Intracellular Transport: The Mannose Phosphate Receptor

项目总结/摘要 本研究的长期目标是确定膜的分子基础 哺乳动物细胞的运输。重点是甘露糖6-磷酸受体（MPR） 将新合成的溶酶体酶从高尔基体传递到前溶酶体， 然后再回到高尔基去拿更多的货物最近发现的几种蛋白质 MPR从晚期内体运输到高尔基体网络所需的：货物 识别晚期内体中MPR的选择蛋白（TIP47），一种途径- 特异性SNARE复合物，用于在TGN融合MPR-囊泡，以及两种蛋白质， 在高尔基体的囊泡束缚中起作用（GCC185和RhoBTB3）。这个的目标 应用是（1）精确定义，货物所采取的不同路线， 从早期内体转运回高尔基体，与比较中的MPR相比， 与霍乱毒素;（2）进行全基因组，自动siRNA筛选蛋白质 用于MPR回收。屏幕将利用这样一个事实， MPR再循环所需的蛋白质导致MPR分散到外周 局部细胞区室。计算机软件可以检测到这种扩散， 允许自动分析22，000个siRNA转染到培养的 细胞(3)还提出了进一步表征两种新型Rab9的实验 对这种运输途径很重要的效应子：RhoBTB3和RUTBC 1。在 总之，这些实验开辟了研究领域的全新领域， MPR贩运，并将提供有关的机制， 受体在人体细胞中的运输。这项工作对我们的 了解许多疾病状态，包括糖尿病、癌症、心脏病 和神经系统疾病。

Project Summary/Abstract 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.