Yeast Mutant with Thermolabile CDP‐choline Synthesis

Abstract

A temperature‐sensitive mutant of Saccharomyces cerevisiae with a lesion in cholinephosphate cytidyltransferase was isolated from a choline auxotroph. This mutant grew in a medium supplemented with ethanolamine or N‐methylethanolamine, but at a much slower rate in a medium supplemented with choline or N,N‐dimethylethanolamine at 37 °C. At 23 °C, mutant cells grew at the same rate in any of these media. Labelling experiments showed that the activity of phosphatidylcholine synthesis from choline in the mutant was thermolabile and rapidly lost on incubation of cells at 37 °C (half‐life, 4 min). Determination of the intermediates and the enzyme activities of the CDP‐choline pathway indicated that mutant cells had a lesion in cholinephosphate cytidyltransferase. Genetic analysis showed that the thermolabile phosphatidylcholine synthesis in the mutant arose from a single mutation in a gene coding for cholinephosphate cytidyltransferase. The mutant strain was found to be defective in dCDP‐choline synthesis. Thus, a single enzyme is responsible for the synthesis of both CDP‐choline and dCDP‐choline. Ethanolaminephosphate cytidyltransferase activity in the mutant was comparable to that in the parental strain. An alteration in cholinephosphate cytidyltransferase did not affect the incorporation of labelled ethanolamine into lipids. These results indicate that ethanolaminephosphate cytidyltranferase is coded for by a distinct gene from that for cholinephosphate cytidyltransferase. The CDP‐choline pathway was not required by cells as long as phosphatidylcholine was supplied via the phosphatidylethanolamine methylation pathway. The phosphatidylethanolamine methylation pathway and the CDP‐choline pathway were found to be complementary to each other in phosphatidylcholine synthesis.

Phosphorylation of CTP Synthetase on Ser36, Ser330, Ser354, and Ser454 Regulates the Levels of CTP and Phosphatidylcholine Synthesis in Saccharomyces cerevisiae

“…S330A, S354A, and S454A mutant CTP synthetase enzymes were within the range of the K m value (1.4 mM) of CTP for the phosphocholine cytidylyltransferase enzyme (84). Thus, phosphocholine cytidylyltransferase activity would be sensitive to the changes in CTP levels brought about by the mutations in CTP synthetase.…”

Isolation and characterization of a yeast mutant defective in cholinephosphotransferase

“…Clearly the last enzymes of the CDP-choline and CDP-ethanolamine pathways are encoded by distinct genes. Genetic evidence has also been presented that choline-phosphate cytidylyltransferase and ethanolamine-phosphate cytidylyltransferase are encoded by different genes in yeast [9]. Although yeast cells incorporate choline and ethanolamine through the same transport system [22], most of the steps, if not all, of the CDP-choline and CDP-ethanolamine pathways are encoded by different genes.…”

“…After washing three times with 5 mlO.145 M NaCl, the water-soluble metabolites were extracted and analyzed as described previously [9].…”

“…The extract was evaporated to dryness and then the radioactivity was counted in a toluene/Triton X-I 00 scintillant. Choline kinase [lo], choline-phosphate cytidylyltransferase [9], phosphatidylserine synthase [12], CDP-diacylgycerol-inositol 3-phosphatidyltransferase [14] and choline transport [8] were assayed as described previously.…”

CTP synthetase and its role in phospholipid synthesis in the yeast Saccharomyces cerevisiae

“…The stimulation of this activity is simply due to increased substrate availability of CTP [20,50]. In wild type cells, the apparent K m of CTP (1.4 mM) for the phosphocholine cytidylyltransferase [51] is 2-fold higher than the cellular concentration of CTP (0.7 mM) [20]. Thus, the activity of the cytidylyltransferase enzyme would be sensitive to the elevation of cellular CTP levels [20].…”