Molecular Mechanisms of Intracellular Sorting and Transport of Membrane Proteins

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。我们发现，Rerlp是一种机制，当ER驻留膜蛋白被错误转运到高尔基体时，通过直接识别其TMD中的极性残基，将其结合到ER。2）凝集素型分选受体的研究。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COPI依赖和独立的方式参与这一过程。

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.