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首页> 外文学位 >Essential GPI mannosyltransferases in Saccharomyces cerevisiae and the pathogenic fungus Candida albicans.
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Essential GPI mannosyltransferases in Saccharomyces cerevisiae and the pathogenic fungus Candida albicans.

机译：酿酒酵母和致病真菌白色念珠菌中的必需GPI甘露糖基转移酶。

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摘要

Diverse glycoproteins are anchored on the outer face of the plasma membrane via a carboxy-terminal glycosylphosphatidylinositol (GPI). The conserved core of the GPI contains three alpha-linked mannoses, but a fourth side-branching alpha-mannose may sometimes be present. Interestingly, yeast GPIs contain at least four mannoses, whereas most mammalian GPIs normally contain only three. My research focused on the mannosyltransferase that adds the fourth GPI mannose in the model yeast Saccharomyces cerevisiae and the pathogenic fungus Candida albicans.; I show that the essential yeast Smp3 protein, a member of a new family of dolichol phosphate mannose-utilizing mannosyltransferases, is responsible for the addition of the fourth mannose. In vivo radiolabeling experiments using temperature-sensitive smp3 mutants, showed that loss of Smp3p function results in the accumulation of a GPI precursor lipid, whose glycan headgroup I structurally characterized as a trimannosyl-GPI. By creating double mutants of smp3 with other gpi mutants, I established that smp3 mutations prevent the formation of tetramannosyl-GPI precursors.; I cloned the C. albicans homologue of SMP3 and found that its product is the functional homologue of ScSmp3p, for it complements null and conditional smp3 mutations. Furthermore, CaSmp3p transfers a fourth mannose to a trimannosyl-GPI precursor in vivo. I determined that CaSMP3 is also an essential gene, as I was unable to recover homozygous null smp3 mutants from C. albicans, an obligate diploid. I created a conditional strain in which expression of CaSMP3 was controlled by the CaMAL2 promoter, and found that depletion of CaSmp3p results in the accumulation of a trimannosyl-GPI precursor and decreased viability. My characterization of this precursor is the first report of a GPI structure from C. albicans . The MAL2 promoter offers a tool to perform functional analysis studies on other C. albicans GPI assembly genes. Several C. albicans GPI anchored proteins have been implicated in virulence and disruption of GPI biosynthesis should globally impair the surface expression of these proteins. Because Smp3p is essential in fungi, yet dispensable in mammalian cells, its function could be exploited as a target for selective inhibitors of pathogenic fungi.
机译：多种糖蛋白通过羧基末端的糖基磷脂酰肌醇（GPI）锚定在质膜的外表面。 GPI的保守核心包含三个α-连接的甘露糖，但有时可能会出现第四个侧枝α-甘露糖。有趣的是，酵母GPI至少包含四个甘露糖，而大多数哺乳动物GPI通常仅包含三个。我的研究集中在甘露糖基转移酶上，该甘露糖基转移酶在模型酵母啤酒酵母和致病真菌白色念珠菌中添加了第四种GPI甘露糖。我表明必需的酵母Smp3蛋白是利用磷酸甘露糖的甘露糖基转移酶的新家族的成员，它负责添加第四种甘露糖。使用对温度敏感的smp3突变体的体内放射性标记实验表明，Smp3p功能的丧失导致GPI前体脂质的积累，该脂质的聚糖头基I在结构上表征为三甘露糖基-GPI。通过与其他gpi突变体一起创建smp3的双突变体，我确定smp3突变可防止四甘露糖基-GPI前体的形成。我克隆了SMP3的白色念珠菌同源物，发现其产物是ScSmp3p的功能同源物，因为它补充了无效和条件性smp3突变。此外，CaSmp3p在体内将第四甘露糖转移至三甘露糖基-GPI前体。我确定CaSMP3也是必不可少的基因，因为我无法从白色念珠菌（专性二倍体）中回收纯合的无效smp3突变体。我创建了一个条件菌株，其中CaSMP3的表达受CaMAL2启动子控制，并且发现CaSmp3p的耗尽导致三甘露糖基GPI前体的积累并降低了生存能力。我对这种前体的表征是白色念珠菌的GPI结构的首次报道。 MAL2启动子提供了对其他白色念珠菌GPI装配基因进行功能分析研究的工具。几种白色念珠菌的GPI锚定蛋白与毒力有关，GPI生物合成的破坏应总体上破坏这些蛋白的表面表达。由于Smp3p在真菌中是必不可少的，但在哺乳动物细胞中却是不可缺少的，因此它的功能可以用作病原性真菌选择性抑制剂的靶标。

著录项

  • 作者

    Grimme, Stephen James.;

  • 作者单位

    University of Illinois at Urbana-Champaign.;

  • 授予单位 University of Illinois at Urbana-Champaign.;
  • 学科 Biology Cell.; Biology Microbiology.; Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2004
  • 页码 181 p.
  • 总页数 181
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 细胞生物学;微生物学;生物化学;
  • 关键词

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