Molecular Mechanisms of Intracellular Sorting and Transport of Membrane Proteins

主要研究了芽殖酵母的膜蛋白分选机制。1)Rerlp分选受体的研究。我们证明了Rerlp是一种机制，当ER驻留的膜蛋白子集被错误地运输到高尔基体时，它通过直接识别它们TMD中的极性残基基序来结合它们，并将它们送回ER2)凝集素类型分类受体的研究。我们认为Emp46/47p在ER-Golgi循环过程中作为糖蛋白亚群的受体，并表明它们的寡聚状态对它们的运输是重要的。3)COPII发芽过程中膜-货物分选的完全重建。我们以一种货物依赖的方式，在一个只包含纯蛋白质和脂类的无细胞系统中重组了COPII囊泡萌发。通过这种分析，我们证明了Sarlp对GTP的水解在货物选择中起到校对作用。4)脂类和泛素在膜蛋白分类中的作用。我们发现，质膜蛋白Tat2p和PMalp到细胞表面的运输受到泛素化的调节，而固醇脂在这一过程中起着重要的作用。5)膜蛋白分选的实时可视化。利用我们研制的高速和高灵敏度激光扫描显微镜，我们证明了货物蛋白质在高尔基体内的运输是通过脑池成熟机制进行的。我们进一步证明，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.