Mechanism and function of ubiquitin binding by conserved endocytic protein Rvs167

描述（由申请人提供）：网格蛋白介导的内吞作用是所有真核细胞中保守的基本过程，并且是许多重要功能所需的，包括营养摄取、膜重塑、突触囊泡再循环和下调活化的信号传导受体。Rvs167是一种与哺乳动物内吞因子endophilin和amphiphysin同源的BAR结构域蛋白，是酵母生长控制和内吞作用所必需的。最近，Rvs167被证明可以结合泛素，初步研究表明，泛素结合是Rvs167功能所必需的。本申请提出通过以下目的来确定Rvs167与泛素结合的机制和功能：1）通过用重组Rvs167的不同片段或突变体进行结合测定来确定Rvs167与泛素之间的新型相互作用的分子基础。2)定义泛素结合在细胞生长，肌动蛋白组织和内吞作用中的作用。将分析泛素结合缺陷细胞对营养剥夺、肌动蛋白丝结构和体相和受体依赖性标志物的内吞作用的生长停滞。此外，实时荧光显微镜将用于监测Rvs167的野生型和泛素结合缺陷型荧光蛋白融合体在共表达其他内吞蛋白与替代荧光标签的菌株中的定位和动力学。3)鉴定和表征通过泛素结合与Rvs167相互作用的蛋白质。通过全基因组酵母双杂交筛选、GST融合蛋白亲和层析或通过酵母裂解物的共免疫沉淀来鉴定相互作用蛋白。一旦鉴定，相互作用蛋白的缺失和Rvs167结合缺陷突变体将进行目的2中所述的测定。这些研究预计将有助于了解负责内吞作用和细胞生长控制的分子机制，并为了解内吞作用的缺陷如何导致人类疾病提供基础。内吞作用是一个基本的细胞过程，从酵母到人类都是保守的。Rvs167是两种人类内吞蛋白的近亲，并且是酵母中生长控制和内吞作用所必需的。这些对Rvs167的研究预计将有助于理解内吞作用的基本分子机制，并为理解内吞作用的缺陷如何导致人类疾病提供基础。

DESCRIPTION (provided by applicant): Clathrin-mediated endocytosis is a fundamental process that is conserved in all eukaryotic cells, and is required for numerous important functions including nutrient uptake, membrane remodeling, synaptic vesicle recycling and down-regulation of activated signaling receptors. Rvs167, a BAR domain protein homologous to the mammalian endocytic factors endophilin and amphiphysin, is required for growth control and endocytosis in yeast. Recently, Rvs167 was demonstrated to bind ubiquitin, and preliminary studies suggest that ubiquitin binding is required for the function of Rvs167. This application proposes to define the mechanism and function of ubiquitin binding by Rvs167 through the following aims: 1) Determine the molecular basis for the novel interaction between Rvs167 and ubiquitin by conducting binding assays with different fragments or mutants of recombinant Rvs167. 2) Define the role of ubiquitin binding in cell growth, actin organization and endocytosis. Ubiquitin binding-defective cells will be analyzed for growth arrest in response to nutrient deprivation, actin filament structure, and endocytosis of both bulk-phase and receptordependent markers. Additionally, real-time fluorescent microscopy will be used to monitor the localization and dynamics of wild-type and ubiquitin binding-defective fluorescent protein fusions of Rvs167 in strains co-expressing other endocytic proteins with alternate fluorescent tags. 3) Identify and characterize protein(s) that interact with Rvs167 via ubiquitin binding. Interacting proteins will be identified by a whole genome yeast-2-hybrid screen, GST fusion protein affinity chromatography, or by coimmunoprecipitation from yeast lysates. Once identified, deletion and Rvs167-binding defective mutants of interacting proteins will be subjected to assays described in aim 2. These studies are anticipated to contribute to understanding of the molecular mechanisms responsible for endocytosis and cell growth control, and to provide a foundation to understand how defects in endocytosis contribute to human disease. Endocytosis is a fundamental cellular process that is conserved from yeast to humans. Rvs167 is a close relative of two human endocytic proteins and is required for growth control and endocytosis in yeast. These studies on Rvs167 are anticipated to contribute to understanding the basic molecular mechanisms responsible for endocytosis and to provide a foundation for understanding how defects in endocytosis contribute to human disease.