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. coli F1F0 ATP synthase was probed by insertion scanning mutagenesis in a region between residues Glu219 and 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.     

Deletions in the second stalk of F1F0-ATP synthase in Escherichia coli

In Escherichia coli F1F0-ATP synthase, the two b subunits form the second stalk spanning the distance between the membrane F0 sector and the bulk of F1. Current models predict that the stator should be relatively rigid and engaged in contact with F1 at fixed points. To test this hypothesis, we constructed a series of deletion mutations in the uncF(b) gene to remove segments from the middle of the second stalk of the subunit. Mutants with deletions of 7 amino acids were essentially normal, and those with deletions of up to 11 amino acids retained considerable activity. Membranes prepared from these strains had readily detectable levels of F1-ATPase activity and proton pumping activity. Removal of 12 or more amino acids resulted in loss of oxidative phosphorylation. Levels of membrane-associated F1-ATPase dropped precipitously for the longer deletions, and immunoblot analysis indicated that reductions in activity correlated with reduced levels of b subunit in the membranes. Assuming the likely alpha-helical conformation for this area of the b subunit, the 11-amino acid deletion would result in shortening the subunit by approximately 16 A. Since these deletions did not prevent the b subunit from participating in productive interactions with F1, we suggest that the b subunit is not a rigid rodlike structure, but has an inherent flexibility compatible with a dynamic role in coupling.     

Interacting helical faces of subunits a and c in the F1Fo ATP synthase of Escherichia coli defined by disulfide cross-linking

Subunits a and c of Fo are thought to cooperatively catalyze proton translocation during ATP synthesis by the Escherichia coli F1Fo ATP synthase. Optimizing mutations in subunit a at residues A217, I221, and L224 improves the partial function of the cA24D/cD61G double mutant and, on this basis, these three residues were proposed to lie on one face of a transmembrane helix of subunit a, which then interacted with the transmembrane helix of subunit c anchoring the essential aspartyl group. To test this model, in the present work Cys residues were introduced into the second transmembrane helix of subunit c and the predicted fourth transmembrane helix of subunit a. After treating the membrane vesicles of these mutants with Cu(1, 10-phenanthroline)2SO4 at 0 degrees, 10 degrees, or 20 degreesC, strong a-c dimer formation was observed at all three temperatures in membranes of 7 of the 65 double mutants constructed, i.e., in the aS207C/cI55C, aN214C/cA62C, aN214C/cM65C, aI221C/cG69C, aI223C/cL72C, aL224C/cY73C, and aI225C/cY73C double mutant proteins. The pattern of cross-linking aligns the helices in a parallel fashion over a span of 19 residues with the aN214C residue lying close to the cA62C and cM65C residues in the middle of the membrane. Lesser a-c dimer formation was observed in nine other double mutants after treatment at 20 degreesC in a pattern generally supporting that indicated by the seven landmark residues cited above. Cross-link formation was not observed between helix-1 of subunit c and helix-4 of subunit a in 19 additional combinations of doubly Cys-substituted proteins. These results provide direct chemical evidence that helix-2 of subunit c and helix-4 of subunit a pack close enough to each other in the membrane to interact during function. The proximity of helices supports the possibility of an interaction between Arg210 in helix-4 of subunit a and Asp61 in helix-2 of subunit c during proton translocation, as has been suggested previously.     

Hydrogen sulfide production and fermentative gas production by Salmonella typhimurium require F0F1 ATP synthase activity

A previously isolated mutant of Salmonella typhimurium lacking hydrogen sulfide production from both thiosulfate and sulfite was shown to have a single mutation which also caused the loss of fermentative gas production and the ability to grow on nonfermentable substrates and which mapped in the vicinity of the atp chromosomal locus. The implication that F0F1 ATP synthase might be essential for H2S and fermentative gas production was explored. The phs plasmid conferring H2S production on wild-type Escherichia coli failed to confer this ability on seven of eight E. coli atp point mutants representing, collectively, the eight genes encoding the subunits of F0F1 ATP synthase. However, it did confer some thiosulfate reductase activity on all except the mutant with a lesion in the ATP synthase catalytic subunit. Localized mutagenesis of the Salmonella atp chromosomal region yielded 500 point mutants unable to reduce thiosulfate to H2S or to produce gas from glucose, but differing in the extents of their ability to grow on succinate, to perform proton translocation as measured in a fluorescence quenching assay, and to reduce sulfite to H2S. Biochemical assays showed that all mutants were completely devoid of both methyl viologen and formate-linked thiosulfate reductase and that N,N'-dicyclohexylcarbodiimide blocked thiosulfate reductase activity by the wild type, suggesting that thiosulfate reductase activity has an absolute requirement for F0F1 ATP synthase. Hydrogenase-linked formate dehydrogenase was also affected, but not as severely as thiosulfate reductase. These results imply that in addition to linking oxidation with phosphorylation, F0F1 ATP synthase plays a key role in the proton movement accompanying certain anaerobic reductions and oxidations.     

