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

参与高尔基体内体运输的网格蛋白接头蛋白的动力学分析

• 批准号: 15590159
• 负责人: WAGURI Satoshi
• 金额: $ 2.3万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590159/
• 关键词: GFP; FRAP; live-cell imaging; trans-Golgi network; endosome; GGA; AP1; mannose 6-phosphate receptor; リソソーム酵素; FRAP6; AP-1

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.

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。

项目成果

Insights into the phosphoregulation of beta-secretase sorting signal by the VHS domain of GGA1

深入了解 GGA1 的 VHS 结构域对 β-分泌酶分选信号的磷酸调节

• 发表时间: 2004
• 期刊: Traffic 5
• 作者: Chiharu Yorikawa;Yuji Tomiyama;Tomoo Shiba;Munehisa Shimamura;Yuji Tomiyama;Tomoo Shiba
• 通讯作者: Tomoo Shiba

Early-phase redistiibution of the cation-independent mannose 6-phosphate receptor by U18666A treatment in HeLa cells

HeLa 细胞中 U18666A 处理对阳离子非依赖性甘露糖 6-磷酸受体的早期再分配

• 发表时间: 2004
• 期刊: Cell and Tissue Research 317
• 作者: Chiharu Yorikawa;Yuji Tomiyama
• 通讯作者: Yuji Tomiyama

GFP changed the world of vesicular transport

GFP 改变了囊泡运输的世界

• 发表时间: 2003
• 期刊: Microscopy 38
• 作者: Chiharu Yorikawa;Yuji Tomiyama;Tomoo Shiba;Munehisa Shimamura;Yuji Tomiyama;Tomoo Shiba;Masaki Wakasugi;和栗 聡;Satoshi Waguri
• 通讯作者: Satoshi Waguri

Insights into the phosphoregulation of beta-secretase soiling signal by the VHS domain of GGA1

深入了解 GGA1 的 VHS 结构域对 β-分泌酶污染信号的磷酸调节

• 发表时间: 2004
• 期刊: Traffic 5
• 作者: Chiharu Yorikawa;Yuji Tomiyama;Tomoo Shiba
• 通讯作者: Tomoo Shiba

Masaki Wakasugi: "Predominant expression of the short form of GGA3 in human cell lines and tissues"Biochemical and Biophysical Research Communications. 306. 687-692 (2003)

Masaki Wakasugi：“短形式 GGA3 在人类细胞系和组织中的主要表达”生物化学和生物物理研究通讯。