TRAFFICKING BY FYVE DOMAIN EFFECTORS OF PI3 KINASE

肌醇磷脂介导的信号调节广泛的细胞通路，包括细胞器之间蛋白质和脂类的分选、细胞增殖和死亡(细胞凋亡)、细胞运动和细胞骨架。磷脂酰肌醇3-激酶(PI3Ks)是肌醇环D-3位的磷酸化产物，在这些途径中都是重要的调节酶。PI3K信号的下游效应至少部分是由直接与PI3K产物结合的效应蛋白介导的，包括最近描述的FYVE环域亚家族。磷脂酰肌醇(PI)与FYVE结构域的结合似乎具有极高的特异性；有两个被证明几乎能与磷脂酰肌醇3-磷酸(PtdIns(3)P)结合，这表明含有FYVE结构域的蛋白质只是PI3Ks的一个子集的效应器。本研究的长期目标是了解PI3K对酿酒酵母中蛋白质和膜运输的调节。这种生物只有一个由Vps34基因编码的PI3K和五个含有FYVE结构域的蛋白质。拟议的研究集中在四个FYVE结构域蛋白上。在具体目标1中，将使用生化、遗传和显微方法来确定Vac1p与受Vps34 PI3K调控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，Vac1p定位到内体细胞器的机制将使用细胞分离和基于荧光显微镜的方法相结合来确定。目前在酵母基因组中编码的FYVE结构域蛋白有两个，Pib1p和Pib2p。在具体目标3中，将确定这些蛋白质所在的细胞内隔间，并将识别和表征由这些蛋白质调控的运输途径。在特定目标4中，将鉴定高亲和力PtdIns(3)P结合所需的人EEA1 FYVE结构域的残基。将确定其他五个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PI结合的相对贡献。这些研究将有助于理解P13Ks如何调控囊泡介导的细胞器间转运，并将为理解新发现的FYVE结构域蛋白的功能提供一个结构/功能框架。

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.