Saito K, Nishijima M, Kuge O
Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162, Japan.
J Biol Chem. 1998 Jul 3;273(27):17199-205. doi: 10.1074/jbc.273.27.17199.
Phosphatidylserine (PS) in mammalian cells is synthesized through the exchange of free L-serine with the base moiety of phosphatidylcholine or phosphatidylethanolamine (PE). The serine base exchange in Chinese hamster ovary (CHO) cells is catalyzed by at least two enzymes, PS synthase (PSS) I and II. A PSS I-lacking mutant of CHO-K1 cells, PSA-3, which exhibits approximately 2-fold lower serine base exchange activity than CHO-K1, is defective in the conversion of phosphatidylcholine to PS but has the ability to convert PE to PS. The PSA-3 mutant requires exogenous PS or PE for cell growth. In the present study, from PSA-3 mutant cells, we isolated a mutant, named PSB-2, with a further decrease in the serine base exchange activity. The activity in the homogenate of PSB-2 mutant cells was approximately 10% that of PSA-3 mutant cells and approximately 5% that of CHO-K1 cells. The PSB-2 mutant exhibited an approximately 80% reduction in the PSS II mRNA level relative to that in PSA-3 mutant and CHO-K1 cells. These results showed that the PSB-2 mutant is defective in PSS II. Like the PSA-3 mutant, the PSB-2 mutant grew well in medium supplemented with PS. However, in the medium supplemented with PE, the PSB-2 mutant was incapable of growth, in contrast to the PSA-3 mutant. In the medium with exogenous PE, the PSB-2 mutant was defective in PS biosynthesis, whereas the PSA-3 mutant synthesized a normal amount of PS. A metabolic labeling experiment with exogenous [32P]PE revealed that the PSB-2 mutant was defective in the conversion of exogenous PE to PS. This defect and the growth and PS biosynthetic defects of the PSB-2 mutant cultivated with exogenous PE were complemented by the PSS II cDNA. In addition, the cDNA of the other PS synthase, PSS I, was shown not to complement the defect in the conversion of exogenous PE to PS of the PSB-2 mutant, implying that PSS I negligibly contributes to the conversion of PE to PS in CHO-K1 cells. These results indicated that PSS II is critical for the growth and PS biosynthesis of PSA-3 mutant cells cultivated with exogenous PE and suggested that most of the PS formation from PE in CHO-K1 cells is catalyzed by PSS II.
哺乳动物细胞中的磷脂酰丝氨酸（PS）是通过游离L-丝氨酸与磷脂酰胆碱或磷脂酰乙醇胺（PE）的碱基部分进行交换而合成的。中国仓鼠卵巢（CHO）细胞中的丝氨酸碱基交换由至少两种酶，即PS合酶（PSS）I和II催化。CHO-K1细胞的一个缺乏PSS I的突变体PSA-3，其丝氨酸碱基交换活性比CHO-K1细胞低约2倍，在磷脂酰胆碱向PS的转化中存在缺陷，但具有将PE转化为PS的能力。PSA-3突变体细胞生长需要外源性PS或PE。在本研究中，我们从PSA-3突变体细胞中分离出一个名为PSB-2的突变体，其丝氨酸碱基交换活性进一步降低。PSB-2突变体细胞匀浆中的活性约为PSA-3突变体细胞的10%，约为CHO-K1细胞的5%。相对于PSA-3突变体和CHO-K1细胞，PSB-2突变体的PSS II mRNA水平降低了约80%。这些结果表明PSB-2突变体在PSS II方面存在缺陷。与PSA-3突变体一样，PSB-2突变体在补充有PS的培养基中生长良好。然而，与PSA-3突变体相反，在补充有PE的培养基中，PSB-2突变体无法生长。在含有外源性PE的培养基中，PSB-2突变体在PS生物合成方面存在缺陷，而PSA-3突变体合成正常量的PS。用外源性[32P]PE进行的代谢标记实验表明，PSB-2突变体在外源性PE向PS的转化方面存在缺陷。PSS II cDNA弥补了这种缺陷以及用外源性PE培养的PSB-2突变体的生长和PS生物合成缺陷。此外，另一种PS合酶PSS I的cDNA不能弥补PSB-2突变体在外源性PE向PS转化方面的缺陷，这意味着PSS I对CHO-K1细胞中PE向PS的转化贡献可忽略不计。这些结果表明，PSS II对于用外源性PE培养的PSA-3突变体细胞的生长和PS生物合成至关重要，并表明CHO-K1细胞中大部分由PE形成PS的过程是由PSS II催化的。
