Analysis of an N-terminal deletion in subunit a of the Escherichia coli ATP synthase.

Subunit a is a membrane-bound stator subunit of the ATP synthase and is essential for proton translocation. The N-terminus of subunit a in E. coli is localized to the periplasm, and contains a sequence motif that is conserved among some bacteria. Previous work has identified mutations in this region that impair enzyme activity. Here, an internal deletion was constructed in subunit a in which residues 6-20 were replaced by a single lysine residue, and this mutant was unable to grow on succinate minimal medium. Membrane vesicles prepared from this mutant lacked ATP synthesis and ATP-driven proton translocation, even though immunoblots showed a significant level of subunit a. Similar results were obtained after purification and reconstitution of the mutant ATP synthase into liposomes. The location of subunit a with respect to its neighboring subunits b and c was probed by introducing cysteine substitutions that were known to promote cross-linking: a_L207C + c_I55C, a_L121C + b_N4C, and a_T107C + b_V18C. The last pair was unable to form cross-links in the background of the deletion mutant. The results indicate that loss of the N-terminal region of subunit a does not generally disrupt its structure, but does alter interactions with subunit b.

Interactions between subunits a and b in the rotary ATP synthase as determined by cross-linking.

The interaction of the membrane traversing stator subunits a and b of the rotary ATP synthase was probed by substitution of a single Cys into each subunit with subsequent Cu(2+) catalyzed cross-linking. Extensive interaction between the transmembrane (TM) region of one b subunit and TM2 of subunit a was indicated by cross-linking with 6 Cys pairs introduced into these regions. Additional disulfide cross-linking was observed between the N-terminus of subunit b and the periplasmic loop connecting TM4 and TM5 of subunit a. Finally, benzophenone-4-maleimide derivatized Cys in the 2-3 periplasmic loop of subunit a were shown to cross-link with the periplasmic N-terminal region of subunit b. These experiments help to define the juxtaposition of subunits b and a in the ATP synthase.

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

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.

Characterization of the first cytoplasmic loop of subunit a of the Escherichia coli ATP synthase by surface labeling, cross-linking, and mutagenesis.

The first cytoplasmic loop of subunit a of the Escherichia coli ATP synthase has been analyzed by cysteine substitution mutagenesis. 13 of the 26 residues tested were found to be accessible to the reaction with 3-(N-maleimidylpropionyl)-biocytin. The other 13 residues predominantly found in the central region of the polypeptide chain between the two transmembrane spans were more resistant to labeling by 3-(N-maleimidylpropionyl)-biocytin while in membrane vesicle preparations. This region of subunit a contains a conserved residue Glu-80, which when mutated to lysine resulted in a significant loss of ATP-driven proton translocation. Other substitutions including glutamine, alanine, and leucine were much less detrimental to function. Cross-linking studies with a photoactive cross-linking reagent were carried out. One mutant, K74C, was found to generate distinct cross-links to subunit b, and the cross-linking had little effect on proton translocation. The results indicate that the first transmembrane span (residues 40-64) of subunit a is probably near one or both of the b subunits and that a less accessible region of the first cytoplasmic loop (residues 75-90) is probably near the cytoplasmic surface, perhaps in contact with b subunits.

The role of transmembrane span 2 in the structure and function of subunit a of the ATP synthase from Escherichia coli.

The importance of the second transmembrane span of subunit a of the ATP synthase from Escherichia coli has been established by two approaches. First, biochemical analysis of five cysteine-substitution mutants, four of which were previously constructed for labeling experiments, revealed that only D119C, found within the second transmembrane span, was deleterious to ATP synthase function. This mutant had a greatly reduced growth yield, indicating inefficient ATP synthesis, but it retained a significant level of ATP-driven proton translocation and sensitivity to N,N(')-dicyclohexyl-carbodiimide, indicating more robust function in the direction of ATP hydrolysis. Second, the entire second transmembrane span was probed by alanine-insertion mutagenesis at six different positions, from residues 98 to 122. Insertions at the central four positions from residues 107 to 117 resulted in the inability to grow on succinate minimal medium, although normal levels of membrane-bound ATPase activity and significant levels of subunit a were detected. Double mutants were constructed with a mutation that permits cross-linking to the b subunit. Cross-linked products in the mutant K74C/114iA were seen, indicating no major disruption of the a-b interface due to the insertion at 114. Analysis of the K74C/110iA double mutant indicated that K74C is a partial suppressor of 110iA. In summary, the results support a model in which the amino-terminal, cytoplasmic end of the second transmembrane span has close contact with subunit b, while the carboxy-terminal, periplasmic end is important for proton translocation.