Intracellular Transport: The Mannose Phosphate Receptor

描述(申请人提供)：这项研究的长期目标是确定哺乳动物细胞膜运输的分子基础。关注的焦点是甘露糖6-磷酸受体(MPR)，它将新合成的溶酶体酶从高尔基体运送到溶酶体前体位，然后返回高尔基体以获得更多货物。最近发现的几种蛋白质对于MPR从晚期内体到跨高尔基体网络的运输是必需的：识别晚期内体中MPR的货物选择蛋白(TIP47)，用于在TGN融合MPR-囊泡的路径特异性SNAR复合体，以及在高尔基体的囊泡拴系中起作用的两种蛋白质(GCC185和RhoBTB3)。这项应用的目标是(1)准确定义从早期内体运回高尔基体的货物所采取的不同路线，重点是与霍乱毒素比较，重点是MPR；(2)对MPR循环所需的蛋白质进行全基因组、自动化的siRNA筛选。该筛选将利用这样一个事实，即MPR循环所需的蛋白质的耗尽导致MPR分散到外围局部的细胞隔间。计算机软件可以检测这种扩散，允许自动分析22,000个siRNA导入培养细胞的影响。(3)还提出了进一步表征两个新的Rab9效应器的实验，这两个效应器对这种转运途径很重要：RhoBTB3和RUTBC1。总之，这些实验开辟了MPR贩运领域的全新研究领域，并将提供有关人体细胞受体贩运机制的基本信息。这项工作对我们理解包括糖尿病、癌症、心脏病和神经疾病在内的许多疾病状态具有广泛的应用。与公共卫生相关：膜交通对于我们分泌和响应胰岛素、清除血液中的胆固醇以及免疫系统细胞杀灭病原体的能力至关重要。膜运输的缺陷是许多疾病状态的基础，病毒感染依赖于这一过程。通过了解负责膜交通的分子事件，我们将能够更好地干预各种疾病状态。

DESCRIPTION (provided by applicant): The long term goal of this research is to determine the molecular basis of membrane traffic in mammalian cells. The focus is on mannose 6-phosphate receptors (MPRs) that deliver newly synthesized lysosomal enzymes from the Golgi to pre-lysosomes, and then return to the Golgi to pick up more cargo. Several recently discovered proteins are needed for MPR transport from late endosomes to the trans Golgi network: a cargo selection protein that recognizes the MPRs in late endosomes (TIP47), a pathway- specific SNARE complex for fusion of MPR-vesicles at the TGN, and two proteins that function in vesicle tethering at the Golgi (GCC185 and RhoBTB3). The goals of this application are (1) to define precisely, the distinct routes taken by cargoes that are transported from early endosomes back to the Golgi, with focus on MPRs in comparison with cholera toxin; (2) to carry out a genome-wide, automated siRNA screen for proteins needed for MPR recycling. The screen will make use of the fact that depletion of proteins needed for MPR recycling leads to dispersal of MPRs into peripherally localized cellular compartments. Computer software can detect this dispersal, permitting automated analysis of the effects of 22,000 siRNAs transfected into cultured cells. (3) Also proposed are experiments to further characterize two novel Rab9 effectors that are important for this trafficking pathway: RhoBTB3 and RUTBC1. In summary, these experiments open up entirely new areas of investigation in the area of MPR trafficking and will provide fundamental information regarding the mechanisms of receptor trafficking in human cells. The work has broad application to our understanding of a number of disease states including diabetes, cancer, heart disease and neurological disorders. PUBLIC HEALTH RELEVANCE: Membrane traffic is essential for our ability to both secrete and respond to insulin, to clear cholesterol from the bloodstream, and for cells of the immune system to kill pathogens. Defects in membrane traffic underlie a number of disease states and virus infection depends upon this process. By understanding the molecular events responsible for membrane traffic, we will be better able to intervene in a variety of disease states.