Rab11-FIPs interact to define distinct functional nodes in the recycling system

描述（由申请人提供）：蛋白质运输和运输是细胞功能的一个重要方面，因为细胞通过细胞表面蛋白质的内化和释放与环境相互作用。细胞表面蛋白的异常加工或转运会导致尿崩症、囊性纤维化和阿尔茨海默病等疾病。虽然特定的货物，如通道和受体对不同的细胞类型是独特的，但质膜回收过程在哺乳动物细胞中是高度保守的，并由包括Rab小gtp酶在内的一组共同的运输蛋白调节。Rab11是一种普遍表达的小GTPase，参与再循环，与Rab11家族相互作用蛋白（FIPs）相互作用。虽然FIPs的功能还没有被完全理解，但它们可能在一定程度上定义了回收系统中不同的子域。此外，初步数据表明FIP2在不同的亚细胞定位中以不同的构象存在。这些数据导致假设与Rab11相互作用的FIPs的离散寡聚物允许Rab11和FIP功能在回收系统中的时间和空间特异性。为了验证这一观点，提出了两个目标。首先，FIP的同二聚化和异二聚化能力将通过酵母双杂交、FRET显微镜和蛋白质共定位来评估，作为了解FIP在循环系统中的功能的一种方式。其次，FIP2构象将在循环系统内的分子内和分子间相互作用方面进行研究。通过阐明FIP复合物的组成及其亚细胞定位，Rab11和FIPs作为质膜循环的一般调节因子的重要性可能会被揭示。

DESCRIPTION (provided by applicant): Protein trafficking and transport is a crucial aspect of cellular function because cells interact with the environment by internalization and release of proteins from the cell surface. Aberrant processing or transport of cell surface proteins contributes to diseases such as diabetes insipidus, cystic fibrosis, and Alzheimer's disease. While the particular cargo such as channels and receptors is unique to different cell types, the process of plasma membrane recycling is highly conserved among mammalian cells and regulated by a common set of trafficking proteins including the Rab small GTPases. Rab11 is a ubiquitously expressed small GTPase involved in recycling that interacts with the Rab11-Family Interacting Proteins (FIPs). Although the functions of FIPs are not fully understood, they may in part define distinct subdomains within the recycling system. Furthermore, preliminary data suggest that FIP2 exists in different conformations in different subcellular localizations. These data lead to the hypothesis that discrete oligomers of FIPs interacting with Rab11 allow for temporal and spatial specificity of Rab11 and FIP function within the recycling system. Two aims are proposed to test this idea. First, homodimerization and heterodimerization capabilities of FIPs will be assessed by yeast two hybrid, FRET microscopy, and protein colocalization as a way to understand FIP function within the recycling system. Second, FIP2 conformation will be studied with regards to intramolecular and intermolecular interactions within the recycling system. By elucidating the composition of FIP complexes and their subcellular localizations, the importance of Rab11 and FIPs as general regulators of plasma membrane recycling may be uncovered. SIGNIFICANCE: Proper protein transport and localization are crucial to maintenance of normal cellular function. Diseases such as cystic fibrosis, diabetes insipidus, and Alzheimer's disease result from mislocalization or aberrant trafficking of vital proteins. This proposal aims to increase the understanding of the basic mechanism of protein trafficking that is common to all cell types by studying an important trafficking protein, Rab11, and its interacting proteins.