Metabolism, regulation and function of phosphatidylserine in mammalian cells.

(1)在哺乳动物细胞中，磷脂酰丝氨酸的合成是通过L-丝氨酸与磷脂酰胆碱和磷脂酰乙醇胺的碱基交换而发生的，该反应分别由PSS(磷脂酰丝氨酸合成酶)1和2催化。外源PtdSer抑制完整细胞和分离的膜组分合成PtdSer，提示反馈调控参与了PtdSer生物合成的调控。PSS1和PSS2的氨基酸序列相似，同源性为32%；但由于与其他已知的酶缺乏同源性，它们的氨基酸序列没有提供关于它们的催化和调节机制的信息。在本研究中，为了确定PSS-1活性和/或调控的关键氨基酸残基，我们系统地将突变引入中国仓鼠PSS-1的cDNA克隆中，即PSS-2共有的66个极性氨基酸残基中的每一个都被一个丙氨酸残基取代。中国仓鼠卵巢细胞的…分析进一步对每个丙氨酸突变克隆进行比较，我们鉴定了8个氨基酸残基(His-172、Glu-197、Glu-200、Asn-209、Glu-212、Asp-216、Asp-221和Asn-226)对于酶反应或维持丝氨酸碱基交换活性所需的正确结构至关重要。在这些残基中，Asn-209可能参与了游离L丝氨酸的识别和/或结合。我们还鉴定了6个氨基酸残基(Arg-95、His-97、Cys-189、Arg-262、Gln-266和Arg-336)对PSS1的调控。此外，我们还发现Tyr-111、Asp-166、Arg-184、Arg-323和Glu-364处的丙氨酸突变影响了中国仓鼠卵巢细胞PSS1的合成和/或稳定性。(2)Sindbis病毒复制子介导的基因表达涉及病毒复制酶在细胞膜上合成RNA。在这里，我们利用磷脂酰丝氨酸生物合成缺陷的中国仓鼠卵巢细胞突变株，研究了膜脂磷脂酰丝氨酸在复制子介导的基因表达中的作用。当将携带病毒复制酶基因的复制子RNA和亚基因组启动子驱动的LacZ基因导入突变细胞时，在磷脂酰丝氨酸缺乏的条件下，复制子亚基因组RNA的β-半乳糖苷酶的表达被抑制。相反，复制子基因组RNA中复制酶蛋白nsP1的表达和亚基因组RNA的积累并未受到磷脂酰丝氨酸缺陷的抑制，表明报告表达的抑制不是由于复制酶的生产和功能缺陷造成的。在相似的条件下，来自SV40启动子驱动的构建体的报告表达被发现是正常的，这意味着磷脂酰丝氨酸缺乏不影响一般翻译和蛋白质降解。这些结果表明，磷脂酰丝氨酸特异性地参与了Sindbis病毒亚基因组启动子驱动的基因表达的转录后事件。较少

(1)PtdSer (phosphatidylserine) synthesis in mammalian cells occurs through the exchange of L-serine with the base moieties of phosphatidylcholine and phosphatidylethanolamine, which is catalysed by PSS (PtdSer synthase) 1 and 2 respectively. PtdSer synthesis in intact cells and an isolated membrane fraction was inhibited by exogenous PtdSer, indicating that feedback control is involved in the regulation of PtdSer biosynthesis. PSS 1 and 2 are similar in amino acid sequence, with an identity of 32%; however, due to a lack of homology with other known enzymes, their amino acid sequences do not provide information on their catalytic and regulatory mechanisms. In the present study, to identify amino acid residues crucial for the activity and/or regulation of PSS 1, we systematically introduced mutations into a Chinese hamster PSS 1 cDNA clone ; namely, each of the 66 polar amino acid residues common to PSS 2 was replaced with an alanine residue. On analysis of Chinese hamster ovary cells t … More ransfected with each of the alanine mutant clones, we identified eight amino acid residues (His-172, Glu-197, Glu-200, Asn-209, Glu-212, Asp-216, Asp-221 and Asn-226) as those crucial for the enzyme reaction or the maintenance of the correct structure required for serine base-exchange activity. Among these residues, Asn-209 was suggested to be involved in the recognition and/or binding of free L-serine. We also identified six amino acid residues (Arg-95, His-97, Cys-189, Arg-262, Gln-266 and Arg-336) as those important for regulation of PSS 1. In addition, we found that the alanine mutations at Tyr-111, Asp-166, Arg-184, Arg-323, and Glu-364 affected the production and/or stability of PSS 1 in Chinese hamster ovary cells.(2)Sindbis virus replicon-mediated gene expression involves RNA synthesis by viral replicase on cytoplasmic membranes. Here we examined the involvement of a membrane lipid, phosphatidylserine, in the replicon-mediated gene expression by using Chinese hamster ovary cell mutants defective in phosphatidylserine biosynthesis. When the mutant cells were transfected with a replicon RNA carrying a viral replicase gene followed by a subgenomic promoter-driven lacZ gene, expression of β-galactosidase from the replicon subgenomic RNA was inhibited under phosphatidylserine-deficient conditions. In contrast, expression of a replicase protein, nsP1, from the replicon genomic RNA and accumulation of the subgenomic RNA were not inhibited by the phosphatidylserine deficiency, indicating that inhibition of the reporter expression was not due to defects in production and function of the replicase. The reporter expression from an SV40 promoter-driven construct was found to be normal under similar conditions, implying the phosphatidylserine deficiency does not affect general translation and protein degradation. These results indicate that phosphatidylserine is specifically involved in a posttranscriptional event of Sindbis virus subgenomic promoter-driven gene expression. Less