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Study on Protein Secretion of Yeast by a Temperature-sensitive Mutant

温度敏感突变体酵母蛋白质分泌的研究

基本信息

批准号：
01560115
负责人：
YODA Koji
金额：
$ 1.09万
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：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01560115/
关键词：
Yeast Secretion Temperature-sensitive Cloning Golgi body Endoplasmic reticulum USO1 Nucleotide sequence フロ-ニング

项目摘要

The uso1 mutant of S. cerevisiae has a defect in the transport of protein from the ER to the Golgi. We cloned the wild-type USO1 gene and determined the nucleotide sequence of 5937 bp. The USO1 gene was found to be essential for growth by gene disruption experiments and was located on the chromosome IV by pulse-field gel electrophoresis. The longest open reading frame starts at ATG (273) and ends at TGA (5607) and codes a protein of 1790 amino acids. This protein is hydrophilic as a whole without any long hydrophobic stretches and has two acidic stretches at 468th and 1771st amino acid. The former acidic stretch contains a calcium-binding motif. The C-terminal half of the protein should form a coiled-coil alpha-helix and the protein should exist as a dimer. We constructed hybrid genes containing codons 36-571 or 1128-1790 of Usolp and expressed them in E. coli. After purifying the fusion roteins, we immunized mice and rabbits with them and obtained anti-Usolp antiserums. The Usolp protein was detected by the rabbit antiserum when USO1 was highly expressed by the GAL7 promoter but not in the wild-type yeasts or yeasts with multicopy USO1 gene. As the mRNA of USO1 was also detected by Nothern blots only when USO1 was expressed by the GAL7 promoter, very small amount of Uso1p should exist and function in yeast cell.

酿酒酵母uso1突变体在蛋白质从内质网到高尔基体的运输过程中存在缺陷。我们克隆了野生型USO1基因，并测定了其5937bp的核苷酸序列。通过基因阻断实验发现USO1基因是生长所必需的，并通过脉冲场凝胶电泳法将其定位在第四染色体上。最长的开放阅读框起始于ATG(273)，终止于TGA(5607)，编码1790个氨基酸的蛋白质。该蛋白整体具有亲水性，没有任何长的疏水伸展，在第468位和1771位氨基酸有两个酸性伸展。先前的酸性拉伸包含一个钙结合基序。蛋白质的C末端的一半应该形成卷曲的α-螺旋，蛋白质应该以二聚体的形式存在。我们构建了含有Usolp密码子36-571或1128-1790的杂交基因，并在大肠杆菌中进行了表达。纯化融合蛋白后，免疫小鼠和兔，获得抗Usolp抗血清。当USO1被GAL7启动子高效表达时，用兔抗血清检测到Usolp蛋白，而在野生型和多拷贝USO1基因酵母中检测不到Usolp蛋白。由于只有当USO1被GAL7启动子表达时，Northerblotts才能检测到USO1的mRNA，因此在酵母细胞中应该存在极少量的Uso1p并发挥作用。