STEROL SENSING AND TRANSPORT IN A MODEL EUKARYOTE

细胞内游离甾醇的浓度被严格调节到 优化所有真核细胞膜的流动性和功能。这 动态平衡是由几种机制赋予的，其关键是 甾醇的感觉和该分子从合成部位的运输 或摄取到细胞内细胞器，在那里它可以被代谢或 被利用了。这些过程中的异常会导致多种疾病。 病理，包括动脉粥样硬化和Niemann Pick C(NPC)病。 在本项目中，将应用酵母中的遗传策略来研究 与人类鼻咽癌疾病有关的类固醇运输和感觉。vbl.使用 一种基于人类间序列保守的候选基因方法 酵母菌，我们已经鉴定出一个与该基因极好的酵母同源物 Niemann Pick C型病(NPC1)缺陷。有两个 原则性针对这一项目。目标1将定义 Niemann-Pick C型1基因家族与细胞内甾醇转运 这将检验NPC1及其酵母同系物Ncr1， 介导囊泡融合事件和/或作为甾醇反应信号 换能器。分子、遗传和生化方法，包括 定点突变和亚细胞蛋白质定位 被应用。AIM 2有两个组件，它们针对相互作用的通路 在鼻咽癌疾病中介导类固醇的运输和感觉。首先， 通过以下方法描述固醇转运途径的组成成分 与影响正常细胞的NCR1基因产物的相互作用 生存能力。第二个组件将分离出 NPC/NCR-Complex验证类固醇关键步骤假说 转运及其调控涉及蛋白质与蛋白质之间的相互作用。 与NCR1和NPC1复杂的因素将使用 酵母双杂交系统。NPC2基因可能来源于这一筛选。 这些研究可能会特别阐明鼻咽癌疾病和 一般情况下，类固醇在细胞内的运输。

The concentration of intracellular free sterol is strictly regulated to optimize the fluidity and function of all eukaryotic membranes. This homeostasis is imparted by several mechanisms, the crux of which is sensing of sterol and transport of this molecule from sites of synthesis or uptake to intracellular organelles where it can be metabolized or utilized. Aberrations in these processes lead to multiple disease pathologies, including atherosclerosis and Niemann Pick C (NPC) disease. In this project, genetic strategies in yeast will be applied to study sterol transport and sensing as it pertains to human NPC disease. Using a candidate gene approach based on sequence conservation between humans and yeast, we have identified an excellent yeast homolog to the gene defective in Niemann Pick type C disease (NPC1). There are two principle aims to this project. Aim 1 will define the role of the Niemann-Pick C type 1 gene family in intracellular sterol transport. This will test the hypothesis that NPC1 and its yeast homolog, Ncr1, mediate vesicle fusion events and/or act as sterol responsive signal transducers. Molecular, genetic and biochemical approaches including site-directed mutagenesis and sub-cellular protein localization, will be applied. Aim 2 has two components that target pathways that interact in NPC disease to mediate sterol transport and sensing. Firstly, to characterize components of sterol transport pathways by virtue of interactions with the NCR1 gene product that effect normal cell viability. The second component will isolate constituents of the NPC/Ncr-complex to test the hypothesis that key steps in sterol transport and its regulation involve protein-protein interactions. Factors that complex with NCR1 and NPC1 will be identified using the yeast two-hybrid system. The NPC2 gene may arise from this screen. These studies will likely elucidate NPC disease in particular and intracellular sterol transport in general.