Molecular Mechanisms of Apical Membrane Traffic in Epithelial Cells

描述(申请人提供)：上皮细胞通过在生物发生和内吞周期中持续分选来建立和维持膜蛋白和脂类的极化分布。蛋白质靶向对大多数上皮功能是至关重要的。因此，它的缺陷被认为会导致各种肾脏和消化系统疾病。我这项研究的主要目的是研究调控Rab 11依赖的顶端蛋白在上皮细胞中的靶向机制。靶向蛋白质是通过使用与特定靶区融合的运输小泡/小管来实现的。囊泡蛋白Rab 11已被证明是参与顶膜运输的关键分子，甚至可能作为囊泡的“地址”标签。一般来说，RABS通过招募效应蛋白来运输囊泡来发挥作用。最近的研究发现了几种新的Rab 11结合蛋白，包括RIP11、FIP2和Eferin/FIP3，也称为FIP。根据我已发表的和初步的数据，我推测：(I)Rab 11/Rip11复合体通过在运输小泡上形成“靶向斑块”来调节顶膜的运输，(Ii)Rab 11/Rip 11的“靶向斑块”是由Rip L 1磷酸化来调节的，以及(Iii)Rab 11/Rip 11复合体的功能是通过招募额外的蛋白质，如激动素II来运输小泡来实现的。为了进一步分析Rab 11/效应器复合体的结构和功能，我们设计了四个特定的目标。首先，将利用结构域图和X射线结晶学研究Rab 11与RIP11相互作用的结构基础。其次，我们将使用AIM#1产生的结构信息来确定RIP11以及其他FIP在体内转运分析中用于心尖和基底外侧靶向的作用。第三，Rip11的磷酸化在极化上皮交通中的作用将被研究。第四，Rab 11/Rip 11复合体相互作用蛋白的鉴定和鉴定。Rab 11/Rip11复合体的特性将为理解蛋白质在上皮细胞中的靶向提供重要的机制信息。

DESCRIPTION (provided by applicant): Epithelial cells establish and maintain the polarized distribution of membrane proteins and lipids by continuously sorting them during biogenesis and endocytic cycles. Protein targeting is critical for most epithelial functions. Consequently, its defects are known to lead to a variety of renal and digestive disorders. The main goal of my research is to investigate the mechanisms that regulate Rab 11-dependent apical protein targeting in epithelial cells. Targeting of proteins is achieved through the use of transport vesicles/tubules that fuse with specific target compartments. The vesicle protein, Rab 11, has been shown to be a key molecule involved in apical membrane traffic and may even function as a vesicle "address" tag. In general, Rabs function by recruiting effector proteins to transport vesicles. Recent studies have identified several novel Rab 11-binding proteins, including Rip 11, FIP2, and Eferin/FIP3, also known as FIPs. Based on my published and preliminary data, I hypothesize that (i) Rab 11/Rip11 complexes regulate apical membrane traffic by forming "targeting patches" on transport vesicles, (ii) Rab 11/Rip 11 "targeting patch" is regulated by Rip l 1 phosphorylation, and (iii) Rab 11/Rip 11 complex function by recruiting additional proteins, such as kinesin II to transport vesicles. Four specific aims are designed to further analyze the structure and function of Rab 11/effector complexes. First, the structural basis for Rab 11 interactions with Rip 11 will be investigated using domain mapping and X-ray crystallography. Second, we will use the structural information generated by Aim #1 to determine the role of Rip 11 as well as other FIPs in apical and basolateral targeting using in in vivo transport assays. Third, the role of Rip11 phosphorylation in polarized epithelial traffic will be investigated. Forth, identification and characterization of the proteins interacting with Rab 11/Rip 11 complexes. Characterization of Rab 11/Rip11 complex will provide critical mechanistic information for understanding protein targeting in epithelial cells.