Characterization of Sacl lipid phosphatases

Sacl 脂质磷酸酶的表征

• 批准号: 7085499
• 负责人: PETER J MAYINGER
• 金额: $ 27.47万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7085499
• 关键词: Golgi apparatus; SDS polyacrylamide gel electrophoresis; biological signal transduction; biological transport; cell growth regulation; density gradient ultracentrifugation; endoplasmic reticulum; enzyme activity; fluorescence microscopy; fluorescent dye /probe; green fluorescent proteins; lipids; mass spectrometry; membrane transport proteins; phosphatidylinositols; phosphoprotein phosphatase; phosphorylation; protein structure function; western blottings; yeast two hybrid system

DESCRIPTION (provided by applicant): Phosphorylated inositol phospholipids (phosphoinositides) regulate a multitude of cellular functions via downstream lipid-binding effector proteins. Phosphoinositide-controlled processes include cytoskeletal organization, gene expression, cell proliferation and membrane trafficking. The increasing interest in phosphoinositides is fueled by evidence that they are related to the development of human diseases. In particular, mutations in genes encoding lipid phosphatases were linked to a variety of severe maladies such as serious congenital disorders, diabetes, and cancer. Thus, the characterization of these enzymes and of the cellular function they perform assumes considerable biomedical relevance. The central hypothesis of this study proposes a pivotal role for the Sac1 lipid phosphatase in coordinating endoplasmic reticulum (ER) and Golgi function in response to nutrients and cell growth rates. Our preliminary data show that dolicholphosphate mannose synthase Dpmlp, an essential ER enzyme involved in glycosylation, recruits Saclp to ER membranes during times of rapid cell division. Nutrient limitation slows cell proliferation and triggers dissociation of Sac1p from Dpm1p, causing accumulation of this lipid phosphatase at the Golgi. The goal of this proposal is to understand how cell growth-specific distribution of Sac1p between ER and Golgi is regulated and how this process coordinates the secretory capacity of these organelles. We will characterize the mechanisms for cell growth-dependent localization of Sac1p using genetic and biochemical analyses. We will also employ fluorescent lipid-binding probes to identify Sac1 -controlled pools of phosphoinositides and examine their role in membrane trafficking and organellar function. Characterization of the specific functions of the Sac1 lipid phosphatase will increase our knowledge of how the specificity of dynamical processes at the membranes of secretory organelles is achieved. Insight into the regulation of lipid signals at ER and Golgi membranes will also improve our understanding of the organization of the secretory pathway.

描述（由申请人提供）：磷酸化肌醇磷脂（磷酸肌醇）通过下游脂质结合效应蛋白调节多种细胞功能。磷酸肌醇控制的过程包括细胞骨架组织、基因表达、细胞增殖和膜运输。有证据表明磷酸肌醇与人类疾病的发展有关，这加剧了人们对磷酸肌醇的兴趣。特别是，编码脂质磷酸酶的基因突变与多种严重疾病有关，例如严重先天性疾病、糖尿病和癌症。因此，这些酶及其执行的细胞功能的表征具有相当大的生物医学相关性。这项研究的中心假设提出，Sac1 脂质磷酸酶在协调内质网 (ER) 和高尔基体功能以响应营养和细胞生长速率方面发挥着关键作用。我们的初步数据表明，多醇磷酸甘露糖合酶 Dpmlp（一种参与糖基化的必需内质网酶）在细胞快速分裂期间将 Saclp 招募到内质网膜上。营养限制会减慢细胞增殖并触发 Sac1p 从 Dpm1p 解离，导致这种脂质磷酸酶在高尔基体积聚。该提案的目的是了解 Sac1p 在 ER 和高尔基体之间的细胞生长特异性分布是如何调节的，以及这个过程如何协调这些细胞器的分泌能力。我们将使用遗传和生化分析来描述 Sac1p 细胞生长依赖性定位的机制。我们还将采用荧光脂质结合探针来识别 Sac1 控制的磷酸肌醇池，并检查它们在膜运输和细胞器功能中的作用。 Sac1 脂质磷酸酶的特定功能的表征将增加我们对如何实现分泌细胞器膜动力学过程的特异性的了解。对内质网和高尔基膜脂质信号调节的深入了解也将提高我们对分泌途径组织的理解。