GENETICS OF THE YEAST MICROTUBULE ORGANIZING CENTER

微管组织中心（MTOC）是细胞的关键细胞器， 调节细胞中微管的结构和功能。 的 本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）的组分，确定其 功能，并建立MTOC组装途径。 识别 保守的MTOC成分应该有重要的影响， 治疗癌症，了解出生缺陷的机制 以及寻找抗真菌药物。 我们有三个具体目标： 首先，我们将研究酵母中心蛋白同系物Cdc31p的功能。 中心蛋白是一种高度保守的蛋白质，存在于植物、动物和哺乳动物的MTOC中， 和真菌。 Cdc31p是启动SPB的关键蛋白 重复。 为了阐明Cdc31p的作用，我们将确定关键的SPB 与Cdc31p相互作用的蛋白质使用遗传和 生物化学方法。 我们将鉴定出蛋白质， 与Cdc31p相互作用，在体内改变其活性或作用于Cdc31p的下游。 SPB组装途径。 反过来，我们将确定 SPB中相互作用的蛋白质。 我们将使用显性负突变体 Cdc31p的形式，以帮助了解中心蛋白的功能，在体外 脊椎动物MTOC的组装。 其次，我们的目标是确定Dsk2p的正常功能， 一种泛素相关蛋白被鉴定为SPB突变的抑制因子 成分 Dsk2p是SPB的一个组件，还是SPB组件的修饰符 还是作用于SPB组装途径的下游？ 使用Dsk2p 特异性抗体，我们将确定Dsk2p的定位， 它是否像泛素一样被加工。 我们将确定Dsk2p是否 与Cdc31p结合或影响Cdc31p或其他SPB组分的稳定性。 泛素依赖性蛋白降解途径的作用将是 评估。 我们将使用各种遗传和物理方法， 鉴定DSK 2相关基因以及相互作用的基因和蛋白质 关于Dsk2p 最后，为了确定SPB是否是保守合成的， 分离，我们将研究分离行为，在体内，标记的， 脉冲合成后的SPB分量。

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.