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膜运输的分子基础。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，然后返回高尔基体以获得更多的货物。我们已经表明，蛋白质，GCC 185是必需的束缚MPR含囊泡在transGolgi。为了验证GCC 185是如何锚定的，并确定GCC 185在高尔基体表面发挥功能的微区的分子组成，我们将使用一种新的生物素化方法来标记最近的邻居。这将有助于我们理解GC 185和Golgi-245在transGolgi上所占据的不同结构域之间的差异。为了研究MPR囊泡在高尔基体的束缚机制，我们将利用原子力显微镜和跟踪荧光相关光谱分析GCC 185的结构，分析核心柔性对束缚功能的重要性。我们还将进行救援实验，以测试可能不灵活的突变蛋白质的功能。我们的结构数据表明，蛋白质可能会在高尔基体上塌陷，而不是在中间弯曲。为了直接区分系链弯曲和系链崩溃，我们将尝试仅生物素化靠近高尔基体表面的GCC 185肽，并将其映射到GCC 185蛋白序列上。如果GCC 185位于高尔基体上，则所有肽都应该是生物素化的。最后，我们将分离荧光标记的，含有甘露糖6-磷酸受体的囊泡，并监测这些囊泡如何与GCC 185系链结合。囊泡结合在哪里？什么模型最能解释GCC 185如何将囊泡拴在高尔基体上？总之，这些实验开辟了MPR运输领域的全新研究领域，并将提供有关人类细胞高尔基体囊泡束缚机制的基本信息。这项工作对我们理解包括糖尿病、癌症、心脏病和神经系统疾病在内的许多疾病状态具有广泛的应用。

 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. We have shown that the protein, GCC185 is needed for tethering of MPR-containing vesicles at the trans Golgi. To verify how GCC185 is anchored and to define the molecular composition of the microdomain where GCC185 functions on the Golgi surface, we will use a novel biotinylation approach to tag nearest neighbors. This will help us understand the difference between the distinct domains occupied by GC185 and Golgi-245 on the trans Golgi. To investigate the mechanism of MPR vesicle tethering at the Golgi, we will analyze the structure of GCC185 using atomic force microscopy and tracking fluorescence correlation spectroscopy, to analyze the importance of core flexibility for tethering function. We will also carry out rescue experiments to test the function of mutant proteins that may not be flexible. Our structural data suggest that the protein may collapse on the Golgi rather than bend in the middle. To distinguish directly between tether bending and tether collapse, we will attempt to biotinylate only GCC185 peptides that are in close proximity to the Golgi surface and map these onto the GCC185 protein sequence. If GCC185 lies down on the Golgi, all peptides should be biotinylatable. Finally, we will isolate fluorescently labeled, mannose 6-phosphate receptor-containing vesicles and monitor how these engage the GCC185 tether. Where do the vesicles bind? What models best explain how GCC185 tethers vesicles at the Golgi? 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 vesicle tethering at the Golgi 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.