Beta-galactosidase gene fusions as probes for the cytoplasmic regions of subunits I and II of the membrane-bound cytochrome d terminal oxidase from Escherichia coli.
The cytochrome d terminal oxidase complex is a component of the aerobic respiratory chain of Escherichia coli. This enzyme catalyzes the oxidation of ubiquinol-8 within the cytoplasmic membrane and the reduction of molecular oxygen to water along with the concomitant generation of a proton-motive force across the membrane. Previous studies have established that the oxidase is composed of one copy of each of two subunits (I and II), and contains four heme prosthetic groups. The hydropathy profiles of the amino acid sequences suggest that each subunit has multiple transmembrane-spanning helical segments. The goal of the current work is to obtain experimental information about which portions of the two polypeptide chains are facing the cytoplasm. This is part of an effort to determine the topological folding of the two subunits across the membrane. A number of random gene fusions were generated in vitro which encode hybrid proteins in which the amino-terminal portion is provided by one of the two subunits of the oxidase, and the carboxyl-terminal portion is beta-galactosidase. Studies from other systems have indicated that the only hybrid proteins which will manifest high beta-galactosidase specific activity and be membrane-bound will be those where the fusion junction is in a region of the cytochrome polypeptides facing the cytoplasm. Fusions were obtained in eight positions within subunit I and 11 positions within subunit II. These identified four cytoplasmic-facing regions within subunit II, consistent with its hydropathy profile showing eight transmembrane helices. The data with subunit I are less conclusive.
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005696:Galactosidases / Q000235:genetics*; D046911:Macromolecular Substances; D008566:Membranes / Q000201:enzymology; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D001616:beta-Galactosidase / Q000235:genetics*
D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D010088:Oxidoreductases; C040077:cytochrome bd terminal oxidase complex, E coli; D005696:Galactosidases; D001616:beta-Galactosidase