Building the stator of the yeast vacuolar-ATPase -: Specific interaction between subunits E and G

被引:45
作者
Féthière, J
Venzke, D
Diepholz, M
Seybert, A
Geerlof, A
Gentzel, M
Wilm, M
Böttcher, B
机构
[1] European Mol Biol Lab, Struct & Computat Biol Programme, D-69117 Heidelberg, Germany
[2] European Mol Biol Lab, Protein Express & Purificat Core Facil, D-69117 Heidelberg, Germany
[3] European Mol Biol Lab, Bioanalyt Res Grp, D-69117 Heidelberg, Germany
关键词
D O I
10.1074/jbc.M407086200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The vacuolar (H+)-ATPase (or V-ATPase) is a membrane protein complex that is structurally related to F-1 and F-0 ATP synthases. The V-ATPase is composed of an integral domain (V-0) and a peripheral domain (V-1) connected by a central stalk and up to three peripheral stalks. The number of peripheral stalks and the proteins that comprise them remain controversial. We have expressed subunits E and G in Escherichia coli as maltose binding protein fusion proteins and detected a specific interaction between these two subunits. This interaction was specific for subunits E and G and was confirmed by co-expression of the subunits from a bicistronic vector. The EG complex was characterized using size exclusion chromatography, cross-linking with short length chemical cross-linkers, circular dichroism spectroscopy, and electron microscopy. The results indicate a tight interaction between subunits E and G and revealed interacting helices in the EG complex with a length of about 220 Angstrom. We propose that the V-ATPase EG complex forms one of the peripheral stators similar to the one formed by the two copies of subunit b in F-ATPase.
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页码:40670 / 40676
页数:7
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