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首页> 外文学位 >New Tools and Approaches for Studying the Role of the Saccharomyces cerevisiae phosphatidylinositol 4-kinase Pik1 in the Yeast Nucleus.
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New Tools and Approaches for Studying the Role of the Saccharomyces cerevisiae phosphatidylinositol 4-kinase Pik1 in the Yeast Nucleus.

机译：研究酿酒酵母磷脂酰肌醇4-激酶Pik1在酵母核中的作用的新工具和方法。

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摘要

Phosphoinositides are a specialized type of glycerophospholipids found in all eukaryotes and their levels undergo dynamic temporal and spatial changes mediated by the actions of dedicated phosphatidylinositol kinases, phosphatases, and lipases. The Saccharomyces cerevisiae genome encodes two distinct Type III PtdIns 4-kinases that share sequence homology to each other: PIK1 and STT4. Together, Pik1 and Stt4 account for synthesis of more than 90% of the PtdIns4P detectable in yeast extracts, each is essential for viability, and the inactivation of either Pik1 or Stt4 via conditional alleles results in distinct non-overlapping phenotypes. It is thought that the independent functions of Pik1 and Stt4 result from their localization to specific cellular compartments. In the budding yeast, Stt4 localizes exclusively to the plasma membrane, whereas Pik1 localizes primarily to the Golgi body, but is also found in the cytoplasm and in the nucleus.;For the first part of my dissertation research (Chapter 3), I have characterized a novel potential specific inhibitor of Pik1. Compound ST016598, dubbed optimistically "pikostatin", specifically inhibits the growth of cells with reduced dosage of PIK1, without affecting Pik1 stability, in vivo protein-protein interactions, or localization of fluorescently-tagged Pik1. Although ST016598 (pikostatin) is only a weak inhibitor of the lipid kinase activity of Pik1 in vitro, pikostatin treatment nevertheless results in a specific, rapid, and reversible depletion of the Pik1-dependent Golgi body-specific pool of PtdIns4P, without affecting the separate pool of PtdIns4P created at the plasma membrane by Stt4. I also found that a subset of pik1ts alleles exhibit hypersensitivity to pikostatin even under permissive conditions, with subsequent implications for the mechanism by which pikostatin might inhibit Pik1 activity at the Golgi body.;Previous work from this lab used two differentially-localized Pik1 constructs to demonstrate that localization of Pik1 both to the Golgi and to the nucleus is required for viability. However, so far, no attempts have been made to separate the function of Pik1 in the nucleus from its now well-characterized essential function in the Golgi compartment. For the second part of my dissertation research (Chapter 4), I have tested directly whether Pik1 is responsible for generating nuclear PtdIns4P that might serve as the precursor for production of a soluble cofactor (InsP6) that is necessary for mRNA export. I also attempted to apply an unbiased genetic selection for dosage suppressors of the lack of nuclear Pik1 as a means to identify potential phosphoinositide- or inositol-polyphosphate-binding effectors in the yeast nucleus.
机译：磷脂酰肌醇是在所有真核生物中发现的一种特殊类型的甘油磷脂，其水平会经历由专用磷脂酰肌醇激酶，磷酸酶和脂肪酶的作用介导的动态时空变化。酿酒酵母基因组编码两个彼此共享序列同源性的不同III型PtdIns 4-激酶：PIK1和STT4。在一起，Pik1和Stt4占了酵母提取物中可检测到的PtdIns4P的90％以上的合成，它们对于生存力都是必不可少的，并且Pik1或Stt4通过条件等位基因的失活会导致明显的非重叠表型。可以认为，Pik1和Stt4的独立功能是由于它们定位于特定的细胞区室。在发芽的酵母中，Stt4仅定位于质膜，而Pik1主要定位于高尔基体，但也存在于细胞质和细胞核中。在论文研究的第一部分（第3章）中，表征了一种新型的潜在的Pik1特异性抑制剂。化合物ST016598被乐观地称为“匹高他汀”，它特异性地抑制PIK1剂量降低的细胞生长，而不会影响Pik1的稳定性，体内蛋白-蛋白相互作用或荧光标记的Pik1的定位。尽管ST016598（匹高他汀）只是体外Pik1的脂质激酶活性的弱抑制剂，但匹高他汀治疗仍然导致Pt1Ins4P依赖于Pik1的高尔基体特异性池的特异性，快速和可逆耗竭，而不影响单独的PtdIns4P。 Stt4在质膜上形成的PtdIns4P库。我还发现，即使在允许的条件下，pik1ts等位基因的一个子集也对pikostatin表现出超敏性，这对pikostatin可能抑制高尔基体Pik1活性的机制产生了潜在的影响;该实验室的先前工作使用了两种差异定位的Pik1构建体证明Pik1既定位于高尔基体又定位于细胞核是生存力所必需的。然而，到目前为止，还没有尝试将Pik1在细胞核中的功能与现在已被充分表征的高尔基体中的基本功能区分开。对于我的论文研究的第二部分（第4章），我直接测试了Pik1是否负责产生核PtdIns4P，该核PtdIns4P可能是生产mRNA出口所必需的可溶性辅因子（InsP6）的前体。我还尝试对缺乏核Pik1的剂量抑制剂进行无偏遗传选择，以此作为鉴定酵母核中潜在的磷酸肌醇或肌醇多磷酸结合效应物的手段。

著录项

  • 作者

    Klimenko, Evguenia S.;

  • 作者单位

    University of California, Berkeley.;

  • 授予单位 University of California, Berkeley.;
  • 学科 Biology Molecular.;Chemistry Biochemistry.;Biology Cell.
  • 学位 Ph.D.
  • 年度 2011
  • 页码 102 p.
  • 总页数 102
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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