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.

磷脂酰丝氨酸合成（PSS）的分析磷脂酰丝氨酸合成酶位于内质网（ER）中。为了研究其靶向ER并维持在那里所必需的结构，（1）通过体外诱变方法繁殖编码该酶的CHO 1基因的缺失突变体。(2)制备了兔抗PSS抗血清。(2)在大肠杆菌中高效表达谷胱甘肽S-转移酶（GST）- PSS融合蛋白，并对其进行亲和纯化，制备兔抗血清。这些材料是非常有用的，以澄清的机制组装PSS到ER membranes.II.分析CTP：磷酸乙醇胺胞苷酰转移酶（ECT）。突变体缺陷ECT活性被分离和DNA克隆，补充突变被分离。对3.2kb的DNA进行序列分析，发现其开放阅读框为972 bp，其推导的氨基酸序列与酵母和大鼠的CTP：磷酸胆碱胞苷酰转移酶有很高的相似性，是一种功能相似的酶。在大肠杆菌中表达的融合蛋白具有ECT活性。我们的结论是，克隆的基因ECT 1编码酶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)。