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Mutagenesis of the L, M, and N subunits of Complex I from Escherichia coli indicates a common role in function.

Michel J, DeLeon-Rangel J, Zhu S, Van Ree K, Vik SB - PLoS ONE (2011)

Bottom Line: The membrane arm of Complex I (NADH:ubiquinone oxidoreductase) contains three large, and closely related subunits, which are called L, M, and N in E. coli.Corresponding mutations in the three subunits were found to have very similar effects on all activities measured.The results show a close correlation with reduced activity among the corresponding mutations, and provide evidence that the L, M, and N subunits have a common role in Complex I.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Southern Methodist University, Dallas, Texas, United States of America.

ABSTRACT

Background: The membrane arm of Complex I (NADH:ubiquinone oxidoreductase) contains three large, and closely related subunits, which are called L, M, and N in E. coli. These subunits are homologous to components of multi-subunit Na(+)/H(+) antiporters, and so are implicated in proton translocation.

Methodology/principal findings: Nineteen site-specific mutations were constructed at two corresponding positions in each of the three subunits. Two positions were selected in each subunit: L_K169, M_K173, N_K158 and L_Q236, M_H241, N_H224. Membrane vesicles were prepared from all of the resulting mutant strains, and were assayed for deamino-NADH oxidase activity, proton translocation, ferricyanide reductase activity, and sensitivity to capsaicin. Corresponding mutations in the three subunits were found to have very similar effects on all activities measured. In addition, the effect of adding exogenous decylubiquinone on these activities was tested. 50 µM decylubiquinone stimulated both deamino-NADH oxidase activity and proton translocation by wild type membrane vesicles, but was inhibitory towards the same activities by membrane vesicles bearing the lysine substitution at the L236/M241/N224 positions.

Conclusions/significance: The results show a close correlation with reduced activity among the corresponding mutations, and provide evidence that the L, M, and N subunits have a common role in Complex I.

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Related in: MedlinePlus

Immunoblots of membrane preparations from all nineteen mutants.Three independent blots were performed with each mutant, using antibodies                        against subunits L, M, or for detection of N, anti-HA antibodies. For                        comparison, the  strain, BA14, and the wild type strain, BA14/pBA400,                        were included in each blot. Each lane contained 40 µg of protein. (A)                        Seven L mutants. (B) Six M mutants. (C) Six N mutants.
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pone-0017420-g002: Immunoblots of membrane preparations from all nineteen mutants.Three independent blots were performed with each mutant, using antibodies against subunits L, M, or for detection of N, anti-HA antibodies. For comparison, the strain, BA14, and the wild type strain, BA14/pBA400, were included in each blot. Each lane contained 40 µg of protein. (A) Seven L mutants. (B) Six M mutants. (C) Six N mutants.

Mentions: In a previous study [32], mutations at numerous positions of subunit N of the E. coli Complex I resulted in the loss of enzyme activity, but several mutations at E154, K158, H224 and Y300 also caused reduced ability to utilize exogenous decylubiquinone in NADH oxidase activity. In this study, the residues homologous to K158 (position 1) and H224 (position 2) were selected in subunit L (K169, Q236) and in subunit M (K173, H241), as shown in Figure 1. These residues are highly conserved among bacterial L, M and N subunits. They were selected in part because of their proximity to two residues that are highly conserved in Complex I subunits from nearly all species, and which might be involved in proton translocation: E (L144, M144, N133) and K (L229, M234, N217) [32], [33], [34], [35], [36]. At least three mutations were constructed at each of the six sites, resulting in 19 in total. Each mutation was transferred to the nuo operon expression vector, pBA400, and the resulting plasmids were used individually to transform the nuo deletion strain BA14. All of the mutants, which are listed in Table 1, could grow on minimal medium plates with acetate as the sole carbon source, indicating a functional, or partially functional, Complex I. Each of the mutant strains was further analyzed by immunoblotting of membrane vesicle preparations for all three subunits L, M, and N. Peptide-based antibodies were used to detect subunits L and M, and subunit N was detected via an engineered HA epitope tag at its C-terminus. The results, shown in Figure 2, indicate that the levels of all of the subunits are very similar to the strain with the wild type plasmid.


