GENETICS OF THE YEAST MICROTUBULE ORGANIZING CENTER

酵母微管组织中心的遗传学

基本信息

批准号：
2701677
负责人：
Mark David Rose
金额：
$ 21.16万
依托单位：
PRINCETON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-05-01 至 2000-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2701677
关键词：
electron microscopy fungal genetics gene mutation microtubules protein biosynthesis protein structure function spindle pole body

项目摘要

The microtubule organizing center (MTOC) is the key organelle for regulating the structure and function of microtubules in the cell. The long term objectives of this proposal are to identify important functional components of the yeast MTOC or spindle pole body (SPB), determine their functions, and establish the pathway of MTOC assembly. The identification of conserved MTOC components should have important implications for the treatment of cancer, the understanding of the mechanism of birth defects and the search for antifungal drugs. We have three specific aims: First, we will examine the function of the yeast centrin homolog, Cdc31p. Centrin is a highly conserved protein found in the MTOCs of plants, animals and fungi. Cdc31p is a key protein required for initiation of SPB duplication. To elucidate Cdc31p's role we will identify critical SPB proteins which interact with Cdc31p using a combination of genetic and biochemical approaches. We will identify proteins that either physically interact with Cdc31p, modify its activity in vivo or act downstream in the pathway of SPB assembly. In turn, we will determine the functions of the interacting proteins in the SPB. We will use dominant negative mutant forms of Cdc31p to help understand centrin's function in the in vitro assembly of vertebrate MTOCs. Second, we aim to determine the normal function of Dsk2p, a novel ubiquitin-related protein identified as a suppressor of a mutation in a SPB component. Is Dsk2p a component of the SPB, a modifier of an SPB component or does it act downstream in the SPB assembly pathway? Using Dsk2p specific antibodies, we will determine the localization of Dsk2p and whether it is processed like ubiquitin. We will determine whether Dsk2p binds to Cdc31p or affects the stability of Cdc31p or other SPB components. The role of the ubiquitin-dependent protein degradation pathway will be assessed. We will use a variety of genetic and physical methods to identify DSK2 related genes as well as genes and proteins that interact with Dsk2p. Finally, to determine whether the SPB is conservatively synthesized and segregated, we will examine the segregation behavior, in vivo, of tagged SPB components after pulse synthesis.

微管组织中心（MTOC）是微管组织的关键细胞器调节细胞内微管的结构和功能。这该提案的长期目标是确定重要的功能酵母 MTOC 或纺锤体极体 (SPB) 的组成部分，确定它们的功能，并建立MTOC组装途径。鉴定保守的 MTOC 成分应该对癌症治疗、出生缺陷机制的理解以及寻找抗真菌药物。我们有三个具体目标：首先，我们将检查酵母中心蛋白同源物 Cdc31p 的功能。 Centrin 是一种高度保守的蛋白质，存在于植物、动物的 MTOC 中和真菌。 Cdc31p 是 SPB 启动所需的关键蛋白复制。为了阐明 Cdc31p 的作用，我们将确定关键的 SPB 使用遗传和基因组合与 Cdc31p 相互作用的蛋白质生化方法。我们将鉴定出物理上的蛋白质与 Cdc31p 相互作用，修改其体内活性或在下游发挥作用 SPB 组装途径。反过来，我们将确定 SPB 中的相互作用蛋白。我们将使用显性失活突变体 Cdc31p 的形式有助于了解中心蛋白在体外的功能脊椎动物 MTOC 的组装。其次，我们的目标是确定 Dsk2p 的正常功能，这是一种新颖的泛素相关蛋白被鉴定为 SPB 突变的抑制因子成分。 Dsk2p是SPB的组件吗，SPB组件的修饰符或者它在 SPB 组装途径的下游起作用吗？使用 Dsk2p 特异性抗体，我们将确定 Dsk2p 的定位和是否像泛素一样被加工。我们将确定是否Dsk2p 与 Cdc31p 结合或影响 Cdc31p 或其他 SPB 组件的稳定性。泛素依赖性蛋白质降解途径的作用将是评估。我们将使用多种遗传和物理方法来识别 DSK2 相关基因以及相互作用的基因和蛋白质与 Dsk2p。最后判断SPB是否是保守合成的分离后，我们将检查标记的体内分离行为脉冲合成后的 SPB 组件。