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.
Hjelmstad
R H
RH
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
Bell
R M
RM
D003001:Cloning, Molecular / Q000379:methods; D002798:Diacylglycerol Cholinephosphotransferase / Q000235:genetics / Q000378:metabolism; D004982:Ethanolaminephosphotransferase / Q000235:genetics* / Q000378:metabolism; D005796:Genes; D005800:Genes, Fungal; D005838:Genotype; D007700:Kinetics; D009154:Mutation; D010770:Phosphotransferases / Q000235:genetics*; D010957:Plasmids; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D013379:Substrate Specificity
D010770:Phosphotransferases; D004982:Ethanolaminephosphotransferase; D002798:Diacylglycerol Cholinephosphotransferase