Steven B. Vik

Membrane Topology of Subunit a of the F1F0 ATP Synthase as Determined by Labeling of Unique Cysteine Residues

The membrane topology of the a subunit of the F1F0 ATP synthase from Escherichia coli has been probed by surface labeling using 3-(N-maleimidylpropionyl) biocytin. Subunit a has no naturally occurring cysteine residues, allowing unique cysteines to be introduced at the following positions: 8, 24, 27, 69, 89, 128, 131, 172, 176, 196, 238, 241, and 277 (following the COOH-terminal 271 and a hexahistidine tag). None of the single mutations affected the function of the enzyme, as judged by growth on succinate minimal medium. Membrane vesicles with an exposed cytoplasmic surface were prepared using a French pressure cell. Before labeling, the membranes were incubated with or without a highly charged sulfhydryl reagent, 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid. After labeling with the less polar biotin maleimide, the samples were solubilized with octyl glucoside/cholate and the subunit a was purified via the oligohistidine at its COOH terminus using immobilized nickel chromatography. The purified samples were electrophoresed and transferred to nitrocellulose for detection by avidin conjugated to alkaline phosphatase. Results indicated cytoplasmic accessibility for residues 69, 172, 176, and 277 and periplasmic accessibility for residues 8, 24, 27, and 131. On the basis of these and earlier results, a transmembrane topology for the subunit a is proposed.

A Novel Labeling Approach Supports the Five-transmembrane Model of Subunit a of the Escherichia coli ATP Synthase

Cysteine mutagenesis and surface labeling has been used to define more precisely the transmembrane spans of subunita of the Escherichia coli ATP synthase. Regions of subunit a that are exposed to the periplasmic space have been identified by a new procedure, in which cells are incubated with polymyxin B nonapeptide (PMBN), an antibiotic derivative that partially permeabilizes the outer membrane of E. coli, along with a sulfhydryl reagent, 3-(N-maleimidylpropionyl) biocytin (MPB). This procedure permits reaction of sulfhydryl groups in the periplasmic space with MPB, but residues in the cytoplasm are not labeled. Using this procedure, residues 8, 27, 37, 127, 131, 230, 231, and 232 were labeled and so are thought to be exposed in the periplasm. Using inside-out membrane vesicles, residues near the end of transmembrane spans 1, 64, 67, 68, 69, and 70 and residues near the end of transmembrane spans 5, 260, 263, and 265 were labeled. Residues 62 and 257 were not labeled. None of these residues were labeled in PMBN-permeabilized cells. These results provide a more detailed view of the transmembrane spans of subunit a and also provide a simple and reliable technique for detection of periplasmic regions of inner membrane proteins in E. coli.

A model for the structure of subunit a of the Escherichia coli ATP synthase and its role in proton translocation.

Most of what is known about the structure and function of subunit a, of the ATP synthase, has come from the construction and isolation of mutations, and their analysis in the context of the ATP synthase complex. Three classes of mutants will be considered in this review. (1) Cys substitutions have been used for structural analysis of subunit a, and its interactions with subunit c. (2) Functional residues have been identified by extensive mutagenesis. These studies have included the identification of second-site suppressors within subunit a. (3) Disruptive mutations include deletions at both termini, internal deletions, and single amino acid insertions. The results of these studies, in conjunction with information about subunits b and c, can be incorporated into a model for the mechanism of proton translocation in the Escherichia coli ATP synthase.

Insertion scanning mutagenesis of subunit a of the F1F0 ATP synthase near His245 and implications on gating of the proton channel.

Subunit a of the E. coliF1F0 ATP synthase was probed by insertion scanning mutagenesis in a region between residues Glu219and His245. A series of single amino acid insertions, of both alanine and aspartic acid, were constructed after the following residues: 225, 229, 233, 238, 243, and 245. The mutants were tested for growth yield, binding of F1 to membranes, dicyclohexylcarbodiimide sensitivity of ATPase activity, ATP-driven proton translocation, and passive proton permeability of membranes stripped of F1. Significant loss of function was seen only with insertions after positions 238 and 243. In contrast, both insertions after residue 225 and the alanine insertion after residue 245 were nearly identical in function to the wild type. The other insertions showed an intermediate loss of function. Missense mutations of His245 to serine and cysteine were nonfunctional, while the W241C mutant showed nearly normal ATPase function. Replacement of Leu162 by histidine failed to suppress the 245 mutants, but chemical rescue of H245S was partially successful using acetate. An interaction between Trp241 and His245 may be involved in gating a “half-channel” from the periplasmic surface of F0 to Asp61 of subunit a.

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.

