Studies employing Saccharomyces cerevisiae cpt1 and ept1 null mutants implicate the CPT1 gene in coordinate regulation of phospholipid biosynthesis.

The Saccharomyces cerevisiae CPT1 and EPT1 genes are structural genes encoding sn-1,2-diacylglycerol choline phosphotransferase and sn-1,2-diacylglycerol choline/ethanolamine phosphotransferase, respectively. Incorporation of 32Pi into phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine in wild type and ept1 strains was decreased in the presence of exogenous inositol. In contrast, inositol did not affect 32Pi incorporation into phospholipid in cpt1 or cpt1ept1 strains. In membranes isolated from wild type and ept1 strains grown in the presence of inositol or inositol/choline, the CPT1-derived cholinephosphotransferase activities were reduced 40-50 and 65%, respectively. Inositol-dependent reductions in CPT1 derived choline-phosphotransferase activity correlated with transcript levels in both wild type and ept- backgrounds. The ethanolaminephosphotransferase activity of the EPT1 gene product in wild type cells was reduced 40% by exogenous inositol alone and 50% by inositol/choline. In the cpt1 strain, however, the ethanolaminephosphotransferase activity was unaffected by exogenous inositol or inositol/choline. The inositol-dependent reduction of ethanolaminephosphotransferase activity observed in wild type cells correlated with reduced levels of EPT1 transcripts; in the cpt1 strain, EPT1 transcript levels were not affected by inositol. These results indicate that 1) a functional CPT1 gene or gene product is required for inositol-dependent regulation of phospholipid synthesis; 2) the enzyme activities of both the CPT1 and EPT1 gene products are repressed by inositol and inositol/choline, and require an intact CPT1 gene; 3) inositol mediates its regulatory effects on phospholipid synthesis via a transcriptional mechanism.

Chimeric enzymes. Structure-function analysis of segments of sn-1,2-diacylglycerol choline- and ethanolaminephosphotransferases.

The Saccharomyces cerevisiae CPT1 and EPT1 genes represent structural genes that encode distinct choline- and choline/ethanolaminephosphotransferases, respectively. To explore the function of linear segments of these enzymes, a series of 14 EPT1-CPT1 chimeric gene constructs and the parental wild-type genes were expressed in a cpt1 ept1 double null mutant background completely devoid of phosphoamino alcohol transferase activity. Eleven of the chimeric genes expressed functional enzymes. The CDP-amino alcohol and sn-1,2-diacylglycerol (DAG) substrate specificities and essential phospholipid cofactor requirements of the parental and chimeric enzymes were investigated using a mixed micellar assay system. Chimeric enzymes exhibited a pattern of CDP-amino alcohol affinities that defined a structural domain sufficient to confer CDP-amino alcohol specificity. When wild-type enzymes were investigated using a chemically defined series of DAGs, each possessed a distinct characteristic pattern of utilization. Chimeric enzymes exhibited DAG acyl chain specificity profiles that either conformed to parental wild-type patterns or represented novel substrate specificities. Correlation of these outcomes with their underlying structural modifications permitted the assignment of an internal, linear region of 218 amino acids sufficient to confer DAG acyl chain specificity; this region contained three predicted transmembrane segments. Neither wild-type enzyme showed significant acyl chain selectivity with respect to phospholipid activation when a homologous series of chemically defined phosphatidylcholines were employed, suggesting that enzyme recognition of the fatty acyl moieties of the DAG substrate and phospholipid activator is fundamentally different. Analysis of chimeric enzymes dependence on phospholipid activators suggested the involvement of discontinuous protein segments participating in the interaction with phospholipid cofactors.

sn-1,2-diacylglycerol choline- and ethanolaminephosphotransferases in Saccharomyces cerevisiae. Mixed micellar analysis of the CPT1 and EPT1 gene products.

