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

为了了解溶酶体酶、网格蛋白接纳蛋白、GGA和AP 1以及甘露糖6-磷酸受体（MPR）的运输的详细机制，将其与GFP融合，通过活细胞成像和FRAP（光漂白后的荧光恢复）分析来检查。通过本课题的研究，取得了以下成果. GGA 1和AP 1在TGN（trans-Golgi network）区域中表现出不同的分布模式，并且大多数离开TGN的运输载体仅包含AP 1。这些结果表明，GGA和AP 1分别参与了芽前和芽后事件. GGA 2比GGA 1和GGA 3更快地与TGN膜重复缔合和运动。此外，GGA 1的铰链-耳部结构域的存在延缓了这种循环动力学.阳离子非依赖性MPR胞外结构域的存在可能促进含有MPR的TGN衍生的转运载体与内体的融合，并使MPR在外周区域保留较长时间。GGA 3的剪接变体主要在除脑以外的几种人体组织和人源性培养细胞系中表达。由于GGA 3的变异体缺少MPR的结合位点，因此GGA 3可能在MPR运输之外发挥新的作用. U18666 A处理导致MPR在异常的内体结构中积累。这种再分布可能与MPR的配体依赖性转运步骤有关。

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.