Cloning of the NM1killer toxin resistant gene in Saccharomyces cervisiae

姆氏汉逊酵母分泌的HM-1杀伤毒素通过干扰β-1，3-葡聚糖合成抑制酿酒酵母细胞生长。克隆了一个过表达可使酿酒酵母细胞对HM-1杀伤毒素产生抗性的基因。该基因命名为HKR 1，含有5.4kb的开放阅读框。HKR 1编码一种富含丝氨酸和苏氨酸的1型膜蛋白，在细胞质domein中具有钙结合的共有序列。HKR 1的无效突变是致命的，并且HKR 1的过表达增加细胞壁中的β-1，3-葡聚糖含量。HKR 1的部分破坏显著降低了β-1，3-葡聚糖合酶活性水平以及细胞壁β-1，3-葡聚糖含量，并改变了单倍体细胞的轴向出芽模式。用大肠杆菌表达的HKR 1抗体进行免疫荧光显微镜观察，发现HKR 1主要定位于细胞表面。这些结果表明，HKR 1编码一种细胞表面活性蛋白，该蛋白调节细胞壁β-1，3-葡聚糖合成和出芽模式。

HM-1killer toxin secreted from Hansenula mrakii inhibits the growth of Saccharomyces cerevisiae cells by interfering with beta-1,3-glucan synthesis. A gene whose overexpression can endow Saccharomyces cerevisiae cells with resistant to HM-1 killer toxin was cloned. The gene, designated HKR1 contains 5.4-kb open reading frame. The HKR1 encodes a serine-and threonine-rich type 1 membrane protein with calcium-binding consensus sequence in the cytoplasmic domein. The null mutation of HKR1 is lethal and overexpression of HKR1 increases beta-1,3-glucan contents in cell wall. The partial disruption of HKR1 significantly reduced the level of beta-1,3-glucan synthase activities as well as cell wall beta-1,3-glucan contents, and altered the axial budding pattern of haploid cells. Immunofluorescednce microscopy with an antibody raised against Hkr1 expreseed in Escherichia coli revealed that HKR1 was predominantly localized on the cell surface. These results demonstrate that HKR1 encodes a cell surfase protein that regulates both cell wall beta-1,3-glucan synthesis and budding pattern.