Mutagenesis of the L, M, and N subunits of Complex I from Escherichia coli indicates a common role in function.

Michel J, DeLeon-Rangel J, Zhu S, Van Ree K, Vik SB - PLoS ONE (2011)

Immunoblots of membrane preparations from all nineteen mutants.Three independent blots were performed with each mutant, using antibodies                        against subunits L, M, or for detection of N, anti-HA antibodies. For                        comparison, the  strain, BA14, and the wild type strain, BA14/pBA400,                        were included in each blot. Each lane contained 40 µg of protein. (A)                        Seven L mutants. (B) Six M mutants. (C) Six N mutants.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3046159&req=5

pone-0017420-g002: Immunoblots of membrane preparations from all nineteen mutants.Three independent blots were performed with each mutant, using antibodies against subunits L, M, or for detection of N, anti-HA antibodies. For comparison, the strain, BA14, and the wild type strain, BA14/pBA400, were included in each blot. Each lane contained 40 µg of protein. (A) Seven L mutants. (B) Six M mutants. (C) Six N mutants.
Mentions: In a previous study [32], mutations at numerous positions of subunit N of the E. coli Complex I resulted in the loss of enzyme activity, but several mutations at E154, K158, H224 and Y300 also caused reduced ability to utilize exogenous decylubiquinone in NADH oxidase activity. In this study, the residues homologous to K158 (position 1) and H224 (position 2) were selected in subunit L (K169, Q236) and in subunit M (K173, H241), as shown in Figure 1. These residues are highly conserved among bacterial L, M and N subunits. They were selected in part because of their proximity to two residues that are highly conserved in Complex I subunits from nearly all species, and which might be involved in proton translocation: E (L144, M144, N133) and K (L229, M234, N217) [32], [33], [34], [35], [36]. At least three mutations were constructed at each of the six sites, resulting in 19 in total. Each mutation was transferred to the nuo operon expression vector, pBA400, and the resulting plasmids were used individually to transform the nuo deletion strain BA14. All of the mutants, which are listed in Table 1, could grow on minimal medium plates with acetate as the sole carbon source, indicating a functional, or partially functional, Complex I. Each of the mutant strains was further analyzed by immunoblotting of membrane vesicle preparations for all three subunits L, M, and N. Peptide-based antibodies were used to detect subunits L and M, and subunit N was detected via an engineered HA epitope tag at its C-terminus. The results, shown in Figure 2, indicate that the levels of all of the subunits are very similar to the strain with the wild type plasmid.

Bottom Line: The membrane arm of Complex I (NADH:ubiquinone oxidoreductase) contains three large, and closely related subunits, which are called L, M, and N in E. coli.Corresponding mutations in the three subunits were found to have very similar effects on all activities measured.The results show a close correlation with reduced activity among the corresponding mutations, and provide evidence that the L, M, and N subunits have a common role in Complex I.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Southern Methodist University, Dallas, Texas, United States of America.

ABSTRACT

Background: The membrane arm of Complex I (NADH:ubiquinone oxidoreductase) contains three large, and closely related subunits, which are called L, M, and N in E. coli. These subunits are homologous to components of multi-subunit Na(+)/H(+) antiporters, and so are implicated in proton translocation.

Methodology/principal findings: Nineteen site-specific mutations were constructed at two corresponding positions in each of the three subunits. Two positions were selected in each subunit: L_K169, M_K173, N_K158 and L_Q236, M_H241, N_H224. Membrane vesicles were prepared from all of the resulting mutant strains, and were assayed for deamino-NADH oxidase activity, proton translocation, ferricyanide reductase activity, and sensitivity to capsaicin. Corresponding mutations in the three subunits were found to have very similar effects on all activities measured. In addition, the effect of adding exogenous decylubiquinone on these activities was tested. 50 µM decylubiquinone stimulated both deamino-NADH oxidase activity and proton translocation by wild type membrane vesicles, but was inhibitory towards the same activities by membrane vesicles bearing the lysine substitution at the L236/M241/N224 positions.

Conclusions/significance: The results show a close correlation with reduced activity among the corresponding mutations, and provide evidence that the L, M, and N subunits have a common role in Complex I.

Show MeSH
Related in: MedlinePlus