Prediction of transmembrane topology of F0 proteins from Escherichia coli F1F0 ATP synthase using variational and hydrophobic moment analyses

The a subunit, a membrane protein from the E. coli F1F0 ATP synthase has been examined by Fourier analysis of hydrophobicity and of amino-acid residue variation. The amino-acid sequences of homologous subunits from Vibrio alginolyticus, Saccharomyces cerevisiae, Neurospora crassa, Aspergillus nidulans, Schizosaccharomyces pombe and Candida parapsilosis were used in the variability analysis. By Fourier analysis of sequence variation, two transmembrane helices are predicted to have one face in contact with membrane lipids, while the other spans are predicted to be more shielded from the lipids by protein. By Fourier analysis of hydrophobicity, six amphipathic alpha-helical segments are predicted in extra-membrane regions, including the region from Glu-196 to Asn-214. Fourier analysis of sequence variation in the b- and the c-subunits of the Escherichia coli F1F0 ATP synthase indicates that the single transmembrane span of the b-subunit and the C-terminal span of the c subunit each have a face in contact with membrane lipids. On the basis of this analysis topographical models for the a- and c-subunits and for the F0 complex are proposed.

Close proximity of a cytoplasmic loop of subunit a with c subunits of the ATP synthase from Escherichia coli

Interactions between subunit a and the c subunits of the Escherichia coli ATP synthase are thought to control proton translocation through the Fo sector. In this study cysteine substitution mutagenesis was used to define the cytoplasmic ends of the first three transmembrane spans of subunit a, as judged by accessibility to 3-N-maleimidyl-propionyl biocytin. The cytoplasmic end of the fourth transmembrane span could not be defined in this way because of the limited extent of labeling of all residues between 186 and 206. In contrast, most of the preceding residues in that region, closer to transmembrane span 3, were labeled readily. The proximity of this region to other subunits in Fo was tested by reacting mono-cysteine mutants with a photoactivated cross-linker. Residues 165, 169, 173, 174, 177, 178, and 182–184 could all be cross-linked to subunit c, but no sites were cross-linked to b subunits. Attempts using double mutants of subunita to generate simultaneous cross-links to two differentc subunits were unsuccessful. These results indicate that the cytoplasmic loop between transmembrane spans 3 and 4 of subunita is in close proximity to at least one csubunit. It is likely that the more highly conserved, carboxyl-terminal region of this loop has limited surface accessibility due to protein-protein interactions. A model is presented for the interaction of subunit a with subunit c, and its implications for the mechanism of proton translocation are discussed.

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 Cu2+ 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.

Constraining the Lateral Helix of Respiratory Complex I by Cross-linking Does Not Impair Enzyme Activity or Proton Translocation

Background: A lateral helix in the membrane arm of Complex I was proposed to function as a piston during proton translocation. Results: Cross-linking the lateral helix to other subunits did not impair proton translocation. Conclusion: The evidence does not support a piston-type role for the lateral helix. Significance: Energy transduction by Complex I must involve communication through other structural features. Complex I (NADH:ubiquinone oxidoreductase) is a multisubunit, membrane-bound enzyme of the respiratory chain. The energy from NADH oxidation in the peripheral region of the enzyme is used to drive proton translocation across the membrane. One of the integral membrane subunits, nuoL in Escherichia coli, has an unusual lateral helix of ∼75 residues that lies parallel to the membrane surface and has been proposed to play a mechanical role as a piston during proton translocation (Efremov, R. G., Baradaran, R., and Sazanov, L. A. (2010) Nature 465, 441–445). To test this hypothesis we have introduced 11 pairs of cysteine residues into Complex I; in each pair one is in the lateral helix, and the other is in a nearby region of subunit N, M, or L. The double mutants were treated with Cu2+ ions or with bi-functional methanethiosulfonate reagents to catalyze cross-link formation in membrane vesicles. The yields of cross-linked products were typically 50–90%, as judged by immunoblotting, but in no case did the activity of Complex I decrease by >10–20%, as indicated by deamino-NADH oxidase activity or rates of proton translocation. In contrast, several pairs of cysteine residues introduced at other interfaces of N:M and M:L subunits led to significant loss of activity, in particular, in the region of residue Glu-144 of subunit M. The results do not support the hypothesis that the lateral helix of subunit L functions like a piston, but rather, they suggest that conformational changes might be transmitted more directly through the functional residues of the proton translocation apparatus.

Direct transfer of NADH from malate dehydrogenase to complex I in Escherichia coli

During aerobic growth of Escherichia coli, nicotinamide adenine dinucleotide (NADH) can initiate electron transport at either of two sites: Complex I (NDH-1 or NADH: ubiquinone oxidoreductase) or a single-subunit NADH dehydrogenase (NDH-2). We report evidence for the specific coupling of malate dehydrogenase to Complex I. Membrane vesicles prepared from wild type cultures retain malate dehydrogenase and are capable of proton translocation driven by the addition of malate+NAD. This activity was inhibited by capsaicin, an inhibitor specific to Complex I, and it proceeded with deamino-NAD, a substrate utilized by Complex I, but not by NDH-2. The concentration of free NADH produced by membrane vesicles supplemented with malate+NAD was estimated to be 1 μM, while the rate of proton translocation due to Complex I was consistent with a some what higher concentration, suggesting a direct transfer mechanism. This interpretation was supported by competition assays in which inactive mutant forms of malate dehydrogenase were able to inhibit Complex I activity.These two lines of evidence indicate that the direct transfer of NADH from malate dehydrogenase to Complex I can occur in the E. coli system.