Characterization of the membrane domain subunit NuoJ (ND6) of the NADH-quinone oxidoreductase from Escherichia coli by chromosomal DNA manipulation.
The ND6 subunit is one of seven mitochondrial DNA-encoded subunits of the proton-translocating NADH-quinone oxidoreductase (complex I). Physiological importance of the ND6 subunit is becoming increasingly apparent because a number of mutations leading to amino acid changes in this subunit have been found to be associated with known mitochondrial diseases. Using the Escherichia coli enzyme (NDH-1), we have investigated the NuoJ subunit (the E. coli counterpart of ND6) by employing a chromosomal DNA manipulation technique. A series of point mutations was constructed directly on the nuoJ gene in the chromosome targeting at highly conserved residues. Analyses with blue-native gel electrophoresis and immunological methods revealed that, in all point mutants, the assembly of NDH-1 was normal and that the deamino-NADH-K(3)Fe(CN)(6) reductase activity of the membrane was essentially the same as that of the wild-type. However, energy-coupled NDH-1 activities were affected to varied extents. Among them, mutants of the Val-65 residue that is located in the most conserved transmembrane segment significantly lost the coupled electron-transfer activities and exhibited diminished membrane potential and proton translocation. This may suggest that Val-65 or the area around it is important for energy transduction of the coupling site 1. Together with the results on mutations related to human diseases, possible functional roles of the NuoJ subunit have been discussed.
Kao
Mou-Chieh
MC
Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Di Bernardo
Salvatore
S
Nakamaru-Ogiso
Eiko
E
Miyoshi
Hideto
H
Matsuno-Yagi
Akemi
A
Yagi
Takao
T
D000595:Amino Acid Sequence; D002876:Chromosomes, Bacterial / Q000737:chemistry* / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D004272:DNA, Mitochondrial / Q000737:chemistry / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D008564:Membrane Potentials / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary / Q000235:genetics; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D011808:Quinone Reductases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial; D004272:DNA, Mitochondrial; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D021122:Protein Subunits; C041831:NADH dehydrogenase (quinone); D011808:Quinone Reductases