgi processes. This effect is accompanied by alterations in inositol phospholipid metabolism and inositol auxotrophy. Elucidation of how sac1 mutants effect "bypass Sec14p" will provide insights into Sec14p function in vivo. We now report that, in addition to a dramatic accumulation of phosphatidylinositol-4-phosphate, sac1 mutants also exhibit a specific acceleration of phosphatidylcholine biosynthesis via the CDP-choline pathway. This phosphatidylcholine metabolic phenotype is sensitive to the two physiological challenges that abolish bypass Sec14p in sac1 strains; i.e. phospholipase D inactivation and expression of bacterial diacylglycerol (DAG) kinase. Moreover, we demonstrate that accumulation of phosphatidylinositol-4-phosphate in sac1 mutants is insufficient to effect bypass Sec14p. These data support a model in which phospholipase D activity contributes to generation of DAG that, in turn, effects bypass Sec14p. A significant fate for this DAG is consumption by the CDP-choline pathway. Finally, we determine that CDP-choline pathway activity contributes to the inositol auxotrophy of sac1 strains in a novel manner that does not involve obvious defects in transcriptional expression of the INO1 gene.
We previously isolated two distinct Saccharomyces cerevisiae myo-inositol transporter genes, ITR1 and ITR2 (Nikawa et al., 1991). Here, we studied the regulation of their expression by measuring steady-state mRNA levels and beta-galactosidase activities of lacZ fusion genes under different conditions. The results show that the expression of the two ITR genes is differently regulated: ITR1 was repressed by inositol and choline whereas ITR2 was constitutive. Deletion analysis of the ITR1 upstream region and comparison with the upstream regions of other genes involved in phospholipid synthesis indicate that the octamer sequence 5'-TTCACATG-3' is important for the expression and inositol/choline regulation of the ITR1 gene.
Long-chain acyl-coenzyme-A synthetase from the microsomes as well as from the mitochondrial fraction of rat liver has been purified to homogeneity as evidenced by dodecylsulfate/polyacrylamide gel electrophoresis, amino-terminal analysis and the elution profile at the final chromatography step. The purification procedure involves resolution of the cellular particles with Triton X-100 and chromatography on Blue-Sepharose, hydroxyapatite and phosphocellulose. The purified enzymes from both sources have a specific activity of 26-29 units/mg protein at 35 'C, which is more than 100-fold higher than those of long-chain acyl-CoA synthetases of animal and bacterial origin hitherto reported. The purified enzymes exhibit a molecular weight of approximately 76 000 as estimated by dodecylsulfate/polyacrylamide gel electrophoresis and catalyze the activation of saturated fatty acids with 10-18 carbon atoms and unsaturated fatty acids with 16-20 carbon atoms most efficiently. The purified enzyme from the microsomes and that from the mitochondrial fraction, which are obtained by essentially identical procedures, are indistinguishable from each other with respect to all molecular and catalytic properties examined, including molecular weight, amino acid composition, amino-terminal residue, heat stability, specific activity, pH optimum and substrate specificity regarding fatty acid, acyl acceptor and nucleoside 5'-triphosphate.
A second form (beta isoform) of rat phosphatidylinositol transfer protein cDNA was cloned by complementation of the yeast sec14 mutation from a rat brain cDNA expression library. The deduced sequence of the protein comprised 271 amino acids with a calculated molecular mass of 31,449 Da. The deduced amino acid sequence showed 77% identity to that of the rat phosphatidylinositol transfer protein, recently reported by Dickeson et al. [Dickeson, S.K., Lim, C.N., Schuyler, G.T., Dalton, T.P., Helmkamp, G.M., Jr., & Yarbrough, L.R. (1989) J. Biol. Chem. 264, 16557-16564]. Northern blot analysis revealed that the cDNA probe of the beta isoform hybridized to an approximately 3-kilobase RNA in various rat tissues. The mRNA was expressed abundantly in brain, kidney, liver, and lung, but in a lesser amount in testis.
The regulation of choline kinase (EC 2.7.1.32), the initial enzyme in the CDP-choline pathway, was examined in Saccharomyces cerevisiae. The addition of myo-inositol to a culture of wild-type cells resulted in a significant decrease in choline kinase activity. Additional supplementation of choline caused a further reduction in the activity. The coding frame of the choline kinase gene, CKI, was joined to the carboxyl terminus of lacZ and expressed in Escherichia coli as a fusion protein, which was then used to prepare an anti-choline kinase antibody. Upon Western (immuno-) and Northern (RNA) blot analyses using the antibody and a CKI probe, respectively, the decrease in the enzyme activity was found to be correlated with decreases in the enzyme amount and mRNA abundance. The molecular mass of the enzyme was estimated to be 66 kilodaltons, in agreement with the value predicted previously from the nucleotide sequence of the gene. The coding region of CKI was replaced with that of lacZ, and CKI expression was measured by assaying ,-galactosidase. The expression of j-galactosidase from this fusion was repressed by myo-inositol and choline and derepressed in a time-dependent manner upon their removal. The present findings indicate that yeast choline kinase is regulated by myo-inositol and choline at the level of mRNA abundance.The cooperation of myo-inositol (inositol) and choline in the control of phospholipid synthesis in the yeast Saccharomyces cerevisiae was first demonstrated for the enzymes in the phosphatidylethanolamine methylation pathway (38). Thereafter, a number of enzymes involved in the synthesis of phospholipids were shown to be subject to regulatory changes in response to inositol and choline. These changes include inhibition of phosphatidylserine synthase (20), induction of phosphatidate phosphatase (28), and inactivation or degradation of phosphatidylglycerophosphate synthase (10), but a more general response is the coordinate repression of phospholipid-synthesizing enzymes, such as CDPdiacylglycerol synthase (13,14), phosphatidylserine synthase (1,5,13,22,31), phosphatidylserine decarboxylase (6), phosphatidylethanolamine methyltransferase (22, 35-37), phospholipid methyltransferase (38), and inositol-1-phosphate synthase (7,8,12). The activities of these enzymes are considerably reduced when cells are cultured in the presence of inositol alone, but maximum reductions occur in the presence of both inositol and choline. The decreases in the activities have been correlated with decreases in the enzyme amounts in the cases of CDP-diacylglycerol synthase (13), phosphatidylserine synthase (13, 31), and inositol-1-phosphate synthase (8). Furthermore, in the case of phosphatidylserine synthase (1), inositol-1-phosphate synthase (12), phosphatidylethanolamine methyltransferase, and phospholipid methyltransferase (23; T. Kodaki, unpublished results), it has been shown that decreases in mRNA abundance are associated with changes in enzyme activities and amounts.In the present study, we investigated the effect...
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