Phosphatidylethanolamine Methylation Pathway Research Articles

2-hydroxyethylhydrazine as a potent inhibitor of phospholipid methylation in yeast

The effect of 2-hydroxyethylhydrazine on the phosphatidylethanolamine methylation pathway in yeast was studied. 2-Hydroxyethylhydrazine inhibited the growth of cells. The concentration required for 50% inhibition was 66 μM. The growth rate decreased by 2-hydroxyethylhydrazine was restored by the addition of a low concentration of choline. Incorporation of radioactivity from l-[3- 14C)serine, l-[ methyl- 14C]methionine and S- adenosyl- l-[ methyl- 14C] methionine into phosphatidylcholine was markedly reduced by 2-hydroxyethylhydrazine. The restoration of growth by choline was not due to the reversal of the inhibition, but to the formation of phosphatidylcholine via the CDPcholine pathway. Thus, the site of action of 2-hydroxyethylhydrazine in vivo was the phosphatidylethanolamine methylation pathway. Experiments with methylation mutants indicated that all three steps of methylation were sensitive to 2-hydroxyethylhydrazine. 2-Hydroxy-ethylhydrazine was shown to inhibit the methyltransferase after it had become chemically or metabolically transformed in cells. 2-Hydroxyethylhydrazine-resistant mutants were obtained and were found to have a defect in choline transport activity. Genetic data indicated that the uptake of 2-hydroxyethylhydrazin into cells is mediated by the choline transport system.

Yeast mutant with thermolabile CDP-choline synthesis. Isolation and characterization of a cholinephosphate cytidyltransferase mutant.

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.

Regulation of the phosphatidylethanolamine methylation pathway in Saccharomyces cerevisiae.

1. Phosphatidyl-N-methylethanolamine methyltransferase mutants of Saccharomyces cerevisiae were isolated. Genetic analysis showed that phosphatidylethanolamine methyltransferase and phosphatidyl-N-methylethanolamine methyltransferase are coded for by separate genes. Phosphatidyl-N-methylethanolamine methyltransferase activity and phosphatidyl-N,N-dimethylethanolamine methyltransferase activity appeared to be catalyzed by the same enzyme. 2. Phosphatidyl-N-methylethanolamine methyltransferase was found to be repressed by myo-inositol and choline. Both myo-inositol and choline at concentrations of 10 micrograms/ml were required for repression. The decreased enzyme level was restored by the removal of myo-inositol or choline or both. 3. Both myo-inositol and choline were required for the maximum repression of phosphatidylethanolamine methyltransferase in wild-type cells. In contrast, choline was not required for the repression of the enzyme in mutant strain 172. This was due to a single nuclear gene mutation in the genome of strain 172. 4. The activity of the phosphatidylethanolamine methylation pathway in cells decreased with time on incubation of cells with myo-inositol and choline, myo-Inositol could not be replaced by other structurally related compounds, such as scyllo-inositol or mannitol. 5. The physiological significance of the repression of the phosphatidylethanolamine methylation pathway is discussed with respect to the mechanism for maintaining the contents of phosphatidylethanolamine and phosphatidylcholine at normal levels.