ANALYSIS OF CIK1P AND RELATED FUNCTIONS IN YEAST

酵母CIK1P及相关功能分析

• 批准号: 2191127
• 负责人: MICHAEL P. SNYDER
• 金额: $ 22.34万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-01-01 至 1998-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2191127
• 关键词: Saccharomyces cerevisiae; binding proteins; electron microscopy; fungal genetics; gel filtration chromatography; gene interaction; immunoprecipitation; kinesin; microtubule associated protein; myosin light chain kinase; polymerase chain reaction; protein biosynthesis; protein isoforms; protein reconstitution; protein structure function; western blottings; Saccharomyces cerevisiae; binding proteins; electron microscopy; fungal genetics; gel filtration chromatography; gene interaction; immunoprecipitation; kinesin; microtubule associated protein; myosin light chain kinase; polymerase chain reaction; protein biosynthesis; protein isoforms; protein reconstitution; protein structure function; western blottings

Kinesin-related proteins represent a large class of microtubule motor proteins with diverse roles in a variety of essential cellular processes. One important component of the motor complex is the light chain, whose function s poorly understood. We have identified a protein called yeast Cik1p which interacts with the non-motor domain of the Kar3 kinesin heavy chain-related protein, and possesses several properties expected of a kinesin light chain. The Cik1p-Kar3p complex has been hypothesized to crosslink and slide microtubules. Cik1p is important for Kar3p localization and function. We plan to use a series of biochemical, genetic and molecular approaches to characterize the structure and function of Cik1p. Biochemical approaches will be used to characterize the subunit composition, size and stability of the Cik1p-Kar3p complex. We will determine if Cik1p is stably associated with Kar3p as expected for a Kar3p light chain. We will attempt to reconstitute the Cik1p-Kar3p complex in vitro and analyze its structure using electron microscopy. To help elucidate the function of the Cik1p-Kar3p complex, we will determine if Cik1p in conjunction with the non-motor domain of Kar3p is capable of binding microtubules. The possibility that the Cik1p-Kar3p complex can bundle microtubules and slide them past one another will also be tested. To dissect how Cik1p functions, different functional domains of Cik1p will be analyzed. Sequences potentially involved in dimerization, and those important for Kar3p interaction, nuclear localization and spindle pole body/microtubule localization will be identified using a variety of biochemical and genetic approaches. Importantly, Cik 1p and Kar3p are thought to be differentially compartmentalized within the cell during the yeast life cycle, and Cik1p is appears to be the critical subunit for mediating the differential localization. We will attempt to determine if different isoforms of Cik1p exist and are responsible for regulating the nuclear/cytoplasmic compartmentalization. Finally, in order to identify other components that either function similarly to Cik1p, or that operate within the same or redundant pathways to those of Cik1p, we will characterize five genes, which when present in high copy, suppress the temperature-sensitive growth defects of Cik1 cells. The suppressor genes will be sequenced and the predicted protein sequences examined for similarities to Cik1p and other components of microtubule motor complexes. Disruption phenotypes of the suppressor genes and the subcellular localization of the encoded products will be determined to examine the potential role of these genes in microtubule-associated processes. We expect these studies to enhance our understanding of how microtubule motor complexes are assembled, targeted to the proper subcellular location, and function.

运动蛋白相关的蛋白代表大型微管电动机 在各种必需细胞过程中具有不同作用的蛋白质。 电动机复合物的一个重要组成部分是轻链，其 功能知之甚少。 我们已经确定了一种称为酵母的蛋白质 与Kar3驱动蛋白重的非运动域相互作用的CIK1P 与链链相关的蛋白质，并具有A 动力学轻链。 CIK1P-KAR3P复合物已假设 交叉链接和滑动微管。 CIK1P对KAR3P很重要 本地化和功能。 我们计划使用一系列生化，遗传 以及分子方法来表征的结构和功能 cik1p。 生化方法将用于表征亚基 CIK1P-KAR3P复合物的组成，大小和稳定性。 我们将 确定CIK1P是否与KAR3P预期的KAR3P稳定相关 轻链。 我们将尝试重建CIK1P-KAR3P复合体 使用电子显微镜分析并分析其结构。 提供帮助 阐明CIK1P-KAR3P复合物的功能，我们将确定是否 CIK1P与KAR3P的非运动域结合了 结合微管。 CIK1P-KAR3P复合物可以 束微管束并将它们滑过彼此也将进行测试。 为了剖析CIK1P的功能，CIK1P的不同功能域将 可以分析。 序列可能参与二聚化，以及 对于KAR3P相互作用，核定位和主轴杆很重要 将使用多种物体/微管定位确定 生化和遗传方法。 重要的是，CIK 1P和KAR3P是 被认为是在细胞中被差异化的 酵母生命周期和CIK1P似乎是关键的亚基 介导差分定位。 我们将尝试确定是否 CIK1P的不同同工型存在并负责调节 核/细胞质分区化。 最后，为了识别其他功能的其他组件 类似于CIK1P或在相同或冗余路径内运行的 对于Cik1p的那些，我们将描述五个基因，当存在于 高拷贝，抑制CIK1细胞温度敏感的生长缺陷。 抑制基因将进行测序，并预测的蛋白质序列 检查了与Cik1p和微管的其他组件的相似性 运动综合体。 抑制基因的破坏表型和 编码产品的亚细胞定位将确定为 检查这些基因在微管相关的潜在作用 过程。 我们希望这些研究能够增强我们对微管的理解 组装运动复合物，针对适当的亚细胞位置， 和功能。