Localization mechanisms of Golgi peripheral membrane proteins

高尔基体由一组囊泡室组成，这些囊泡室由它们的标记蛋白来区分。在酿酒酵母细胞中，这些隔室在物理上是分开的。为了更广泛地表征它们，我们之前开发了一种方法，通过分别携带SNAREs Sed5或Tlg2（早期和晚期高尔基区室的标记物）的免疫分离囊泡，分别分离早期高尔基区室和晚期高尔基区室，并分析膜跨越蛋白。高尔基体的细胞器身份不仅由驻留的跨膜蛋白决定，而且外周膜、蛋白也对确定早期或晚期高尔基亚室的功能有重要贡献。基于这些事实，本研究的目的是改进这种外周高尔基膜蛋白分析的免疫分离方法，并揭示外周膜蛋白被募集到特定高尔基亚室的机制。我们主要分析的蛋白是Gea1和Sec7，它们都是小g蛋白Arf的GDP/GTP交换因子，但具有不同的定位，Gea1特异性定位于早期高尔基亚室，而Sec7特异性定位于晚期高尔基亚室。我们发现，通过多次修改，免疫分离程序也可用于确定外周膜蛋白的定位，因为Gea1在sed5 -室（富集于早期高尔基蛋白）中被回收，Sec7在tlg2 -室（富集于晚期高尔基蛋白）中被回收。通过改变缓冲液和破坏细胞的方法，该方法回收的外周膜蛋白数量进一步增加，这使得该免疫分离方法可用于高尔基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驻留在酵母高尔基膜上的外周膜蛋白。少

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