Molecular Mechanisms of Apical Membrane Traffic in Epithelial Cells

描述（由申请人提供）：上皮细胞在生物发生和内吞周期中通过不断分选膜蛋白和脂质来建立和维持膜蛋白和脂质的极化分布。蛋白质靶向对大多数上皮功能至关重要。因此，已知它的缺陷会导致各种肾脏和消化系统疾病。我研究的主要目的是研究在上皮细胞中调节Rab 11依赖的根尖蛋白靶向的机制。蛋白质的靶向是通过使用与特定靶室融合的运输囊泡/小管来实现的。囊泡蛋白rab11已被证明是参与顶膜运输的关键分子，甚至可能起囊泡“地址”标签的作用。一般来说，Rabs通过招募效应蛋白来运输囊泡。最近的研究发现了几种新的Rab 11结合蛋白，包括Rip 11、FIP2和Eferin/FIP3，也被称为FIPs。根据我发表的和初步的数据，我假设(I) Rab 11/Rip11复合物通过在运输囊泡上形成“靶向斑块”来调节顶膜交通，（ii） Rab 11/Rip11“靶向斑块”由rip1磷酸化调节，以及（iii） Rab 11/Rip11复合物通过招募额外的蛋白质（如运动蛋白ii）来运输囊泡。设计了四个具体目标来进一步分析Rab 11/效应复合物的结构和功能。首先，利用区域映射和x射线晶体学研究Rab 11与Rip 11相互作用的结构基础。其次，我们将使用Aim #1生成的结构信息，通过体内转运试验确定Rip 11以及其他FIPs在根尖和基底外侧靶向中的作用。第三，将研究Rip11磷酸化在极化上皮运输中的作用。第四，与Rab 11/Rip 11复合物相互作用蛋白的鉴定和表征。Rab 11/Rip11复合物的鉴定将为理解上皮细胞中的蛋白靶向提供关键的机制信息。

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.