Post-Golgi trafficking of the mannose 6-phosphate receptor in living cells

1. 溶酶体酶的分选由甘露糖6-磷酸受体（M6PR）进行，该受体在反式高尔基网络（TGN）、核内体和质膜之间运输。在本研究项目中，我们检测了M6PR胞质外结构域在其细胞内运输中的功能，得到以下结论。(1) GFP-M6PR的延时成像和FRAP（光漂白后荧光恢复）分析以及氯喹处理表明，M6PR的胞质外结构域需要与内吞室紧密相互作用并被它们保留。(2)使用药物U18666A和来自i细胞疾病患者的成纤维细胞进行的实验表明，与M6P配体的结合参与了M6PR的转运步骤，但尚未确定。U18666A可以作为研究M6PR.2新传输步骤的有用工具。为了研究网格蛋白接头GGAs调节M6PR转运的功能，我们对GGA2进行了RNAi和显性阴性实验。虽然单纯GGA2的缺失并不会导致M6PR的明显重新分布，但溶酶体酶组织蛋白酶D的分泌明显错误。这些结果表明，溶酶体酶的错误分选并不总是伴随着剧烈的M6PR再分布。我们将详细分析哪些运输步骤受gga监管。

1. Sorting of lysosomal enzymes is conducted by mannose 6-phosphate receptors (M6PR), which are transported between the trans-Golgi network (TGN), endosomes, and plasma membrane. In this research project, we examined functions of the extracytoplasmic domain of M6PR in its intracellular trafficking, and obtained the following conclusions.(1) Time-lapse imaging and FRAP (fluorescence recovery after photobleaching) analyses of GFP-M6PR, together with a chloroquine treatment suggest that the extracytoplasmic domain of M6PR is required for tight interaction with endocytic compartments and retention by them.(2) Experiments using a drug, U18666A, and fibroblasts derived from I-cell disease patients, suggest that the binding to the M6P ligands is involved in yet unidentified transport steps of M6PR. U18666A could be a useful tool to investigate new transport steps of M6PR.2. To investigate the function of clathrin adaptors, GGAs, that regulate the M6PR trafficking, we performed RNAi and dominant-negative experiments for GGA2. Although the depletion of GGA2 alone did not cause an apparent re distribution of M6PR, a lysosomal enzyme, cathepsin D, was significantly missecreted. These results suggest that missorting of lysosomal enzymes is not always accompanied by the drastic M6PR redistribution. We will perform detailed analyses as to which transport steps are regulated by GGAs.