PHOSPHOLIPASE C AND KINETOCHORE FUNCTION

磷脂酶 C 和着丝粒功能

• 批准号: 6225812
• 负责人: Ales Vancura
• 金额: $ 14.86万
• 依托单位: ST. JOHN'S UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-01 至 2004-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6225812
• 关键词: Saccharomyces cerevisiae; cell cycle; centromere; chromosome movement; enzyme activity; gene mutation; microtubules; phospholipase C; protein binding; site directed mutagenesis

DESCRIPTION (applicant's description) The long-term goal of our laboratory is to identify and characterize eukaryotic cellular processes regulated by phospholipase C (PLC), an enzyme which plays vital roles in signal transduction pathways. PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to produce two important second messengers: inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] which triggers release of calcium from internal stores, and diacylglycerol (DG) which activates the phospholipid- and Ca2+-dependent protein kinase C. Our recent results (Lin et al., 2000) demonstrate that PLC in Saccharomyces cerevisiae (Plc1p protein encoded by the PLC1 gene) associates with kinetochores and affects their ability to bind microtubules. The kinetochore is a specialized organelle that mediates chromosome attachment to spindle microtubules and hence is essential for proper chromosome segregation and cell cycle progression. We found that cells with deletion of PLC1 gene (plc1delta) display higher frequency of chromosome loss, nocodazole sensitivity, and mitotic delay. Furthermore, chromatin extracts from p1c1delta cells exhibit reduced microtubule binding to minichromosomes. However, it remains unknown whether the enzymatic activity of Plc1p is required for proper mitotic function, or whether the mere binding of this protein to kinetochores is sufficient for normal behavior, or whether Plc1p's kinetochore-binding and its enzymatic activity are both required. Our Specific Aims will resolve these alternative possibilities. We will prepare two types of Plc1p mutants: enzymatically inactive mutants with preserved ability to interact with kinetochores, and (ii) mutants which retain enzymatic activity but are unable to interact with kinetochores. Each mutant Plc1p protein will be characterized biochemically and the yeast strains expressing these mutant Plc1p proteins will be fully characterized in terms of fidelity of chromosome transmission, mitotic delay, sensitivity to nocodazole, and the ability of minichromosomes to bind microtubules. The results will lead to better understanding of the molecular mechanisms regulating chromosome segregation during mitosis, cell proliferation, and oncogenesis.

描述（申请人的描述） 我们实验室的长期目标是鉴定和表征真核生物 磷脂酶C（PLC）调节的细胞过程， 在信号转导通路中的重要作用。PLC水解 磷脂酰肌醇4，5-二磷酸[PtdIns（4，5）P2]产生两个重要的 第二信使：三磷酸肌醇[Ins（1，4，5）P3]， 释放钙从内部存储，和二酰基甘油（DG）， 激活磷脂和Ca 2+依赖性蛋白激酶C。 我们最近的结果（Lin等人，2000）证明了PLC在酵母中 酿酒酵母（由PLC 1基因编码的Plc 1 p蛋白）与 影响其结合微管的能力。动粒是 介导染色体附着到纺锤体上的特殊细胞器 微管，因此是必要的适当的染色体分离和细胞 循环进展我们发现PLC 1基因缺失的细胞（plc 1 delta） 显示较高的染色体丢失频率，诺考达唑敏感性，和 有丝分裂延迟此外，从p1 c1 delta细胞中提取的染色质显示， 减少微管与微型染色体的结合。然而， Plc 1 p的酶活性是否是正常有丝分裂所必需的， 功能，或者这种蛋白质与动粒的结合是否仅仅是 是否足以维持正常行为，或者Plc 1 p的运动舞蹈结合及其 酶活性都是必需的。 我们的具体目标将解决这些替代可能性。我们将准备 两种类型的Plc 1 p突变体：酶失活突变体， 与动粒相互作用的能力，和（ii）保留酶促的突变体 活动，但不能与动粒相互作用。每个突变体Plc 1 p 蛋白质将进行生物化学表征，并且表达 这些突变的Plc 1 p蛋白质将在保真度方面得到充分表征， 染色体传递、有丝分裂延迟、对诺考达唑的敏感性，以及 微型染色体结合微管的能力。结果将导致 更好地了解调控染色体的分子机制 在有丝分裂、细胞增殖和肿瘤发生期间的分离。

项目成果

Phospholipase C interacts with Sgd1p and is required for expression of GPD1 and osmoresistance in Saccharomyces cerevisiae.

磷脂酶 C 与 Sgd1p 相互作用，是酿酒酵母中 GPD1 表达和渗透阻力所必需的。

• DOI: 10.1007/s00438-002-0647-8
• 发表时间: 2002
• 期刊: Molecular genetics and genomics : MGG.
• 作者: Lin,H;Nguyen,P;Vancura,A
• 通讯作者: Vancura,A

Interaction of Pik1p and Sjl proteins in membrane trafficking.

Pik1p 和 Sjl 蛋白在膜运输中的相互作用。

• DOI: 10.1016/j.femsyr.2004.09.007
• 发表时间: 2005
• 期刊: FEMS yeast research
• 影响因子: 3.2
• 作者: Nguyen,PeterH;Hasek,Jiri;Kohlwein,SeppD;Romero,Carlos;Choi,JaeH;Vancura,Ales
• 通讯作者: Vancura,Ales

Expression of FLR1 transporter requires phospholipase C and is repressed by Mediator.

FLR1 转运蛋白的表达需要磷脂酶 C，并受到介体的抑制。

• DOI: 10.1074/jbc.m506728200
• 发表时间: 2006
• 期刊: The Journal of biological chemistry
• 作者: Romero,Carlos;Desai,Parima;DeLillo,Nicholas;Vancura,Ales
• 通讯作者: Vancura,Ales

Plc1p is required for SAGA recruitment and derepression of Sko1p-regulated genes.

Plc1p 是 SAGA 招募和 Sko1p 调节基因去抑制所必需的。

• DOI: 10.1091/mbc.e06-10-0946
• 发表时间: 2007
• 期刊: Molecular biology of the cell
• 影响因子: 3.3
• 作者: Guha,Nilanjan;Desai,Parima;Vancura,Ales
• 通讯作者: Vancura,Ales