Nakamata Kosuke, Kurita Tomokazu, Bhuiyan M Shah Alam, Sato Keisuke, Noda Yoichi, Yoda Koji
Department of Biotechnology, University of Tokyo, Yayoi, Bunkyo-Ku, Tokyo, Japan.
J Biol Chem. 2007 Nov 23;282(47):34315-24. doi: 10.1074/jbc.M706486200. Epub 2007 Sep 24.
KEG1/YFR042w of Saccharomyces cerevisiae is an essential gene that encodes a 200-amino acid polypeptide with four predicted transmembrane domains. The green fluorescent protein- or Myc(6)-tagged Keg1 protein showed the typical characteristics of an integral membrane protein and was found in the endoplasmic reticulum by fluorescence imaging. Immunoprecipitation from the Triton X-100-solubilized cell lysate revealed that Keg1 binds to Kre6, which has been known to participate in beta-1,6-glucan synthesis. To analyze the essential function of Keg1 in more detail, we constructed temperature-sensitive mutant alleles by error-prone polymerase chain reaction. The keg1-1 mutant cells showed a common phenotype with Deltakre6 mutant including hypersensitivity to Calcofluor white, reduced sensitivity to the K1 killer toxin, and reduced content of beta-1,6-glucan in the cell wall. These results suggest that Keg1 and Kre6 have a cooperative role in beta-1,6-glucan synthesis in S. cerevisiae.
酿酒酵母的KEG1/YFR042w是一个必需基因，它编码一个含有四个预测跨膜结构域的200个氨基酸的多肽。绿色荧光蛋白或Myc(6)标记的Keg1蛋白表现出整合膜蛋白的典型特征，通过荧光成像发现其存在于内质网中。从经Triton X-100溶解的细胞裂解物中进行免疫沉淀显示，Keg1与Kre6结合，已知Kre6参与β-1,6-葡聚糖的合成。为了更详细地分析Keg1的必需功能，我们通过易错聚合酶链反应构建了温度敏感突变等位基因。keg1-1突变细胞表现出与Δkre6突变体相同的表型，包括对荧光增白剂超敏、对K1杀伤毒素敏感性降低以及细胞壁中β-1,6-葡聚糖含量降低。这些结果表明，Keg1和Kre6在酿酒酵母的β-1,6-葡聚糖合成中具有协同作用。
