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Transduction of killing action of killer protein to sensitive yeast strains

将杀伤蛋白的杀伤作用转导至敏感酵母菌株

基本信息

批准号：
16580068
负责人：
KITAMOTO Hiroko
金额：
$ 2.05万
依托单位：
National Institute of Agro-environmental Science (2006)National Institute of Agrobiological Sciences (2004-2005)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

项目摘要

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

在自然栖息地中，酵母菌之间分泌的蛋白质毒素的拮抗相互作用似乎相当高。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（Saccharomycescerevisiae）突变株，这些突变株分别缺失了4901个酵母基因，以改变对纯化的乳酸克鲁维酵母（Kluyveromyceslactis）杀伤蛋白（Zymocin）的敏感性。酿酒酵母细胞我们分析了KTI 6突变体，它们抵抗外源性的zymocin，但对抑制性γ-toxin亚单位的细胞内表达敏感，表明KTI 6编码毒素进入细胞所需的因子。与改变的细胞表面性质一致，kti 6细胞抵抗潮霉素B、红霉素E和制霉菌素，这些抗生素需要完整的膜电位或引起膜破坏。KTI 6与PT 1等位，编码甲甘露糖基-二肌醇磷酸-神经酰胺[M（IP）2C]合酶，其产生M（IP）2C， ...更多信息主要质膜鞘脂。kti 6膜缺乏M（IP）2C和具有降低水平的M（IP）2C前体的鞘脂突变体，包括鞘脂结构单元神经酰胺存活酶蛋白。此外，kti 6/ipt 1细胞允许酶蛋白对接，但阻止其毒性γ亚基的输入。遗传分析表明，Kti 6可能作用于脂筏质子泵Kti 10/Pma 1的上游，这是一个以前鉴定的酶敏感性因子。总之，M（IP）2C在质膜的步骤，随后的细胞壁几丁质的识别由zymocin，但之前的延伸，潜在的毒素target.To看到zymocin处理的细胞上的zymocin亚基的定位，我们分级的细胞裂解物的参与。我们通过分别用抗a、B、g亚基抗体的Western印迹分析了每个组分上的每个a、B和g亚基的存在。A和G亚基分别在细胞壁部分和膜部分的不溶性部分中检测到。但未检测到β亚基。这些数据表明，a和y亚基都通过膜侵入敏感细胞。少