GENETICS OF CENTRIN AND THE SPINDLE POLE BODY IN YEAST

酵母中心蛋白和纺锤体的遗传学

• 批准号: 6519646
• 负责人: Mark David Rose
• 金额: $ 26.52万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-05-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6519646
• 关键词: Saccharomyces cerevisiae; fungal genetics; fungal proteins; gene mutation; microtubule associated protein; microtubules; protein biosynthesis; protein structure function; spindle pole body; suppressor mutations; temperature sensitive mutant; yeast two hybrid system

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.

微管组织中心（MTOC，称为纺锤体体或酵母中的SPB）是调节细胞中微管的结构和功能的关键细胞器。 从MTOC发出的微管形成了染色体分离的纺锤体设备，并与皮质位点相互作用，以方向细胞内的细胞核和纺锤体。 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，CDC31P是SPB重复最早步骤所需的SPB蛋白。 Cdc31p是一种高度保守的钙调蛋白样MTOC蛋白的酵母同源物。 KAR1P是SPB处的积分膜蛋白，它是定位Cdc31p所需的。 但是，CDC31P在细胞中具有其他功能。除了在SPB复制中作用外，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。 Dynactin复合物的细胞质动力蛋白和SPB/核取向途径的一部分。 ACT5P是哺乳动物肌动蛋白相关蛋白ARP1的酵母同源物，ARP1是Dynactin复合物中最丰富的亚基。 这些成分如何相互相互作用以及与其他蛋白质相互作用以复制SPB并将其定向在细胞中。我们的远程目标是识别SPB的组成部分，了解它们在重复和取向方面的功能，以及如何在细胞周期中调节和集成这些过程。我们将使用各种遗传和分子技术来测试有关SPB重复和方向的四个特定假设。首先，我们将PKC1途径确定为潜在的钥匙调节剂，该调节器与细胞周期协调SPB重复。我们将检验以下假设：PKC1途径通过调节Cdc31 p的活性来起作用。 其次，我们已经确定了两个编码核包膜蛋白的Kar1相互作用基因NEM1和斑点。 我们将测试NEM1P和SPO7P与KAR1P或CDC31P相互作用的假设，用于组装SPB半桥。 第三，我们提出CDC31的特定区域介导了其不同的功能。 我们将通过大量CDC31突变的表型和生化表征进行测试。 第四，我们已经开始进行遗传分析，以识别Dynactin复合物的其余成分和调节剂。各种ACT5和抑制突变体将用于了解Dynactin复合物的组成部分如何相互作用以帮助SPB/Nucleus定向。 这项研究应提供有关MTOC的基本结构和功能的基本知识，并且与人类健康的重要领域有关，包括先天缺陷和癌症的机制。 此处鉴定的MTOC蛋白可能是抗溶毒药物治疗癌症和真菌感染的重要靶标。