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首页> 外文学位 >Human hsp90-sgt1 and skp1 form a conserved client-adaptor complex regulating global kinetochore structure through the balance of mis12 complex assembly and turnover.
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Human hsp90-sgt1 and skp1 form a conserved client-adaptor complex regulating global kinetochore structure through the balance of mis12 complex assembly and turnover.

机译：人类hsp90-sgt1和skp1通过守恒mis12复杂的组装和营业额的平衡形成一个保守的客户-适配器复合物，调节全局的线粒体结构。

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摘要

The kinetochore is a large multiprotein complex that links chromosomes to the mitotic spindle. Correct assembly ensures the faithful segregation of genetic material to resultant daughter cells during division. The task of assembling a multiprotein complex composed of 60-80 subunits, with high fidelity, represents a major cellular challenge. Failures of assembly reduce the affinity of microtubule-binding sites and impair the ability to monitor, integrate signals of, and correct chromosome mis-attachment. The consequences of this phenotype include chromosome breakage, mutation, and/or the inheritance of abnormal chromosome complements, which are associated with tumorigenesis. Understanding the mechanisms governing kinetochore assembly, regulation, and organization are therefore major issues in kinetochore biology.;Kinetochore assembly is best understood in the budding yeast Saccharomyces cerevisiae. In yeast a kinetochore keystone exists, the centromere binding factor 3 (CBF3), which is assembled by Hsp90, the co-chaperone Sgt1, and the SCF (Skp1-Cullin-F-Box) component Skp1. Assembly of the CBF3 is required for higher order kinetochore. Failure of CBF3 localization, either through CBF3 or Hsp90-Sgt1 loss of function mutants, result in kinetochore assembly defects and thus reduced mitotic fidelity. In contrast to the yeast kinetochore, which binds a single microtubule (i.e. a 'point' kinetochore), the mammalian kinetochore is multivalent and is composed of multiple kinetochore repeat structures -a 'regional' kinetochore. Because of the regional nature of the mammalian kinetochore, it forms an intricate network-like structure whereby proteins form many lateral attachments and outer and inner proteins influence each other's behavior. This level of complexity suggests that mechanisms must be in place to ensure proper formation and function of the kinetochore structure. Interestingly, the core regulatory subunits (Hsp90, Sgt1, and Skp1) involved in yeast CBF3 assembly are also conserved in mammalian cells and are required for kinetochore assembly. Hsp90 or Sgt1 loss of function results in aberrant kinetochore network formation resembling failed CBF3 assembly. However, mammalian cells do not contain a CBF3 homolog and the targets Hsp90-Sgt1 regulation are unknown.;To address this question we took a combined genetic and biochemical approach to identify the mammalian kinetochore targets of Hsp90-Sgt1. Here, we