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

氨基磷脂合成在酵母内质网生物合成中的作用

• 批准号: 04660080
• 负责人: OHTA Akinori
• 金额: $ 1.28万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04660080/
• 关键词: Phosphatidylserine; Phosphatidylethanolamine; Phospholipid; Kennedy Pathway; Saccharomyces cerevisiae; Yeast; Biomembrane; Endoplasmic Reticulum; Saccharomyces cerevisiae

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.

二磷脂酰丝氨酸合成（PSS）的分析。磷脂酰丝氨酸合酶位于内质网（ER）中。为了研究其靶向ER并维持在其中的结构，（1）通过体外诱变方法传播了编码酶的CHO1基因的缺失突变体。 （2）还制备了针对PSS的兔抗血清。 （2）PSS蛋白被过量生产为谷胱甘肽S-转移酶（GST）-PSS融合蛋白在大肠杆菌中，然后亲和纯化以制备兔抗血清。这些材料对于阐明PSS组装到ER膜中的机制非常有用。II.CTP的分析：磷酸乙醇胺细胞丁基转移酶（ECT）（ECT）。在ECT活性中有缺陷并分离了DNA克隆以分离出突变的DNA克隆。对互补区域的3.2 kb DNA的测序分析显示，一个972 bp的开放式阅读框，推导的氨基酸序列与CTP具有显着相似之处：酵母和大鼠的磷胆碱细胞溶解酶，这是相似功能的酶。在大肠杆菌中以融合蛋白向GST表达的基因产物表现出ECT活性。我们得出的结论是，克隆的基因ECT1代码为酶ECT的结构亚基。

项目成果

Shioka Hamamatsu: ""Regulation of phospholipid synthesis in response to nutritional environments in mitochondria of Saccharomyces cerevisiae"" Biosci.Biotech.Biochem.57. 1849-1853 (1993)

Shioka Hamamatsu：“酿酒酵母线粒体中磷脂合成响应营养环境的调节”Biosci.Biotech.Biochem.57。

Rho,Min-Soek ら: "酵母のCDP-ethanolamineの合成に関わる遺伝子のクローン化とその構造の解析" 脂質生化学研究. 35. 278-281 (1993)

Rho, Min-Soek 等人：“酵母中 CDP-乙醇胺合成相关基因的克隆及其结构分析”Lipid Biochemistry Research 35. 278-281 (1993)。

Shioka Hamamatsu ら: "Regulation of Phospholipid Synthesis in Response to Nutritional Environments in Mitochondria of Saccharomyces cerevisiae" Bioscience,Biotechnology and Biochemistry. 57. 1849-1853 (1993)

Shioka Hamamatsu 等人：“酿酒酵母线粒体中磷脂合成响应营养环境的调节”《生物科学、生物技术和生物化学》57。1849-1853 (1993)。

Shioka Hamamatsu: "Regulation of Phospholipid Synthesis in Response to Nutritional Environments in Mitochendria of Saccharomyces cerevisiae" Bioscience,Biotechnology and Biochemistry. 57. 1849-1853 (1993)

Shioka Hamamatsu：“酿酒酵母线粒体中磷脂合成响应营养环境的调节”生物科学、生物技术和生物化学。

Rho,Min-Soek: "酵母のCDP-ethanolamineの合成に関わる遺伝子のクローン化とその構造の解析" 脂質生化学研究. 35. 278-281 (1993)

Rho, Min-Soek：“酵母中参与 CDP-乙醇胺合成的基因的克隆及其结构分析”脂质生物化学研究 35. 278-281 (1993)。