Intracellular Transport: The Mannose Phosphate Receptor

描述（由申请人提供）：本研究的长期目标是确定哺乳动物细胞中膜运输的分子基础。重点是甘露糖6-磷酸受体（MPRs），它将新合成的溶酶体酶从高尔基体传递到前溶酶体，然后返回高尔基体以获取更多的货物。最近发现的MPR从晚期核内体转运到反高尔基网络需要几种蛋白质：识别晚期核内体中MPR的货物选择蛋白（TIP47）， TGN中MPR-囊泡融合的通路特异性SNARE复合体，以及两个在高尔基体中囊泡系结起作用的蛋白质（GCC185和RhoBTB3）。该应用程序的目标是：(1)精确定义从早期内体运输回高尔基体的货物所采取的不同路线，重点是将MPRs与霍乱毒素进行比较；(2)对MPR回收所需的蛋白质进行全基因组的siRNA自动筛选。筛选将利用这样一个事实，即MPR再循环所需的蛋白质耗尽导致MPR分散到周围定位的细胞室中。计算机软件可以检测到这种扩散，允许自动分析22000个sirna转染到培养细胞中的影响。(3)还提出了进一步表征Rab9转运途径中两个重要的新型效应物的实验：RhoBTB3和RUTBC1。总之，这些实验在MPR贩运领域开辟了全新的研究领域，并将提供有关人类细胞中受体贩运机制的基本信息。这项工作广泛应用于我们对许多疾病状态的理解，包括糖尿病、癌症、心脏病和神经系统疾病。公共卫生相关性：膜交通对于我们分泌胰岛素和对胰岛素作出反应，清除血液中的胆固醇，以及免疫系统细胞杀死病原体的能力至关重要。膜传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.