Transduction of killing action of killer protein to sensitive yeast strains

在自然生境中，酵母菌与酵母菌之间分泌的蛋白性毒素具有很高的拮抗作用。为了了解酵母杀伤蛋白的分子机制，我们筛选了一组酿酒酵母突变体，单独删除4901个酵母基因，以改变对纯化的Kluyveromyces lactis （zymocin）杀伤蛋白的敏感性。三聚体（α,β,γ）蛋白毒素复合物Zymocin抑制酿酒葡萄球菌细胞增殖。我们对kti6突变体进行了分析，这些突变体抵抗外源酶，但对其抑制γ-毒素亚基的细胞内表达敏感，这表明kti6编码毒素进入细胞所需的一个因子。与改变的细胞表面特性一致，kti6细胞抵抗湿霉素b、紫霉素和制霉菌素，这些抗生素需要完整的膜电位或引起膜破坏。KTI6是i PT1的等位基因，编码壬基二肌醇磷酸神经酰胺[M(IP)2C]合成酶，产生M(IP)2C，这是最主要的质膜鞘脂。kti6膜缺乏M(IP)2C和鞘脂突变体，其M(IP)2C前体水平降低，包括鞘脂构建块神经酰胺存活酶。此外，kti6/ipt1细胞允许酶素对接，但阻止其毒性γ-亚基的输入。遗传分析表明，Kti6可能作用于脂筏质子泵Kti10/Pma1的上游，Kti10/Pma1是先前发现的酶敏因子。在这种情况下，M(IP)2C在酶酵素识别细胞壁几丁质的质膜步骤中起作用，但在潜在毒素靶点伸长子参与之前。为了观察酶促素亚基在酶促素处理细胞上的定位，我们对细胞裂解液进行了分离。我们分别用抗a、b、g亚基抗体进行Western-blotting，分析各组分中a、b、g亚基的存在。细胞壁和细胞膜的不溶性部分分别检测到A和g亚基。但未检测到β亚基。这些数据表明，a亚基和y亚基都通过细胞膜侵入敏感细胞。少

Antagonistic interactions between yeasts by secreted proteinaceous toxins appear to be quite high in natural habitat. To know the molecular mechanisms of yeast killer proteins, we screened a set of Saccharomyces cerevisiae mutants, individually deleted for 4901 yeast genes, for altered sensitivity against purified killer proteins of Kluyveromyces lactis (zymocin).Zymocin, atrimeric(α,β,γ)protein toxin complex, inhibits proliferation of S. cerevisiae cells. We present an analysis of kti6 mutants, which resist exogenous zymocin but are sensitive to intracellular expression of itsinhibitory γ- toxinsubunit, suggesting that KTI6 encodes a factor needed for toxin entry into the cell. Consistent with altered cell surface properties, kti6 cells resist hygromycinB, syringomycinE, and nystatin, antibiotics that require intact membrane potentials or provok emembrane disruption. KTI6 is allelic toI PT1, coding formannosyl-diinositolphospho-ceramide [M(IP)2C] synthase, which produces M(IP)2C, the … More major plasma membrane sphingolipid. kti6 membranes lack M(IP)2C and sphingolipid mutants that have reduced levels of M(IP)2C precursors, including the sphingolipid building block ceramide survive zymocin. Inaddition, kti6/ipt1 cells allow zymocin docking but prevent import of its toxic γ-subunit. Genetic analysis indicates that Kti6 is likely to act upstream of lipid raft proton pump Kti10/Pma1, apreviously identified zymocin sensitivity factor. Insum, M(IP)2C operates in a plasma membrane step that follows recognition of cell wall chitin by zymocin but precedes the involvement of elongator, the potential toxin target.To see the localization of zymocin subunits on the zyocin treated cell, we fractionated the cell lysate. We analyzed the presence of each a, b and g subunits on each fractions by Western-blotting with anti-a, b, g subunit antibody, respectively. a and g subunits were detected in the insoluble fraction of both in cell wall fraction and membrane fraction, respectively. However, β subunit was not detected. These data shows that both a and y subunits invade in the sensitive cell, through membrane. Less