The Saccharomyces cerevisiae CPT1 and EPT1 genes are structural genes encoding distinct sn-1,2-diacylglycerol choline- and ethanolaminephosphotransferases. A haploid cpt1 ept1 double null mutant lacked detectable choline- and ethanolaminephosphotransferase activity but was viable for growth, establishing that these enzymes are nonessential. The activities of the CPT1 and EPT1 gene products were independently studied in membranes prepared from strains mutant in the cognate locus using mixed micellar assays. Both enzymes absolutely required phospholipid cofactors; half-maximal activation was observed at low mole fractions, suggesting that a small number of phospholipid molecules are required. The activities of the CPT1 and EPT1 gene products were compared with respect to dioleoylglycerol dependence, CDP-aminoalcohol specificity, phospholipid activation, and inhibition by CMP. The EPT1 gene product utilized CDP-ethanolamine, -monomethylethanolamine, -dimethylethanolamine, and -choline to significant extents, while the CPT1 gene product manifested relative specificity for CDP-choline and -dimethylethanolamine. The CPT1 and EPT1 gene products exhibited differing properties with respect to phospholipid activation, but this difference was dependent on the CDP-aminoalcohol substrate. In contrast, the two enzymes could be distinguished on the basis of their dioleoylglycerol dependencies, activation by Mg2+, and CMP inhibition profiles regardless of the CDP-aminoalcohol substrate employed. These studies provide the first definitive kinetic properties of individual choline- and ethanolaminephosphotransferases.

sn-1,2-diacylglycerol choline- and ethanolaminephosphotransferases in Saccharomyces cerevisiae. Nucleotide sequence of the EPT1 gene and comparison of the CPT1 and EPT1 gene products.

The complete nucleotide sequence of the Saccharomyces cerevisiae EPT1 gene, a structural gene encoding an sn-1,2-diacylglycerol ethanolamine- and cholinephosphotransferase (Hjelmstad, R. H., and Bell, R. M. (1988) J. Biol. Chem. 263, 19748-19757), was determined. The 2123-nucleotide extent of DNA sequenced contained an open reading frame encoding 391 amino acids interrupted by an intron near its 5' end. Northern hybridization analysis detected a single 1.4-kilobase transcript. The inferred 44,525-dalton EPT1 gene product exhibited 54% amino acid sequence homology to the cholinephosphotransferase product of the yeast CPT1 gene. Predictive structural analysis of the EPT1 gene product revealed close structural similarity to the CPT1 gene product with respect to membrane topography, features of secondary structure, and transmembrane asymmetry. Regional protein homologies were identified between the EPT1 gene product and several enzymes as well as the nicotinic acetylcholine receptor. Comparative analysis of this set of protein homologies and the related set of protein homologies to the CPT1 gene product permitted identification of a presumptive active site region which contains highly conserved and divergent subregions and a common mononucleotide binding site.

The sn-1,2-diacylglycerol cholinephosphotransferase of Saccharomyces cerevisiae. Nucleotide sequence, transcriptional mapping, and gene product analysis of the CPT1 gene.

The complete nucleotide sequence of the Saccharomyces cerevisiae CPT1 gene, a structural gene for the sn-1,2-diacylglycerol cholinephosphotransferase (Hjelmstad, R. H., and Bell, R. M. (1987) J. Biol. Chem. 262, 3909-3917), was determined. The 2,100-nucleotide extent of DNA sequenced contained an open reading frame encoding 407 amino acids interrupted by an intron near its 5'-end. Northern hybridization analysis detected the presence of 1.4- and 1.7-kilobase transcripts corresponding to the CPT1 gene. S1 nuclease mapping experiments indicated that the 1.4-kilobase transcript was initiated 80 nucleotides upstream from the translational start site near a poly(dA-dT) promoter element and established that the predicted intron was removed in vivo. The previously constructed cpt1::LEU2 insertional mutation was shown to involve disruption of the CPT1 open reading frame approximately in the middle; this construct did not support the production of a stable transcript. The CPT1 promoter region contained several elements homologous to the promoter regions of other phospholipid biosynthetic structural genes. A model for the membrane topography of the predicted 46,305-dalton cholinephosphotransferase was constructed on the basis of predictive methods. The presence of seven transmembrane helices and an asymmetric distribution of hydrophilic regions were predicted. Regional protein homologies to the acetylcholine receptor, phosphoglycerate kinase, and several cytidine diphosphate utilizing enzymes suggested a functional asymmetry which precisely correlated with the predicted topological asymmetry.

