Functional Roles of Four Conserved Charged Residues in the Membrane Domain Subunit NuoA of the Proton-translocating NADH-Quinone Oxidoreductase from Escherichia coli

Kao, Mou-Chieh; Bernardo, Salvatore Di; Perego, Marta; Nakamaru‐Ogiso, Eiko; Matsuno‐Yagi, Akemi; Yagi, Takao

doi:10.1074/jbc.m403885200

Cited by 57 publications

(59 citation statements)

References 56 publications

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“…PCR product, gel extraction, and plasmid purification kits and anti-histidine antibodies (Penta⅐His antibody) were from Qiagen. All of the antibodies against E. coli NDH-1 subunits NuoM-1, NuoM-7, NuoAc, NuoB, NuoCD, NuoE, NuoF, and NuoG were obtained previously in our lab (10,35,36,46). The secondary antibodies, antimouse IgG (horseradish peroxidase conjugate) and anti-rabbit IgG (horseradish peroxidase conjugate) were from Calbiochem (La Jolla, CA) and GE Healthcare, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…Then the cells were harvested at 5800 ϫ g for 10 min and frozen at Ϫ80°C. Inside-out (ISO) membrane vesicles were prepared according to the method described previously (33)(34)(35)(36). Right-side-out (RSO) membrane vesicles were prepared following the method described by Kao et al (48).…”

Section: Mutagenesis Of E Coli Nuom Gene and Preparation Of Mutant Cmentioning

confidence: 99%

“…Blue native PAGEs were performed according to the method described previously (35). The assembly of NDH-1 was confirmed by immunoblotting using anti-NuoM-1 antibodies and NADH dehydrogenase activity staining.…”

Section: Mutagenesis Of E Coli Nuom Gene and Preparation Of Mutant Cmentioning

confidence: 99%

“…The possibility of generating chromosomal deletions and the introduction of site-directed mutations in the nuo genes by homologous recombination has allowed functional studies on various hydrophobic subunits, such as NuoA (ND3), NuoJ (ND6), NuoK (ND4L), NuoN (ND2), NuoM (ND4), and NuoH (ND1) (32)(33)(34)(35)(36)(37)(38)(39). The NuoM subunit, counterpart of the eukaryotic ND4 subunit, is one of the largest polypeptides in the hydrophobic arm of NDH-1.…”

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confidence: 99%

“…Interestingly, in the case of the transmembrane conserved Glu 144 , its conservative mutation to Asp did not show any effect on NDH-1 activity. In this regard, highly conserved transmembrane carboxyl residues in NuoK (ND4L) and NuoA (ND3) (34,35,38) and in some other membrane proteins were reported to play important roles in proton translocation (41)(42)(43)(44)(45). Thus, it was suggested that the essential Glu 144 of the NuoM subunit could play a key role in the proton pump activity of NDH-1.…”

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confidence: 99%

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Features of Subunit NuoM (ND4) in Escherichia coli NDH-1

Torres‐Bacete

Sinha

Castro-Guerrero

et al. 2009

Journal of Biological Chemistry

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The bacterial H ؉ -pumping NADH-quinone oxidoreductase (NDH-1) is an L-shaped membrane-bound enzymatic complex. Escherichia coli NDH-1 is composed of 13 subunits (NuoA-N). NuoM (ND4) subunit is one of the hydrophobic subunits that constitute the membrane arm of NDH-1 and was predicted to bear 14 helices. We attempted to clarify the membrane topology of NuoM by the introduction of histidine tags into different positions by chromosomal site-directed mutagenesis. From the data, we propose a topology model containing 12 helices (helices I-IX and XII-XIV) located in transmembrane position and two (helices X and XI) present in the cytoplasm. We reported previously that residue Glu 144 of NuoM was located in the membrane (helix V) and was essential for the energy-coupling activities of NDH-1 (Torres-Bacete, J., Nakamaru-Ogiso, E., Matsuno-Yagi, A., and Yagi, T. (2007) J. Biol. Chem. 282, 36914 -36922). Using mutant E144A, we studied the effect of shifting the glutamate residue to all sites within helix V and three sites each in helix IV and VI on the function of NDH-1. Twenty double site-directed mutants including the mutation E144A were constructed and characterized. None of the mutants showed alteration in the detectable levels of expressed NuoM or on the NDH-1 assembly. In addition, most of the double mutants did not restore the energy transducing NDH-1 activities. Only two mutants E144A/ F140E and E144A/L147E, one helix turn downstream and upstream restored the energy transducing activities of NDH-1. Based on these results, a role of Glu 144 for proton translocation has been discussed.The proton-translocating NADH-quinone oxidoreductase (designated complex I and NDH-1 for mitochondrial and bacterial enzymes, respectively) constitutes the first energy-coupling site in the respiratory chain in most eukaryotic and prokaryotic cells. It catalyzes the reduction of quinone using NADH as electron donor. This activity is coupled to the translocation of protons through the inner mitochondrial/cytoplasmic membranes (1-4). Complex I is one of the largest and most intricate proteins of the respiratory chain, e.g. bovine complex I was reported to contain 45 different subunits (5, 6). In contrast, NDH-1 has relatively simpler structure and seems suitable to study the coupling site 1. In case of Escherichia coli, NDH-1 is made up of only 13 subunits (designated NuoA-N), which are homologous to the 14 subunits that compose the central core of mitochondrial complex I (7, 8). Electron microscopic images of both mitochondrial complex I and bacterial NDH-1 have revealed a characteristic L-shaped form with two distinct domains, a hydrophilic peripheral arm projected into the mitochondrial matrix (or bacterial cytoplasm) and a transmembrane hydrophobic arm (9). The hydrophilic domain catalyzes electron transfer from NADH to quinone and carries all of the cofactors (one FMN and eight or nine iron-sulfur clusters) (10 -13). In contrast, the membrane domain is considered to participate in proton translocation and quinone binding (14 -18).T...

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Section: Methodsmentioning

confidence: 99%

Section: Mutagenesis Of E Coli Nuom Gene and Preparation Of Mutant Cmentioning

confidence: 99%

Section: Mutagenesis Of E Coli Nuom Gene and Preparation Of Mutant Cmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Features of Subunit NuoM (ND4) in Escherichia coli NDH-1

Torres‐Bacete

Sinha

Castro-Guerrero

et al. 2009

Journal of Biological Chemistry

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The clinically relevant triple mutation in the mtND1 gene inactivates Escherichia coli complex I

2022

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NADH:ubiquinone oxidoreductase (respiratory complex I) plays a major role in cellular energy metabolism. Complex I deficiencies are the most common cause of mitochondrial dysfunction. Patients suffering from a variety of neurodegenerative diseases carry numerous mutations in the mitochondrially encoded subunits of the complex. The biochemical consequences of these mutations are largely unknown because these genes are difficult to access experimentally. Here, we use Escherichia coli as a model system to characterize the effect of a 7 bps inversion in mtND1 (m.3902‐3908inv7) that results in a triple mutation. The triple mutant grew poorly but contained a normal amount of the stably assembled variant. The variant showed no enzymatic activity, which might contribute to the deleterious effect of the mutation in humans.

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