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Localization mechanisms of Golgi peripheral membrane proteins

高尔基体外周膜蛋白的定位机制

基本信息

批准号：
18580068
负责人：
NODA Yoichi
金额：
$ 2.49万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18580068/
关键词：
Golgi apparatus vesicular transport budding yeast 応用微生物蛋白質

项目摘要

The Golgi apparatus consists of a set of vesicular compartments which are distinguished by their marker proteins. These compartments are physically separated in the yeast Saccharomyces cerevisiae cell. To characterize them extensively, we previously developed a method to isolate early Golgi compartments and late Golgi compartments separately, by immuonisolating vesicles carrying either of the SNAREs Sed5 or Tlg2, the markers of the early and late Golgi compartments, respectively, and analyzed the membrane-spanning proteins. Organelle identity of the Golgi apparatus is determined not only by the resident membrane-spanning proteins, but peripheral membranes, proteins also makes significant contributions to specify the functions of early or late Golgi subcompartment. Based on these facts, our goal in this study is to improve this immunoisolation procedure for the analysis of the peripheral Golgi membrane proteins and reveal the mechanism by which peripheral membrane proteins are recruited … More to the specific Golgi subcompartments. The proteins we mainly analyzed were Gea1 and Sec7, both of which are GDP/GTP exchange factors for the small G-protein Arf, but have distinct localizations with Gea1 being specifically localized to the early Golgi subcompartment and with Sec7 being specifically localized to the late Golgi subcompartment. We found, by making several modifications, that the immunoisolation procedure can be also used to determine localization of peripheral membrane proteins, as Gea1 was recovered in the Sed5-compartment (enriched in early Golgi proteins) and Sec7 was recovered in the Tlg2-compartment (enriched in late Golgi proteins). Amount of peripheral membrane proteins recovered by this procedure was further increased by altering buffers and mothods to disrupt cells, which made this immunoisolation method useful for comprehensive proteomics analysis of Golgi peripheral membrane proteins. Thus, we conclude that we successfully devised a simple method to analyse peripheral membrane proteins that reside on the yeast Golgi membranes. Less

高尔基体由一组囊泡室组成，这些囊泡室由其标记蛋白来区分。这些隔间在酿酒酵母细胞中物理上是分开的。为了更广泛地表征它们，我们先前开发了一种分离早期高尔基体和晚期高尔基体的方法，通过免疫分离分别携带早期高尔基体和晚期高尔基体标记的Sed5或Tlg2的囊泡，并分析了膜跨膜蛋白。高尔基体的细胞器特性不仅由驻留的跨膜蛋白决定，也由外周膜决定，蛋白质对早期或晚期高尔基体小室的功能也有重要的贡献。基于这些事实，我们在这项研究中的目标是改进这种免疫分离程序来分析外周高尔基体膜蛋白，并揭示外周膜蛋白招募…的机制更多的是关于特定的高尔基小舱。我们主要分析的蛋白质是Gea1和sec7，它们都是小G蛋白Arf的GDP/GTP交换因子，但都有明显的定位，Gea1特异地定位于早期高尔基亚室，sec7特异地定位于晚期高尔基亚室。我们发现，通过几次修改，免疫分离程序也可以用于确定外周膜蛋白的定位，因为Gea1在Sed5-隔室(富含早期高尔基体蛋白)中恢复，Sec7在Tlg2-隔室(富含晚期高尔基体蛋白)中恢复。通过改变缓冲液和破坏细胞的方法，进一步增加了回收的外周膜蛋白的量，使这种免疫分离方法适用于高尔基体外周膜蛋白的全面蛋白质组学分析。因此，我们得出结论，我们成功地设计了一种简单的方法来分析酵母高尔基体膜上的外周膜蛋白。较少