Isolation and characterization of the Saccharomyces cerevisiae EKI1 gene encoding ethanolamine kinase
Isolation and characterization of the Saccharomyces cerevisiae EKI1 gene encoding ethanolamine kinase
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DOI：
10.1074/jbc.274.21.14857
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发表时间：
1999-05-21
影响因子：
4.8
通讯作者：
Carman, GM
中科院分区：
文献类型：
--
作者：
Kim, KS;Kim, KH;Carman, GM
乙醇胺激酶（ATP：乙醇胺O-磷酸转移酶，EC 2.7.1.82）通过CDP-乙醇胺途径催化磷脂酰乙醇胺合成的关键步骤。编码乙醇胺激酶（EKI 1）的基因是从酵母基因组数据库（基因座YDR 147 W）中鉴定的，基于其与酿酒酵母CKI 1编码的胆碱激酶的同源性，后者也表现出乙醇胺激酶活性。分离EKI 1基因并用于构建eki 1 Delta和eki 1 Delta cki 1 Delta突变体。含有EKI 1基因的多拷贝质粒指导野生型、eki 1 Delta突变体、cki 1 Delta突变体和eki 1 Delta cki 1 Delta双突变体细胞中乙醇胺激酶活性的过表达。异源表达的S.酿酒酵母EKI 1基因在Sf-9昆虫细胞中的表达导致乙醇胺激酶活性相对于对照昆虫细胞的165，500倍过表达，EKI 1基因产物也表现出胆碱激酶活性。在昆虫细胞中表达的酶，在eki 1三角洲突变体，并在CKI 1三角洲突变体的生化分析表明，乙醇胺是优选的底物。eki 1 Delta突变体没有表现出生长表型。对eki 1 Delta、cki 1 Delta和eki 1 Delta cki 1 Delta突变体的生化分析表明，EKI 1和CKI 1基因产物编码酿酒酵母中所有的乙醇胺激酶和胆碱激酶活性。EKI 1基因产物主要负责通过CDP-乙醇胺途径合成磷脂酰乙醇胺，而CKI 1基因产物主要负责通过CDP-胆碱途径合成磷脂酰胆碱。与cki 1 Delta突变体不同，eki 1 Delta突变体不抑制Sec 14 p的基本功能。
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 eki1 Delta and eki1 Delta cki1 Delta mutants. A multicopy plasmid containing the EKI1 gene directed the overexpression of ethanolamine kinase activity in wild-type, eki1 Delta mutant, cki1 Delta mutant, and eki1 Delta cki1 Delta 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 eki1 Delta mutants, and in cki1 Delta mutants indicated that ethanolamine was the preferred substrate. The eki1 Delta mutant did not exhibit a growth phenotype, Biochemical analyses of eki1 Delta, cki1 Delta, and eki1 Delta cki1 Delta mutants showed that the EKI1 and CKI1 gene products encoded all of the ethanolamine kinase and choline kinase activities in S, cerevisiae, lit 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 cki1 Delta mutants, eki1 Delta mutants did not suppress the essential function of Sec14p.