Reconstitution of active transport in proteoliposomes containing cytochrome o oxidase and lac carrier protein purified from Escherichia coli.
Most active transport across the bacterial cell membrane is driven by a proton electrochemical gradient (delta-muH+, interior negative and alkaline) generated via electron transfer through a membrane-bound respiratory chain. This phenomenon is now reproduced in vitro with proteoliposomes containing only two proteins purified from the membrane of Escherichia coli. An o-type cytochrome oxidase was extracted from membranes of a cytochrome d terminal oxidase mutant with octyl beta-D-glucopyranoside after sequential treatment with urea and cholate and was purified to homogeneity by ion-exchange chromatography. The purified oxidase contains four polypeptides (MrS 66,000, 35,000, 22,000, and 17,000), two b-type cytochromes (b558 and b563), and 16-17 nmol of heme b per mg of protein, and it catalyzes the oxidation of ubiquinol and other electron donors with specific activities 20- to 30-fold higher than crude membranes. The lac carrier protein was purified as described. Proteoliposomes were formed in the presence of the oxidase and lac carrier protein by detergent dilution, followed by freeze-thaw/sonication. The system generates a delta-muH+ (interior negative and alkaline) with ubiquinol as electron donor and the magnitude of delta-muH+ is dependent on the concentration of cytochrome o in the proteoliposomes. Furthermore, the proteoliposomes transport lactose against a concentration gradient to an extent that is commensurate with the magnitude of delta-muH+ generated. The results provide powerful additional support for the "chemiosmotic hypothesis" and demonstrate that purified lac carrier protein retains the ability to function in a physiological manner.
D001693:Biological Transport, Active; D003573:Cytochrome b Group; D003580:Cytochromes / Q000302:isolation & purification / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D046911:Macromolecular Substances; D026901:Membrane Transport Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins; D011510:Proteolipids; D027981:Symporters
D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D046911:Macromolecular Substances; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011510:Proteolipids; D027981:Symporters; C020485:proteoliposomes; C017168:cytochrome bo, E coli; C021499:lactose permease; C038862:cytochrome o oxidase; D003576:Electron Transport Complex IV; D007785:Lactose