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首页> 外文学位 >Construction of a new model system in Saccharomyces cerevisiae to study YopO and identify its targets.
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Construction of a new model system in Saccharomyces cerevisiae to study YopO and identify its targets.

机译：在酿酒酵母中构建新的模型系统以研究YopO并确定其目标。

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摘要

Yersinia species use a type three secretion system (TTSS) to inject Yersinia outer proteins (Yops) into the target cell. The effector YopO is a serine/threonine kinase which upon association with actin becomes active. However, activation of the kinase is not required for actin disruption. YopO also contains a Rho/Rac binding domain, but it is unclear how it affects these proteins.;We have established an expression system in Saccharomyces cerevisiae to study YopO. The expression vector contains a GAL1 promoter for inducible expression of YopO as well as a V5 epitope used for visualization of each Yop. We have shown that YopO is lethal in yeast. YopO containing strains were visualized using immunofluorescent microscopy which showed YopO localization near the cell membrane. YopO exerts an effect over the actin cytoskeleton, causing a loss of actin cables and a decrease in the amount of cortical patches by hour three post induction.;Co-immunoprecipitation assays were used to elucidate the cellular targets of YopO. Utilizing the V5 epitope present on the YopO expression vector, and an HA epitope present on the Rho construct expression vector, we were able to precipitate the protein complex. The co-immunoprecipitation assay showed that Rho1, Rho2, Rho3, Rho5 and Cdc42 all bind physically to YopO.
机译：耶尔森氏菌种使用三型分泌系统（TTSS）将耶尔森氏菌外蛋白（Yops）注入靶细胞。效应物YopO是丝氨酸/苏氨酸激酶，其在与肌动蛋白结合后变得活跃。但是，肌动蛋白破坏并不需要激活激酶。 YopO还含有Rho / Rac结合结构域，但尚不清楚它如何影响这些蛋白质。我们已经在酿酒酵母中建立了一个表达系统来研究YopO。表达载体包含用于可诱导表达YopO的GAL1启动子，以及用于可视化每个Yop的V5表位。我们已经证明，YopO在酵母中具有致命性。使用免疫荧光显微镜观察含有YopO的菌株，其显示YopO定位在细胞膜附近。 YopO在肌动蛋白的细胞骨架上产生作用，导致肌动蛋白电缆丢失，诱导后三小时，皮质斑块的数量减少。;共免疫沉淀法用于阐明YopO的细胞靶标。利用存在于YopO表达载体上的V5表位和存在于Rho构建体表达载体上的HA表位，我们能够沉淀蛋白质复合物。免疫共沉淀试验表明，Rho1，Rho2，Rho3，Rho5和Cdc42都与YopO物理结合。

著录项

  • 作者

    Nejedlik, Laura.;

  • 作者单位

    Western Michigan University.;

  • 授予单位 Western Michigan University.;
  • 学科 Biology Molecular.
  • 学位 Ph.D.
  • 年度 2008
  • 页码 129 p.
  • 总页数 129
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 分子遗传学;
  • 关键词

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