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广泛的细胞途径，包括细胞器之间蛋白质和脂质的分类、细胞增殖和死亡（细胞凋亡）、细胞运动和细胞骨架。 磷酸肌醇 3-激酶 (PI3K) 可磷酸化肌醇环的 D-3 位置，已被认为是这些途径中的重要调节酶。 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 RING 结构域的 FYVE 亚家族。 FYVE 结构域与磷酸肌醇 (PI) 的结合似乎具有极高的特异性；两个已被证明几乎完全结合磷脂酰肌醇 3-磷酸 (PtdIns(3)P)，这表明含有 FYVE 结构域的蛋白质仅是 PI3K 子集的效应器。 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。 该生物体具有由 VPS34 基因编码的单个 PI3K 和五个包含 FYVE 结构域的蛋白质。 拟议的研究重点关注四种 FYVE 结构域蛋白。 在具体目标 1 中，将使用生化、遗传和微观方法来识别 Vac1p 与受 Vps34 PI3K 调节的高尔基体到内体运输机制的其他组件之间的相互作用。 在具体目标 2 中，将使用细胞分级分离和基于荧光显微镜的方法相结合来确定 Vac1p 定位于内体细胞器的机制。 酵母基因组中编码有两种目前尚未表征的 FYVE 结构域蛋白：Pib1p 和 Pib2p。 在具体目标 3 中，将鉴定这些蛋白质定位的细胞内区室，并鉴定和表征由这些蛋白质调节的运输途径。 在具体目标 4 中，将鉴定高亲和力 PtdIns(3)P 结合所需的人 EEA1 FYVE 结构域残基。 将确定其他五种 FYVE 结构域蛋白的 PI 结合特异性，并检查它们的 FYVE 结构域对总体 PI 结合的相对贡献。 拟议的研究将有助于了解 P13K 如何调节囊泡介导的细胞器间运输，并将为了解新发现的 FYVE 结构域蛋白的功能提供结构/功能框架。