| Budget Amount *help |
¥3,890,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥390,000)
Fiscal Year 2007: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2006: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
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
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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
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