Differential biochemical regulation of the URA7- and URA8-encoded CTP synthetases from Saccharomyces cerevisiae.

The URA7- and URA8-encoded CTP synthetases (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming) are functionally overlapping enzymes responsible for the biosynthesis of CTP in the yeast Saccharomyces cerevisiae. URA8-encoded CTP synthetase was purified to apparent homogeneity by ammonium sulfate fractionation of the cytosolic fraction followed by chromatography with Q-Sepharose, Affi-Gel Blue, Mono Q, and Superose 6. The subunit molecular mass (67 kDa) of purified URA8-encoded CTP synthetase was in good agreement with the predicted size of the URA8 gene product. Antibodies raised against a fusion protein constructed from the coding sequences of the URA8 gene and expressed in Escherichia coli reacted with purified URA8-encoded CTP synthetase. Native URA8-encoded CTP synthetase existed as a dimer which oligomerized to a tetramer in the presence of its substrates UTP and ATP. Maximum URA8-encoded CTP synthetase activity was dependent on Mg2+ ions (Ka = 2.4 mM) and 2-mercaptoethanol at the pH optimum of 7.5. The enzyme followed saturation kinetics toward UTP (Km = 74 microM), ATP (Km = 22 microM), and glutamine (Km = 0.14 mM). GTP stimulated (Ka = 26 microM) URA8-encoded CTP synthetase activity 12-fold. CTP potently inhibited (IC50 = 85 microM) URA8-encoded CTP synthetase activity and, in addition, caused the dependence of activity toward UTP to become cooperative. The URA8-encoded CTP synthetase and the previously purified URA7-encoded CTP synthetase differed significantly with respect to several biochemical properties including turnover number, pH optimum, substrate dependences, and sensitivity to inhibition by CTP. The URA7-encoded CTP synthetase mRNA was 2-fold more abundant when compared with URA8-encoded CTP synthetase mRNA. Both CTP synthetase isoforms were maximally expressed in the exponential phase of growth.

Specificity of unsaturated fatty acid-regulated expression of the Saccharomyces cerevisiae OLE1 gene.

The Saccharomyces cerevisiae OLE1 gene encodes the delta-9 fatty acid desaturase, an enzyme which forms the monounsaturated palmitoleic (16:1) and oleic (18:1) fatty acids from palmitoyl (16:0) or stearoyl (18:0) CoA. Previous studies demonstrated that OLE1 mRNA levels and desaturase enzyme activity are repressed when either 16:1 delta-9 and 18:1 delta-9 are added to the growth medium (1). The polyunsaturate, linoleic acid (18:2, delta-9,12), which is not a product of the enzyme, is also a strong repressor. The specificity of the OLE1 transcriptional regulatory sensor was examined by testing the response of OLE1 promoter-lacZ fusion reporter genes to fatty acids that differ in chain length, degree of unsaturation and double bond positions. Monounsaturated and polyunsaturated fatty acids that contain a delta-9 double bond are strong repressors of reporter gene activity and native OLE1 mRNA levels. Monounsaturated fatty acids containing double bonds in the delta-10, delta-11, or delta-5 positions showed no repression of reporter enzyme levels although they were rapidly incorporated into membrane lipids and some supported growth of an OLE1 gene disrupted strain. Although 17:1 delta-10 does not repress OLE1 transcription, lipid analysis showed that it replaces almost all of the endogenous 16:1 delta-9 and 18:1 delta-9 in cellular lipids and OLE1 mRNA levels are strongly repressed. This suggests that additional systems regulate desaturase activity by post-transcriptional mechanisms that differ from the transcriptional sensor in their responses to specific fatty acids.

The OLE1 gene of Saccharomyces cerevisiae encodes the delta 9 fatty acid desaturase and can be functionally replaced by the rat stearoyl-CoA desaturase gene.

Strains of Saccharomyces cerevisiae bearing the ole1 mutation are defective in unsaturated fatty acid (UFA) synthesis and require UFAs for growth. A previously isolated yeast genomic fragment complementing the ole1 mutation has been sequenced and determined to encode the delta 9 fatty acid desaturase enzyme by comparison of primary amino acid sequence to the rat liver stearoyl-CoA desaturase. The OLE1 structural gene encodes a protein of 510 amino acids (251 hydrophobic) having an approximate molecular mass of 57.4 kDa. A 257-amino acid internal region of the yeast open reading frame aligns with and shows 36% identity and 60% similarity to the rat liver stearoyl-CoA desaturase protein. This comparison disclosed three short regions of high consecutive amino acid identity (greater than 70%) including one 11 of 12 perfect residue match. The predicted yeast enzyme contains at least four potential membrane-spanning regions and several shorter hydrophobic regions that align exactly with similar sequences in the rat liver protein. An ole1 gene-disrupted yeast strain was transformed with a yeast-rat chimeric gene consisting of the promoter region and N-terminal 27 codons of OLE1 fused to the rat desaturase coding sequence. Fusion gene transformants displayed near equivalent growth rates and modest lipid composition changes relative to wild type yeast control implying a significant conservation of delta 9 desaturase tertiary structure and efficient interaction between the rat desaturase and yeast cytochrome b5.

Isolation and characterization of OLE1, a gene affecting fatty acid desaturation from Saccharomyces cerevisiae.

The unsaturated fatty acid (ufa) requiring ole1 mutant of Saccharomyces cerevisiae appears to produce a defective delta-9 fatty acid desaturase. This enzyme catalyzes double bond formation between carbons 9 and 10 of palmitoyl and stearoyl coenzyme A. A DNA fragment isolated by complementation of an ole1 strain repairs the ufa requirement in mutant cells. Genetic analysis of the cloned DNA fragment indicates that it is allelic to the OLE1 gene. Disruption of a single copy of the wild type gene in a diploid strain produces both wild type and nonreverting ufa-requiring haploid progeny upon sporulation. Membrane lipids of the disrupted haploid strains contain only ufas supplied in the growth medium. The recovery of activity in both wild type and disrupted segregants was examined after removal of ufas from the growth medium. Following ufa deprivation disruptant cells grew normally for about three generations and then at a slower rate for at least 0.6 generations. During that time cellular ufas dropped from 63 to 7.3 mol % of the total fatty acids. No production of the 16:1 and 18:1 products of the desaturase was observed in disruptant cells, whereas desaturation in wild type control cells was evident 2 h after deprivation. These results indicate that 1) the OLE1 gene is essential for production of monounsaturated fatty acids and is probably the structural gene for the delta-9 desaturase enzyme. 2) A large part of membrane ufas present under normal culture conditions are not essential for growth and cell division.