Edwards S R, Braley R, Chaffin W L
Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, Lubbock 79430, USA.
FEMS Microbiol Lett. 1999 Aug 15;177(2):211-6. doi: 10.1111/j.1574-6968.1999.tb13734.x.
Non-covalently attached or soluble cell wall proteins of Saccharomyces cerevisiae were extracted using a high pH/2-mercaptoethanol procedure and were separated for peptide sequencing using 2D-PAGE. A partial N-terminal sequence of a major protein spot was obtained and showed high identity with enolase gene products. Western blotting with an anti-enolase antibody confirmed that enolase was present in the cell wall extract. Biotinylation of cells prior to protein extraction with a membrane impermeable biotinylating agent confirmed that the detection was not owing to cell lysis during extraction. Transmission immunoelectron microscopy showed enolase to be present in the cell wall. Enolase contains no known secretion signal that would localize it to the cell wall. Thus S. cerevisiae must have further mechanisms for targeting proteins to the cell wall.
使用高pH/2-巯基乙醇法提取酿酒酵母非共价连接的或可溶性细胞壁蛋白，并用二维聚丙烯酰胺凝胶电泳（2D-PAGE）分离以进行肽测序。获得了一个主要蛋白斑点的部分N端序列，该序列与烯醇化酶基因产物具有高度同源性。用抗烯醇化酶抗体进行的蛋白质印迹证实细胞壁提取物中存在烯醇化酶。在用膜不透性生物素化剂进行蛋白质提取之前对细胞进行生物素化，证实检测结果并非由于提取过程中的细胞裂解。透射免疫电子显微镜显示烯醇化酶存在于细胞壁中。烯醇化酶不包含已知的可将其定位到细胞壁的分泌信号。因此，酿酒酵母必定具有将蛋白质靶向细胞壁的其他机制。
