GENETICS OF CENTRIN AND THE SPINDLE POLE BODY IN YEAST

微管组织中心（Microtubule organizing centers，MTOC）是调控细胞微管结构和功能的重要细胞器。 从MTOC发出的微管形成用于染色体分离的纺锤体装置，并与皮质位点相互作用以定向细胞内的核和纺锤体。 SPB的几种蛋白质和定向纺锤体的蛋白质复合物在真核生物中高度保守。 例如，Cdc 31 p是SPB复制中最早步骤所需的SPB蛋白。 Cdc 31 p是中心蛋白的酵母同源物，中心蛋白是一种高度保守的钙调蛋白样MTOC蛋白。 Kar 1 p是SPB上的一种膜蛋白，是定位Cdc 31 p所必需的。 然而，Cdc 31 p在细胞中具有额外的功能;除了在SPB复制中的作用外，Cdc 31 p还与细胞质激酶Kic 1 p相关联，并且是细胞形态和完整性所需的。 细胞质动力蛋白和动力肌动蛋白复合物的组分形成SPB/核取向的一个途径的一部分。 Act 5 p是哺乳动物肌动蛋白相关蛋白Arp 1的酵母同源物，Arp 1是动力肌动蛋白复合物中最丰富的亚基。 关于这些成分如何相互作用以及与其他蛋白质相互作用以复制SPB并在细胞中定位它，我们知之甚少。我们的长期目标是确定SPB的组成部分，了解它们在复制和定向中的功能，以及这些过程如何在细胞周期中进行调节和整合。我们将使用各种遗传和分子技术来测试四个具体的假设SPB复制和方向。首先，我们确定了PKC 1通路作为一个潜在的关键调节器，协调SPB复制与细胞周期。我们将测试的假设，PKC 1通路的行为通过调节Cdc 31 p的活性。第二，我们已经确定了两个KAR 1相互作用的基因，NEM 1和SPOT编码核膜蛋白。 我们将检验Nem 1 p和Spo 7 p与Kar 1 p或Cdc 31 p相互作用以组装SPB半桥的假设。 第三，我们提出CDC 31的特定区域介导其不同的功能。 我们将通过大量CDC 31突变的表型和生化特征来测试这一点。 第四，我们已经开始进行基因分析，以确定动力蛋白复合体的其余成分和调节因子。各种ACT 5和抑制突变体将被用来了解动力肌动蛋白复合物的组分如何相互作用，以帮助定位SPB/细胞核。 这项研究应提供有关MTOC的基本结构和功能的基本知识，并与人类健康的重要领域有关，包括出生缺陷和癌症的机制。 在此鉴定的MTOC蛋白可能是用于治疗癌症和真菌感染的抗有丝分裂药物的重要靶点。

Microtubule organizing centers (MTOC, called the spindle pole body or SPB in yeast) are the key organelles for regulating the structure and function of microtubules in the cell. Microtubules emanating from the MTOC form the spindle apparatus for chromosome segregation and interact with cortical sites to orient the nucleus and spindle within the cell. Several proteins of the SPB and the protein complexes that orient the spindle are highly conserved in eukaryotes. For example, Cdc31p is an SPB protein required for the earliest step in SPB duplication. Cdc31p is the yeast homologue of centrin, a highly conserved calmodulin-like MTOC protein. Kar1p is an integral membrane protein at the SPB that is required to localize Cdc31p. However, Cdc31p has additional functions in the cell; in addition to a role in SPB duplication, Cdc31p associates with a cytoplasmic kinase, Kic1p, and is required for cell morphology and integrity. Cytoplasmic dynein and components of the dynactin complex form part of one pathway for SPB/nuclear orientation. Act5p is the yeast homologue of the mammalian actin-related protein, Arp1, the most abundant subunit of the dynactin complex. Little is known about how these components interact with each other and with other proteins to duplicate the SPB and orient it in the cell. Our long-range objectives are to identify components of the SPB, understand their functions in duplication and orientation and how these processes are regulated and integrated in the cell cycle. We will use a variety of genetic and molecular techniques to test four specific hypotheses about SPB duplication and orientation. First, we identified the PKC1 pathway as a potential key regulator that coordinates SPB duplication with the cell cycle. We will test the hypothesis that the PKC1 pathway acts by regulating the activity of Cdc31 p. Second, we have identified two KAR1 interacting genes, NEM1 and SPOT that encode nuclear envelope proteins. We will test the hypothesis that Nem1p and Spo7p interact with Kar1p or Cdc31p for assembly of the SPB half- bridge. Third, we propose that specific regions of CDC31 mediate its different functions. We will test this by the phenotypic and biochemical characterization of a large number of CDC31 mutations. Fourth, we have begun a genetic analysis to identify the remaining components and regulators of the dynactin complex. A variety of ACT5 and suppressor mutants will be used to understand the how the components of the dynactin complex interact to help orient the SPB/nucleus. This research should provide fundamental knowledge about the basic structure and function of the MTOC and is relevant to significant areas of human health, including the mechanisms of birth defects and cancer. The MTOC proteins identified here may be important targets for anti-mitotic drugs for the treatment of cancer and fungal infections.