Cloning of the NM1killer toxin resistant gene in Saccharomyces cervisiae

汉草分泌的hm -1杀伤毒素通过干扰β -1,3-葡聚糖的合成抑制酿酒酵母细胞的生长。克隆了一种基因，该基因的过表达可使酿酒酵母细胞具有对HM-1杀伤毒素的抗性。该基因被命名为HKR1，包含5.4 kb的开放阅读框。HKR1编码一种富含丝氨酸和苏氨酸的1型膜蛋白，在细胞质结构域中具有钙结合的一致序列。HKR1的零突变是致命的，HKR1的过表达增加了细胞壁中β -1,3-葡聚糖的含量。HKR1的部分破坏显著降低了β -1,3-葡聚糖合成酶活性水平和细胞壁β -1,3-葡聚糖含量，改变了单倍体细胞的轴向出芽模式。在大肠杆菌中表达Hkr1的抗体的免疫荧光显微镜显示，Hkr1主要定位在细胞表面。这些结果表明，HKR1编码一种细胞表面酶蛋白，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.