show that human Hsp90-Sgt1 interacts with the Mis12 complex, a so-called keystone complex required to assemble a large fraction of the kinetochore. Inhibition of Hsp90 or Sgt1 destabilizes the Mis12 complex and delays proper chromosome alignment due to inefficient formation of microtubule binding sites. Interestingly, co-inhibition of Sgt1 and the SCF subunit, Skp1, increases Mis12 complexes at kinetochores and restores timely chromosome alignment but forms less robust microtubule binding sites. These results suggest that Sgt1 functions to link Mis12 complexes to Hsp90 assembly and Skp1-directed turnover pathways. Consistent with this idea we find that Skp1 interacts directly with Mis12 complexes and that this interaction is important for Mis12 complex turnover. The interaction of Skp1 and Mis12 subunits is directed through a previously unidentified F-Box motif in Dsn1. Mutation of the Dsn1 F-box motif reduces Dsn1 turnover, leading to chromosome congression defects, and a reduction in the stability of kinetochore-microtubule contacts. We propose that Skp1 directed turnover of Dsn1 is required for maintain the fidelity of kinetochore-microtubule binding sites through the removal of non-functional Mis12 complexes. Taken together, these findings support a novel role for Hsp90-Sgt1 and Skp1 chaperones in ensuring the fidelity of multi-protein complexes through the balance of assembly and turnover.
机译：线粒体是一种大型的多蛋白复合物，它将染色体与有丝分裂纺锤体相连。正确组装可确保在分裂过程中将遗传物质忠实地分离到最终的子细胞中。具有高保真度的由60-80个亚基组成的多蛋白复合物的组装代表了一项重大的细胞挑战。组装失败降低了微管结合位点的亲和力，并削弱了监测，整合信号和纠正染色体错接的能力。该表型的后果包括染色体断裂，突变和/或异常染色体补体的遗传，这与肿瘤发生有关。因此，了解控制线粒体组装，调控和组织的机制是线粒体生物学中的主要问题。动植物的组装在酿酒酵母中是最好的理解。在酵母中，有一个线粒体的关键蛋白，着丝粒结合因子3（CBF3），由Hsp90，辅分子伴侣Sgt1和SCF（Skp1-Cullin-F-Box）组分Skp1组装而成。 CBF3的组装对于更高阶的动球体是必需的。 CBF3本地化失败，无论是通过CBF3还是Hsp90-Sgt1功能突变体的丧失，都将导致线粒体装配缺陷，从而降低有丝分裂保真度。与结合单个微管（即``点''动粒）的酵母动粒相比，哺乳动物的动粒是多价的，并且由多个动粒重复结构-一个``区域性''动粒组成。由于哺乳动物的线粒体具有区域性，因此它形成了复杂的网络状结构，从而蛋白质形成许多侧向附着，外部和内部蛋白质相互影响。这种复杂性水平表明必须有适当的机制来确保线粒体结构的正确形成和功能。有趣的是，参与酵母CBF3装配的核心调节亚基（Hsp90，Sgt1和Skp1）在哺乳动物细胞中也保守，并且是动线粒装配所必需的。 Hsp90或Sgt1的功能丧失导致类似于CBF3组装失败的动球体网络形成。然而，哺乳动物细胞不包含CBF3同源物，并且靶标Hsp90-Sgt1的调控是未知的。为了解决这个问题，我们采取了遗传和生化相结合的方法来鉴定Hsp90-Sgt1的哺乳动物动粒靶。在这里，我们证明了人类Hsp90-Sgt1与Mis12复合物相互作用，而Mis12复合物是组装大部分动线粒所需的所谓的梯形失真复合物。 Hsp90或Sgt1的抑制作用会破坏Mis12复合物的稳定性，并由于微管结合位点形成效率低而延迟正确的染色体排列。有趣的是，Sgt1和SCF亚基Skp1的共抑制作用增加了动植物的Mis12复合物，并恢复了及时的染色体排列，但形成了较不牢固的微管结合位点。这些结果表明Sgt1的功能将Mis12复合体链接到Hsp90大会和Skp1指导的营业额途径。与这个想法一致，我们发现Skp1与Mis12复合体直接交互，并且这种交互对于Mis12复合体更新很重要。 Skp1和Mis12亚基的相互作用是通过Dsn1中以前未知的F-Box基序进行的。 Dsn1 F-box基序的突变减少了Dsn1转换，导致染色体国会缺陷，并减少了线粒体-微管接触的稳定性。我们建议通过去除无功能的Mis12复合物来维持Skpochore-微管结合位点的保真度需要Skp1定向的Dsn1转换。综上所述，这些发现支持Hsp90-Sgt1和Skp1分子伴侣在通过组装和周转的平衡确保多蛋白复合物保真度方面的新作用。

著录项

  • 作者

    Davies, Alexander Eugene.;

  • 作者单位

    University of California, Davis.;

  • 授予单位 University of California, Davis.;
  • 学科 Cellular biology.;Molecular biology.
  • 学位 Ph.D.
  • 年度 2010
  • 页码 131 p.
  • 总页数 131
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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