Fo portion of Escherichia coli ATP synthase. Further resolution of trypsin-generated fragments from subunit b.
F1-stripped everted membrane vesicles of the ATP synthase-overproducing Escherichia coli strain KY 7485 were treated with trypsin for different lengths of time. Subsequently, the Fo complex was isolated and analyzed by sodium dodecyl sulfate-gel electrophoresis, as well as immunoblotting using antibodies raised against subunit b. By these techniques 3 degradation products with apparent molecular masses of about 16 kDa could be detected in accordance with previous findings (Perlin, D.S., and Senior, A.E. (1985) Arch. Biochem. Biophys. 236, 603-611). Labeling of isolated trypsin-treated Fo fractions with the thiol-specific reagent N-(7-dimethylamino-4-methylcoumarinyl)-maleimide, which has been demonstrated recently to specifically modify subunit b (Schneider, E., and Altendorf, K. (1985) Eur. J. Biochem. 153, 105-109) revealed that the 16-kDa digestion products were degraded into two stable fragments of 12 and 8.3 kDa. These polypeptides do not react with the anti-b antibodies. Treatment of purified liposome-integrated Fo with trypsin resulted in a similar cleavage pattern. In both cases protease digestion inhibited F1 binding while proton-translocating activity remained unaffected. However, liposomes reconstituted with Fo isolated from trypsin-treated membranes were impaired in both binding of F1 and proton translocation. These activities could be restored when reconstitution was carried out in the presence of native subunit b. From this we conclude that the C-terminal region of subunit b is necessary for proper reconstitution of Fo into liposomes.
D002462:Cell Membrane / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology*; D008081:Liposomes; D046911:Macromolecular Substances; D011510:Proteolipids; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D014357:Trypsin
D008081:Liposomes; D046911:Macromolecular Substances; D011510:Proteolipids; C020485:proteoliposomes; D014357:Trypsin; D006180:Proton-Translocating ATPases