The sn-1,2-diacylglycerol ethanolaminephosphotransferase activity of Saccharomyces cerevisiae. Isolation of mutants and cloning of the EPT1 gene.

A colony autoradiographic assay was used to identify nine Saccharomyces cerevisiae mutants defective in in situ ethanolaminephosphotransferase activity (ept mutants). Genetic analysis revealed five complementation groups. The EPT1 gene was cloned by complementation of ept1 using a yeast genomic library and was localized to a 2.1-kilobase region of DNA. An ept1 deletional mutant was constructed and introduced into the chromosome by integrative transformation. The ethanolaminephosphotransferase activities in membranes prepared from ept1 and ept2 mutants were reduced 30- to 90-fold and 2- to 3-fold compared with wild-type activity, respectively; the other ept mutants had activities similar to wild type. In strains transformed with a multicopy EPT1-bearing plasmid, a 22- to 33-fold overproduction of ethanolaminephosphotransferase activity was observed. The sn-1,2-diacylglycerol cholinephosphotransferase activities in membranes prepared from ept1 mutants were reduced 3.5- to 7-fold. In contrast to the residual CMP-sensitive cholinephosphotransferase activity observed in cpt1 mutants (Hjelmstad, R. H., and Bell, R. M. (1987) J. Biol. Chem. 262, 3909-3917), the residual cholinephosphotransferase activity of ept1 mutants was CMP-insensitive. The cholinephosphotransferase activities in strains bearing the EPT1 gene on multicopy plasmids were elevated 13- to 23-fold and were CMP-sensitive. The data indicate that 1) the cloned EPT1 gene most likely represents the structural gene for the yeast ethanolaminephosphotransferase, 2) the EPT1 gene product possesses both ethanolamine- and cholinephosphotransferase activities, and 3) the EPT1 gene is nonessential for growth.

Mutants of Saccharomyces cerevisiae defective in sn-1,2-diacylglycerol cholinephosphotransferase. Isolation, characterization, and cloning of the CPT1 gene.

A colony autoradiographic assay for the sn-1,2-diacylglycerol cholinephosphotransferase activity in Saccharomyces cerevisiae was developed. Twenty-two mutants defective in cholinephosphotransferase activity were isolated. Genetic analysis revealed that all of these mutations were recessive, and three complementation groups were identified. The cholinephosphotransferase activities in membranes prepared from cpt1 mutants were reduced 2-10-fold compared to wild-type activity. The cholinephosphotransferase activities of two cpt1 isolates differed from wild-type activity with respect to their apparent KM for CDP-choline. The residual cholinephosphotransferase activities of cpt1 isolates were more sensitive to inhibition by CMP than the wild-type activity. The CPT1 gene was cloned by genetic complementation of cpt1 using a yeast genomic library. In strains transformed with the CPT1-bearing plasmid, a 5-fold overproduction of cholinephosphotransferase activity with wild-type kinetic properties was observed. The CPT1 gene was localized to a 1.2-2.4-kilobase region of DNA by transposon Tn5 mutagenesis and deletion mapping. An insertional mutant of the CPT1 gene was constructed and introduced into the chromosome by integrative transformation. The resulting cpt insertional mutant fell into the cpt1 complementation group. The cholinephosphotransferase activity in membranes prepared from the cpt1 insertional mutant was reduced 5-fold and exhibited CMP sensitivity. The sn-1,2-diacylglycerol ethanolaminephosphotransferase activities in membranes from all of the cpt1 isolates including the insertional mutant were normal. The data indicate that the cloned CPT1 gene represents the yeast cholinephosphotransferase structural gene, that the yeast choline- and ethanolaminephosphotransferase activities are encoded by different genes, and that the CPT1 gene is nonessential for growth.