The isolation and characterization in yeast of a gene for Arabidopsis S-adenosylmethionine:phospho-ethanolamine N-methyltransferase.

Saccharomyces cerevisiae opi3 mutant strains do not have the phospholipid N-methyltransferase that catalyzes the two terminal methylations in the phosphatidylcholine (PC) biosynthetic pathway. This results in a build up of the intermediate phosphatidylmonomethylethanolamine, causing a temperature-sensitive growth phenotype. An Arabidopsis cDNA library was used to isolate three overlapping plasmids that complemented the temperature-sensitive phenotype. Phospholipid analysis showed that the presence of the cloned cDNA caused a 65-fold reduction in the level of phosphatidylmonomethylethanolamine and a significant, though not equivalent, increase in the production of PC. Sequence analysis established that the cDNA was not homologous to OPI3 or to CHO2, the only other yeast phospholipid N-methyltransferase, but was similar to several other classes of methyltransferases. S-adenosyl-Met:phospho-base N-methyltransferase assays revealed that the cDNA catalyzed the three sequential methylations of phospho-ethanolamine to form phospho-choline. Phospho-choline is converted to PC by the CDP-choline pathway, explaining the phenotype conferred upon the yeast mutant strain by the cDNA. In accordance with this the gene has been named AtNMT1. The identification of this enzyme and the failure to isolate a plant phospholipid N-methyltransferase suggests that there are fundamental differences between the pathways utilized by yeast and by some plants for synthesis of PC.

Regulation of inositol transport in Saccharomyces cerevisiae involves inositol-induced changes in permease stability and endocytic degradation in the vacuole.

Uptake of inositol by Saccharomyces cerevisiae is mediated by a specific inositol permease encoded by the ITR1 gene. Removal of inositol from the growth medium results in an increase in ITR1 mRNA abundance. The increase in ITR1 mRNA is accompanied by an increase in de novo synthesis of the Itr1 permease leading to an increased capacity for uptake. When inositol is added to the growth medium inactivation of uptake activity occurs, and both transcription of ITR1 and uptake activity are repressed to a basal level of function. The transcriptional regulation of ITR1 depends on the INO2, INO4, and OPI1 genes. In addition, repression is also achieved by regulation of ITR1 expression at the post-translational level. In this study, we show that there is a change in the stability of the Itr1 permease after the addition of inositol to the growth medium. Immunoblot analysis using a monoclonal antibody against an epitope attached to the Itr1 permease showed that the addition of inositol causes a dramatic increase in the rate of degradation of the permease. After the repressed (basal) level is achieved, turnover continues to be rapid. The increased rate of degradation was also observed in strains with mutations that block conjugation to ubiquitin. Degradation was not observed in strains defective in the END3/END4 endocytic pathway or in the production of vacuolar proteases (PEP4). Thus, inactivation of the Itr1 permease is accompanied by endocytic internalization followed by degradation in the vacuole. Inactivation may be a separate process that precedes and signals endocytic degradation. Since the end3/end4 mutations did not affect uptake activity under derepressed conditions, endocytosis is not required for normal inositol uptake.