Mutagenesis of subunit N of the Escherichia coli complex I. Identification of the initiation codon and the sensitivity of mutants to decylubiquinone.
The last gene in the nuo operon of Escherichia coli, nuoN, encodes a membrane-bound subunit of Complex I (NADH:ubiquinone oxidoreductase). In this report, the gene for subunit N was disrupted by a 163 bp deletion in the chromosome, resulting in the loss of Complex I function, as measured by deamino-NADH oxidase activity. This activity could be recovered after transformation of the mutant strain by a plasmid that contains the previously identified nuoN gene and the upstream intergenic region between nuoM and nuoN. Mutagenesis of the first ATG downstream of nuoM led to a loss of function, indicating that this is the likely initiation codon for nuoN, and predicting a protein of 485 amino acids and 52 044 Da. Thirty site-specific mutations in nuoN at 19 different positions were constructed in a vector that expresses the full-length subunit N with both an octahistidine tag and an HA epitope tag at the carboxyl terminus. Highly conserved charged and aromatic residues were selected for mutagenesis, as well as a substitution that occurs as a secondary mutation in Leber's hereditary optic neuropathy (LHON). Membranes from the mutant strains were tested for production of subunit N by immunoblots and for NADH-linked activities. Mutants with substitutions at six different positions (K158, K217, H224, K247, Y300, and K395) had rates of deamino-NADH oxidase activity that were no more than 50% of that of the wild type and had reduced rates of proton translocation. These mutants also showed enhanced inhibition by decylubiquinone, indicating that subunit N interacts with quinones. The mutation associated with LHON, G391S, had little effect on these functions.
Amarneh
Bilal
B
Department of Biological Sciences, Southern Methodist University, Dallas, Texas 75275-0376, USA.
Vik
Steven B
SB
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D018387:Codon, Initiator / Q000235:genetics; D042967:Electron Transport Complex I / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000494:pharmacology*
D018387:Codon, Initiator; D029968:Escherichia coli Proteins; C000589385:NuoN protein, E coli; D021122:Protein Subunits; D011994:Recombinant Proteins; D014451:Ubiquinone; C060262:2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I