Characterization of the methyltransferases in the yeast phosphatidylethanolamine methylation pathway by selective gene disruption.
PEM1 and PEM2 are structural genes for the yeast phosphatidylethanolamine methylation pathway which mediates the three-step methylation of phosphatidylethanolamine to phosphatidylcholine. Selective disruption of each locus in the yeast genome was performed using the in-vitro-inactivated gene with insertion of yeast LEU2 or HIS3. Complementation test and spore analysis indicated that the disruptants were allelic with our previous mutants that were isolated by chemical mutagenesis and used for the cloning of PEM1 and PEM2. The methyltransferase activities of the disruptants were assayed using their membrane fractions. When the PEM1 locus was disrupted, the activity for the first methylation was greatly decreased but was still detectable, while the activities for the second and third methylations were well retained. The remaining three activities exhibited nearly identical pH optima and apparent Km values for S-adenosyl-L-methionine. The disruptant incorporated radioactivity from L-[methyl-14C]Met into phosphatidylcholine at a low but measurable rate and required choline for optimal growth. When choline was omitted from the culture medium, the phosphatidylcholine content of the cells significantly decreased, but was restored by the addition of N-monomethylethanolamine or choline. When the PEM2 locus was disrupted, the activities for the second and third methylations were totally lost, but that for the first methylation remained. This activity could be distinguished from those remaining in the pem1 disruptant by its different pH optimum and apparent Km for S-adenosyl-L-methionine. When incubated with [methyl-14C]Met, the pem2 disruptant accumulated the radioactivity in phosphatidylmonomethylethanolamine. This disruptant also required choline for optimal growth. In the absence of choline, it accumulated phosphatidylmonomethylethanolamine with a concomitant decrease in phosphatidylcholine and phosphatidylethanolamine. When both loci were disrupted, all phospholipid-methylating activities were lost and cells absolutely required choline for growth. The flux through the pathway became negligible. Thus, the PEM1-encoded methyltransferase was strictly specific to the first step while the PEM2-encoded methyltransferase exhibited a somewhat broader specificity with a preference for the second and third steps of the pathway. These two enzymes accounted for all the activities in the yeast phosphatidylethanolamine methylation pathway.
Kodaki
T
T
Department of Biochemistry, Gunma University School of Medicine, Maebashi, Japan.
Yamashita
S
S
D003001:Cloning, Molecular; D015723:Gene Library; D005800:Genes, Fungal / Q000502:physiology*; D005821:Genetic Techniques; D005838:Genotype; D007700:Kinetics; D008745:Methylation; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009710:Nucleotide Mapping; D010714:Phosphatidylethanolamines / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D010714:Phosphatidylethanolamines; D010743:Phospholipids; D008780:Methyltransferases