GENETICS OF CENTRIN AND THE SPINDLE POLE BODY IN YEAST

微管组织中心（microtule organizing centers， MTOC，酵母中称为纺锤极体或SPB）是调控细胞内微管结构和功能的关键细胞器。从MTOC发出的微管形成染色体分离的纺锤体装置，并与皮质位点相互作用，使细胞核和纺锤体在细胞内定向。SPB的一些蛋白质和定位纺锤体的蛋白质复合物在真核生物中是高度保守的。例如，Cdc31p是SPB复制的最早步骤所必需的SPB蛋白。Cdc31p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素样MTOC蛋白。Kar1p是位于SPB的完整膜蛋白，是定位Cdc31p所必需的。然而，Cdc31p在细胞中具有其他功能；除了在SPB复制中发挥作用外，Cdc31p还与细胞质激酶Kic1p相关，并且是细胞形态和完整性所必需的。细胞质动力蛋白和动力蛋白复合物的组成部分构成了SPB/核取向的一个途径的一部分。Act5p是哺乳动物肌动蛋白相关蛋白Arp1的酵母同源物，Arp1是肌动蛋白复合物中最丰富的亚基。对于这些成分如何相互作用以及如何与其他蛋白质相互作用来复制SPB并使其在细胞中定向，人们知之甚少。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被调节和整合。我们将使用各种遗传和分子技术来测试关于SPB复制和取向的四个特定假设。首先，我们确定PKC1通路是一个潜在的关键调节因子，它协调SPB复制与细胞周期。我们将测试PKC1途径通过调节cdc31p活性起作用的假设。其次，我们已经确定了两个KAR1相互作用的基因，NEM1和SPOT编码核膜蛋白。我们将验证Nem1p和Spo7p与Kar1p或Cdc31p相互作用以组装SPB半桥的假设。第三，我们提出CDC31的特定区域介导其不同的功能。我们将通过大量CDC31突变的表型和生化特征来测试这一点。第四，我们已经开始进行遗传分析，以确定动力蛋白复合体的剩余成分和调节因子。各种ACT5和抑制突变体将被用来了解动力蛋白复合物的成分如何相互作用，以帮助定位SPB/核。这项研究应提供关于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。这里鉴定的MTOC蛋白可能是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.