Building the stator of the yeast vacuolar-ATPase: specific interaction between subunits E and G.
The vacuolar (H+)-ATPase (or V-ATPase) is a membrane protein complex that is structurally related to F1 and F0 ATP synthases. The V-ATPase is composed of an integral domain (V0) and a peripheral domain (V1) 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 angstroms. 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.
Structural and Computational Biology Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
James
J
Féthière
David
D
Venzke
Meikel
M
Diepholz
Anja
A
Seybert
Arie
A
Geerlof
Marc
M
Gentzel
Matthias
M
Wilm
Bettina
B
Böttcher
D002845:Chromatography; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D008854:Microscopy, Electron; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D006180:Proton-Translocating ATPases / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism
D003432:Cross-Linking Reagents; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases