Characterization of Sacl lipid phosphatases

说明(申请人提供)：磷酸化肌醇磷脂(磷脂酰肌醇)通过下游脂结合效应蛋白调节多种细胞功能。肌醇磷脂调控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，肌醇磷脂与人类疾病的发展有关，这推动了人们对肌醇磷脂日益增长的兴趣。特别是，编码脂肪磷酸酶的基因突变与各种严重疾病有关，如严重的先天性疾病、糖尿病和癌症。因此，这些酶的特性和它们执行的细胞功能具有相当大的生物医学相关性。这项研究的中心假设是SAC1脂磷酸酶在协调内质网(ER)和高尔基体功能对营养物质和细胞生长速度的反应中起着关键作用。我们的初步数据显示，Dpmlp是参与糖基化的一种重要的ER酶，在细胞快速分裂期间将Saclp招募到ER膜上。营养限制会减缓细胞增殖，并引发Sac1p和Dpm1p的解离，导致这种脂质磷酸酶在高尔基体积累。这个建议的目标是了解细胞生长特异性的Sac1p在内质网和高尔基体之间的分配是如何调节的，以及这个过程是如何协调这些细胞器的分泌能力的。我们将利用遗传和生化分析来表征Sac1p依赖细胞生长的定位机制。我们还将使用荧光脂质结合探针来识别SAC1控制的肌醇磷脂池，并检测它们在膜运输和细胞器功能中的作用。对SAC1脂磷酸酶特定功能的表征将增加我们对分泌细胞器膜上动态过程的特异性是如何实现的了解。深入了解内质网和高尔基体膜上脂质信号的调节也将提高我们对分泌途径组织的理解。

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.