title: Characterization of the membrane domain Nqo11 subunit of the proton-translocating NADH-quinone oxidoreductase of Paracoccus denitrificans.
abstract: The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans consists of at least 14 unlike subunits (designated Nqo1-14). The NDH-1 is composed of two segments (the peripheral and membrane segments). The membrane domain segment appears to be made up of seven subunits (Nqo7, -8, -10-14). In this report, the characterization of the Paracoccus Nqo11 subunit has been investigated. An antibody against the C-terminal 12 amino acid residues of the Paracoccus Nqo11 subunit (Nqo11c) has been raised. The Nqo11c antibody reacted with a single band (11 kDa) of the Paracoccus membranes and cross-reacted with Rhodobactor capsulatus membranes. The Nqo11 subunit was not able to be extracted from the Paracoccus membranes by NaI or alkaline treatment, unlike the peripheral subunits (Nqo1 and Nqo6). The C-terminal region of the Paracoccus Nqo11 is exposed to the cytoplasmic phase. For further characterization of the Paracoccus Nqo11 subunit, the subunit was overexpressed in Escherichia coli by using the maltose-binding protein (MBP) fusion system. The MBP-fused Nqo11 subunit was expressed in the E. coli membranes (but not in soluble phase) and was extracted by Triton X-100. The isolated MBP-fused Nqo11 subunit interacted with the phospholipid vesicles and suppressed their membrane fluidity. Topological studies of the Nqo11 subunit expressed in E. coli membranes have been performed by using cysteine mapping and immunochemical analyses. The data suggest that the Nqo11 subunit has three transmembrane segments and its C-terminus protrudes into the cytoplasmic phase.
authors.affiliation: Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: Mou-Chieh
authors.initials: MC
authors.lastname: Kao
authors.forename: Salvatore
authors.initials: S
authors.lastname: Di Bernardo
authors.forename: Akemi
authors.initials: A
authors.lastname: Matsuno-Yagi
authors.forename: Takao
authors.initials: T
authors.lastname: Yagi
mesh_terms: D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry*; D002352:Carrier Proteins / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D029968:Escherichia coli Proteins; D015151:Immunoblotting; D007120:Immunochemistry; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D017830:Octoxynol / Q000494:pharmacology; D010230:Paracoccus / Q000201:enzymology*; D017434:Protein Structure, Tertiary; D011522:Protons; D011808:Quinone Reductases / Q000096:biosynthesis / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011522:Protons; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D017830:Octoxynol; C041831:NADH dehydrogenase (quinone); D011808:Quinone Reductases; D003545:Cysteine