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

酿酒葡萄球菌的uso1突变体在将蛋白质从内质网转运到高尔基体方面存在缺陷。克隆了野生型USO1基因，确定了5937 bp的核苷酸序列。通过基因破坏实验发现USO1基因对生长至关重要，并通过脉冲场凝胶电泳定位在第4号染色体上。最长的开放阅读框开始于ATG(273)，结束于TGA(5607)，编码1790个氨基酸的蛋白质。该蛋白整体亲水，没有任何长疏水延伸，在第468和1771个氨基酸上有两个酸性延伸。前者的酸性拉伸含有钙结合基序。蛋白质的c端应该形成一个螺旋状的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和1128-1790的杂交基因，并在大肠杆菌中表达。纯化融合蛋白后，用其免疫小鼠和家兔，获得抗usolp抗血清。当GAL7启动子高表达USO1时，兔抗血清检测到Usolp蛋白，而在野生型酵母或多拷贝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，northern blot才检测到USO1的mRNA，因此在酵母细胞中应该有非常少量的Uso1p存在并发挥作用。

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.