Abstract
Ethanolamine kinase (ATP:ethanolamine O-phosphotransferase, EC 2.7.1. 82) catalyzes the committed step of phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway. The gene encoding ethanolamine kinase (EKI1) was identified from the Saccharomyces Genome Data Base (locus YDR147W) based on its homology to the Saccharomyces cerevisiae CKI1-encoded choline kinase, which also exhibits ethanolamine kinase activity. The EKI1 gene was isolated and used to construct eki1Delta and eki1Delta cki1Delta mutants. A multicopy plasmid containing the EKI1 gene directed the overexpression of ethanolamine kinase activity in wild-type, eki1Delta mutant, cki1Delta mutant, and eki1Delta cki1Delta double mutant cells. The heterologous expression of the S. cerevisiae EKI1 gene in Sf-9 insect cells resulted in a 165,500-fold overexpression of ethanolamine kinase activity relative to control insect cells. The EKI1 gene product also exhibited choline kinase activity. Biochemical analyses of the enzyme expressed in insect cells, in eki1Delta mutants, and in cki1Delta mutants indicated that ethanolamine was the preferred substrate. The eki1Delta mutant did not exhibit a growth phenotype. Biochemical analyses of eki1Delta, cki1Delta, and eki1Delta cki1Delta mutants showed that the EKI1 and CKI1 gene products encoded all of the ethanolamine kinase and choline kinase activities in S. cerevisiae. In vivo labeling experiments showed that the EKI1 and CKI1 gene products had overlapping functions with respect to phospholipid synthesis. Whereas the EKI1 gene product was primarily responsible for phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway, the CKI1 gene product was primarily responsible for phosphatidylcholine synthesis via the CDP-choline pathway. Unlike cki1Delta mutants, eki1Delta mutants did not suppress the essential function of Sec14p.
Highlights
The major membrane phospholipids in the yeast Saccharomyces cerevisiae are PC,1 PE, PI, and PS [1, 2]
EKI1 Gene Encoding Ethanolamine Kinase from S. cerevisiae ing the enzymes catalyzing the last two steps in the CDPethanolamine pathway have been isolated and characterized [17, 18], little is known about the enzyme ethanolamine kinase (ATP:ethanolamine O-phosphotransferase, EC 2.7.1.82) that catalyzes the committed step in this pathway
Isolation of the S. cerevisiae EKI1 Gene Encoding Ethanolamine Kinase and the Deduced Primary Structure of Its Encoded Protein—The EKI1 gene was identified in the Saccharomyces Genome Data Base on the basis that its deduced amino acid sequence showed 40% sequence identity to the C-terminal amino acid sequence of the CKI1-encoded choline kinase of S. cerevisiae [45, 46] (Fig. 2)
Summary
The major membrane phospholipids in the yeast Saccharomyces cerevisiae are PC, PE, PI, and PS [1, 2]. The activation of the CDP-choline pathway, due to the unregulated synthesis of CTP, results in significant increases in the synthesis of PC and PA and a decrease in the synthesis of PS [14]. These changes are accompanied by an increase in total neutral lipid content at the expense of total phospholipids [14]. The role of the CDP-ethanolamine pathway in phospholipid metabolism and cell physiology has not been studied as extensively as the CDP-choline pathway. Eki1⌬ mutants did not suppress the essential function of Sec14p
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