TRAFFICKING BY FYVE DOMAIN EFFECTORS OF PI3 KINASE

磷酸肌醇介导的信号传导调节了广泛的细胞通路，包括细胞器之间的蛋白质和脂质分选、细胞增殖和死亡（凋亡）、细胞运动和细胞骨架。磷酸化肌醇环D-3位置的磷酸肌醇3激酶（PI3Ks）在这些途径中都是重要的调节酶。PI3K信号的下游作用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。磷酸肌肽（PI）与FYVE结构域的结合似乎是非常特异性的；其中两种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。本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。该生物具有一个由VPS34基因编码的PI3K和五个含有FYVE结构域的蛋白质。提出的研究重点是四个FYVE结构域蛋白。在Specific Aim 1中，生化、遗传和显微方法将用于鉴定Vac1p和其他由Vps34 PI3K调控的高尔基到核内体运输机制的组分之间的相互作用。在特异性目标2中，Vac1p定位于内体细胞器的机制将使用细胞分离和荧光显微镜相结合的方法来确定。酵母基因组中编码有两个目前尚未鉴定的FYVE结构域蛋白，Pib1p和Pib2p。在Specific Aim 3中，将鉴定这些蛋白质定位的细胞内区室，并鉴定和表征这些蛋白质调节的运输途径。在Specific Aim 4中，将鉴定高亲和力PtdIns(3)P结合所需的人类EEA1 FYVE结构域残基。另外5种FYVE结构域蛋白的pi结合特异性将被确定，并检测它们的FYVE结构域对pi结合的相对贡献。这些研究将有助于理解P13Ks如何调节囊泡介导的细胞器间运输，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.