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Molecular Mechanisms of Intracellular Sorting and Transport of Membrane Proteins

膜蛋白细胞内分选和运输的分子机制

基本信息

批准号：
14104023
负责人：
NAKANO Akihiko
金额：
$ 72.47万
依托单位：
The University of Tokyo (2003-2006)The Institute of Physical and Chemical Research (2002)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (S)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-14104023/
关键词：
protein secretion cargo sorting COPII vesicles cell-free reconstitution Sarl GTPase the Golgi apparatus cisternal maturation imaging ゴルジ体内輸送 Sarlp GTPase Arf GTPase GTP加水分解 GTPase活性化因子メンブレントラフィック Emp46 47p オリゴマー形成膜タンパク質

项目摘要

Mainly working on budding yeast, we obtained the following results regarding sorting mechanisms of membrane proteins.1)Study on Rerlp sorting receptor. We showed that Rerlp is the machinery that binds a subset of ER-resident membrane proteins when they are mistransported to the Golgi, by directly recognizing polar residue motifs in their TMD, and send them back to the ER.2)Study on lectin-type sorting receptors. We suggested that Emp46/47p act as receptors for a subset of glycoproteins during ER-Golgi recycling and showed that their oligomerization status is important for their trafficking.3)Complete reconstitution of membrane-cargo sorting during COPII budding. We reconstituted COPII vesicle budding in a cell-free system comprising only pure proteins and lipids in a cargo-dependent manner. By this assay, we demonstrated that GTP hydrolysis by Sarlp plays a proof-reading role in cargo selection.4)Roles of lipids and ubiquitin in membrane protein sorting. We showed that transport of plasma-membrane proteins, Tat2p and Pmalp, to the cell surface is regulated by ubiquitination and that sterol lipid plays an important role in this process.5)Real-time visualization of membrane-protein sorting. Using a high-speed and high-sensitivity confocal laser scanning microscope we developed, we demonstrated that intra-Golgi transport of cargo proteins is performed by the cisternal maturation mechanism. We further showed that dynamic sorting and segregation of membrane are involved in this process in COPI-dependent and independent ways.

本研究主要以芽殖酵母为研究对象，在膜蛋白分选机制方面取得了以下研究成果：1）Rerlp分选受体的研究。我们表明，Rerlp是一种机制，当ER驻留膜蛋白被错误转运到高尔基体时，它通过直接识别其TMD中的极性残基序来结合ER驻留膜蛋白的一个子集，并将它们送回ER。2）关于凝集素的研究-类型分选受体。我们认为Emp 46/47 p在ER-高尔基体再循环过程中作为糖蛋白亚类的受体，并表明它们的寡聚化状态对其运输很重要。3）在COPII出芽过程中膜货物分选的完全重建。我们重建COPII囊泡出芽在一个无细胞的系统，仅包括纯蛋白质和脂质在货物依赖性的方式。通过该测定，我们证明了Sarlp的GTP水解在货物选择中起校对作用。4）脂质和泛素在膜蛋白分选中的作用。我们发现质膜蛋白Tat 2 p和Pmalp向细胞表面的转运受泛素化的调节，并且固醇脂质在此过程中起重要作用。5）膜蛋白分选的实时可视化。使用高速和高灵敏度的共聚焦激光扫描显微镜，我们开发的，我们证明了货物蛋白质的高尔基体内运输是由脑池成熟机制。我们进一步表明，膜的动态分选和分离以依赖于COPI和独立的方式参与了这一过程。