TRAFFICKING BY FYVE DOMAIN EFFECTORS OF PI3 KINASE

磷酸肌醇介导的信号传导调节广泛的细胞途径，包括细胞器之间的蛋白质和脂质的分选、细胞增殖和死亡（凋亡）、细胞运动和细胞骨架。 磷酸肌醇3-激酶（PI 3 Ks），其磷酸化肌醇环的D-3位置，已经被认为是这些途径中的每一个中的重要调节酶。 PI 3 K信号传导的下游效应至少部分地由直接结合PI 3 K产物的效应蛋白介导，包括最近描述的RING结构域的FYVE亚家族。 FYVE结构域与磷脂酰肌醇（PI）的结合似乎具有极高的特异性;有两种结构域几乎只与磷脂酰肌醇3-磷酸（PtdIns（3）P）结合，这表明含有FYVE结构域的蛋白质仅是PI 3 K亚组的效应物。 本研究的长期目标是了解PI 3 K在酿酒酵母中对蛋白质和膜运输的调控。 该生物体具有由VPS 34基因编码的单个PI 3 K和含有FYVE结构域的五种蛋白质。 这些研究主要集中在四个FYVE结构域蛋白上。 在特定目标1中，将使用生物化学、遗传学和显微镜方法来鉴定Vac 1 p与高尔基体至内体运输机制的其他组分之间的相互作用，这些组分受Vps 34 PI 3 K调节。 在特定目标2中，将使用细胞分级分离和基于荧光显微镜的方法的组合来确定Vac 1 p定位于内体细胞器的机制。 在酵母基因组中编码有两种目前未表征的FYVE结构域蛋白，Pib 1 p和Pib 2 p。 在特定目标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。 在特定目标4中，将鉴定高亲和力PtdIns（3）P结合所需的人EEA 1 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P13 Ks如何调节囊泡介导的细胞器间运输，并将提供一个结构/功能框架，了解新发现的FYVE结构域蛋白的功能。

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.