TRAFFICKING BY FYVE DOMAIN EFFECTORS OF PI3 KINASE

通过 PI3 激酶的 FYVE 域效应器进行贩运

• 批准号: 6658929
• 负责人: Christopher G Burd
• 金额: $ 24.3万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6658929
• 关键词: Saccharomyces cerevisiae; fluorescence microscopy; intracellular transport; membrane activity; microorganism metabolism; phosphatidylinositol 3 kinase; phosphatidylinositols; protein binding; protein localization; protein structure function; vesicle /vacuole

Phosphoinositide-mediated signaling regulates a broad range of cellular pathways, including sorting of proteins and lipids between organelles, cell proliferation and death (apoptosis), cell motility, and the cytoskeleton. Phosphoinositide 3-kinases (PI3Ks), which phosphorylate the D-3 position of the inositol ring, have been implicated as important regulatory enzymes in each of these pathways. The downstream effects of PI3K signaling are mediated, at least in part, by effector proteins which bind PI3K products directly, including the recently described FYVE sub-family of RING domains. Phosphoinositide (PI) binding by FYVE domains appears to be exquisitely specific; two have been shown to bind phosphatidylinositol 3-phosphate (PtdIns(3)P) nearly exclusively, suggesting that proteins containing FYVE domains are effectors of only a subset of PI3Ks. The long term goal of this research is to understand regulation of protein and membrane trafficking by PI3K in the yeast Saccharomyces cerevisiae. This organism has a single PI3K, encoded by the VPS34 gene, and five proteins containing FYVE domains. The proposed studies focus on four FYVE domain proteins. In Specific Aim 1, biochemical, genetic, and microscopic methods will be used to identify interactions between Vac1p and other components of the Golgi-to-endosome trafficking machinery which are regulated by Vps34 PI3K. In Specific Aim 2, the mechanism by which Vac1p is localized to endosomal organelles will be determined using a combination of cell fractionation and fluorescence microscopy- based methods. There are two currently uncharacterized FYVE domain proteins encoded in the yeast genome, Pib1p and Pib2p. In Specific Aim 3, the intracellular compartments where these proteins are localized will be identified, and trafficking pathways regulated by these proteins will be ientified and characterized. In Specific Aim 4, residues of the human EEA1 FYVE domain required for high affinity PtdIns(3)P binding will be identified. The PI-binding specificities of five other FYVE domain proteins will be determined, and the relative contribution of their FYVE domains to overall PI-binding examined. The proposed studies will contribute to an understanding of how P13Ks regulate vesicle-mediated interorganelle trafficking, and will provide a structure/function framework for understanding the functions of newly discovered FYVE domain proteins.

磷酸肌醇介导的信号传导调节了广泛的细胞途径，包括细胞器之间的蛋白质和脂质分类，细胞增殖和死亡（凋亡），细胞运动性和细胞骨架。 磷酸化3-激酶（PI3KS）磷酸化肌醇环的D-3位置，已与这些途径中的每一个中的重要调节酶表示为重要的调节酶。 PI3K信号传导的下游效应至少部分是由直接结合PI3K产物的效应蛋白介导的，包括最近描述的fyve sub-fyve sub-fimily of Ring域。 FYVE结构域的磷酸肌醇（PI）结合似乎是精确的特异性。几乎只有两种结合3-磷酸磷脂酰肌醇（PTDINS（3）P）的磷脂酰肌醇，这表明含有FYVE结构域的蛋白质仅是PI3KS子集的效应子。 这项研究的长期目标是了解PI3K在酿酒酵母中对蛋白质和膜运输的调节。 该生物具有一个单个PI3K，由VPS34基因编码，五种含有FYVE结构域的蛋白质。 拟议的研究集中于四个FYVE结构域蛋白。 在特定的目标1中，将使用生化，遗传和微观方法来识别VAC1P与高尔基体到粘体运输机械的其他组件之间的相互作用，该组件受VPS34 PI3K的调节。 在特定的目标2中，将使用细胞分馏和基于荧光显微镜的方法的组合确定VAC1P定位到内体细胞器的机制。 目前有两个未表征的FYVE结构域蛋白，它们在酵母基因组，PIB1P和PIB2P中编码。 在特定的目标3中，将确定这些蛋白质所在的细胞内室，并且这些蛋白质调节的运输途径将被赋予和表征。 在特定的目标4中，将确定高亲和力PTDIN所需的人EEA1 FYVE结构域的残基（3）P结合。 将确定其他五个FYVE结构域蛋白的PI结合特异性，并研究其FYVE域对总体PI结合的相对贡献。 拟议的研究将有助于理解P13K如何调节囊泡介导的Interganelle贩运，并提供一个结构/功能框架，以了解新发现的FYVE结构域蛋白的功能。