Role of Amino-phospholipid Synthesis in Biogenesis of Endoplasmic Reticulum of Yeast

1 .磷脂酰丝氨酸合成（PSS）分析。磷脂酰丝氨酸合成酶存在于内质网中。为了研究其靶向内质网并在内质网维持所必需的结构，(1)采用体外诱变方法扩增编码该酶的CHO1基因的缺失突变体。(2)制备了兔抗PSS血清。(2)在大肠杆菌中以谷胱甘肽s -转移酶(GST) - PSS融合蛋白的形式过量生产PSS蛋白，通过亲和纯化制备家兔抗血清。这些材料对阐明PSS在内质膜上的组装机制具有重要意义。二。磷酸乙醇胺胞基转移酶（ECT）的分析。分离出有ECT活性缺陷的突变体，并分离出与该突变互补的DNA克隆。对3.2 kb的互补区DNA进行测序分析，发现一个972 bp的开放阅读框，其中推断出的氨基酸序列与酵母和大鼠的CTP：磷酸胆碱胞基转移酶具有显著的相似性，这是一种功能相似的酶。该基因产物以GST融合蛋白的形式在大肠杆菌中表达，表现出ECT活性。我们得出结论，克隆基因ECT1编码酶ECT的一个结构亚基。

I.Analysis of phosphatidylserine synthesis (PSS).Phosphatidylserine synthase resides in endoplasmic reticulum (ER). To investigate its structure that is necessary to be targeted to ER and maintained there, (1) deletion mutants of the CHO1 gene that encodes the enzyme were propagated by in vitro mutagenic methods. (2) Rabit antiserum against PSS was also prepared. (2) PSS protein was overproduced as a glutathione S-transferase (GST) - PSS fusion protein in Escherichia coli, then affinity-purified to prepare rabit antiserum. These materials are very useful to clarify the mechanism of assembly of PSS into the ER membrane.II.Analysis of CTP : phosphoethanolamine cytidylyltransferase (ECT).Mutants defective in ECT activity were isolated and DNA clones that complemented the mutation were isolated. Sequencing analysis of 3.2 kb DNA of the complementary region revealed a 972 bp open reading frame of which deduced amino acid sequence had a significant similarity to CTP : phosphocholine cytidylyltransferases of yeast and rat, which are the enzymes of similar function. The gene product which was expressed in E.coli as a fusion protein to GST exhibited ECT activity. We concluded that the cloned gene ECT1 codes for a structural subunit of the enzyme ECT.