Kinetic analyses of clathrin adaptor proteins involved in the Golgi-endosome trafficking

为了了解溶酶体酶转运的详细机制，将笼状蛋白采取器蛋白、GGAS和AP1以及甘露糖6-磷酸受体(MPR)与GFP融合，并通过活细胞成像和FRAP(荧光漂白后恢复)分析对其进行检测。通过本课题的研究，取得了以下成果。GGA1和AP1在TGN(Trans-Golgi Network)区域表现出不同的分布格局，离开TGN的大多数运输载体仅含有AP1。这些结果表明，GGAs和AP1分别参与了萌发前和萌发后的事件。GGA2与TGN膜的重复结合和运动比GGA1和GGA3更快。此外，GGA1的铰链耳结构域的存在延缓了这种循环动力学。阳离子非依赖性MPR胞外区的存在可能促进含有MPR的TGN来源的转运体与内体的融合，并使MPR在外周区域保留更长时间。GGA3的剪接变异体主要在除脑和人源性培养细胞系以外的几个人体组织中表达。由于该变异形式缺少MPR的结合位点，GGA3可能在MPR交易之外发挥新的作用。U18666A处理可导致MPR在异常的内体结构中积聚。这种重新分布可能与MPR的配体依赖的转运步骤有关。

To understand detailed mechanisms of the transport of lysosomal enzymes, clathrin adopter proteins, the GGAs and AP1, and the mannose 6-phosphate receptor (MPR) were fused with GFP, which were examined by live-cell imaging and FRAP (fluorescence recovery after photobleaching) analysis. Through this research project, following results were obtained.1. GGA1 and AP1 showed different distribution patterns in the TGN (trans-Golgi network) region, and most transport carriers that left the TGN contained AP1 alone. These results suggest that GGAs and AP1 are involved in the pre- and post-budding events, respectively.2. GGA2 repeated association and motion with the TGN membrane more rapidly than GGA1 and GGA3. Moreover, the presence of the hinge-ear domain of GGA1 retarded this cycling kinetics.3. The presence of the extracellular domain of the cation-independent MPR may facilitate the fusion of the TGN-derived transport carvers containing the MPR with endosomes, and retain the MPR in the peripheral region for longer periods.Followings are results obtained in relation to this research project.1. A splice-variant of GGA3 was predominantly expressed in several human tissues except brain, and human-derived culture cell lines. Because the variant form lacks a binding site for MPR, GGA3 may play a new role other than the MPR trafficking.2. U18666A treatment caused an accumulation of MPR in aberrant endosomal structures. This redistribution may associate with ligand-dependent transport steps of MPR.