Cross-linking of the delta subunit to one of the three alpha subunits has no effect on functioning, as expected if delta is a part of the stator that links the F1 and F0 parts of the Escherichia coli ATP synthase

A mutant of the Escherichia coli F1F0-ATPase has been generated (alphaQ2C) in which the glutamine at position 2 of the alpha subunit has been replaced with a cysteine residue. Cu2+ treatment of ECF1 from this mutant cross-linked an alpha subunit to the delta subunit in high yield. Two different sites of disulfide bond formation were involved, i.e. between Cys90 (or the closely spaced Cys47) of alpha with Cys140 of delta, and between Cys2 of alpha and Cys140 of delta. Small amounts of other cross-linked products, including alpha-alpha, delta internal, and alpha-alpha-delta were obtained. In ECF1F0, there was no cross-linking between the intrinsic Cys of alpha and Cys140. Instead, the product generated between Cys2 of alpha and Cys140 of delta was obtained at near 90% yield. Small amounts of alpha-alpha and delta internal were present, and under high Cu2+ concentrations, alpha-alpha-delta was also formed. The ATPase activity of ECF1 and ECF1F0 was not significantly affected by the presence of these cross-links. When Cys140 of delta was first modified with N-ethylmaleimide in ECF1F0, an alpha-delta cross-link was still produced, although in lower yield, between Cys64 of delta and Cys2 of alpha. ATP hydrolysis-linked proton pumping of inner membranes from the mutant alpha2QC was only marginally affected by cross-linking of the alpha to the delta subunit. These results indicate that Cys140 and Cys64 of the delta subunit and Cys2 of the alpha subunit are in close proximity. This places the delta subunit near the top of the alpha-beta hexagon and not in the stalk region. As fixing the delta to the alpha by cross-linking does not greatly impair either the ATPase function of the enzyme, or coupled proton translocation, we argue that the delta subunit forms a portion of the stator linking F1 to F0.     

Percent cholesterol absorption in normal women and men quantified with dual stable isotopic tracers and negative ion mass spectrometry

Amino acid substitutions at many positions in the a subunit of F1F0 ATP synthase result in impaired proton translocation and altered catalytic activity. In this work, we demonstrate that amino acid substitutions in the a subunit affect the epsilon subunit. In mutant F1F0 ATP synthases, the epsilon subunit was studied by determining its sensitivity to proteolysis and by chemical crosslinking under conditions of active turnover and in quiescent enzyme. Like native F1F0 ATP synthase, the epsilon subunit in enzymes carrying either the aarg-210-->ile or agly-218-->asp substitutions proved resistant to trypsin digestion during ATP hydrolysis. In each case, the epsilon subunit was rapidly digested in the presence of a nonhydrolyzable ligand, but this did not result in the activation of hydrolytic activity typically seen in wild-type enzyme. In enzyme carrying the aala-217-->arg substitution, the trypsin digestion of the epsilon subunit occurred regardless of ligand and was accompanied by a limited hydrolytic activation. Relative to the native F1F0 ATP synthase, the aala-217-->arg substitution resulted in reduced efficiency of crosslinking between the epsilon and beta subunits using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. These observations indicate that the structural changes resulting from amino acid substitutions in the a subunit are propagated to the epsilon subunit and are specific to the individual substitutions.     

Topological and functional relationship of subunits F1-gamma and F0I-PVP(b) in the mitochondrial H+-ATP synthase

Diamide treatment of the F0F1-ATP synthase in "inside out" submitochondrial particles (ESMP) in the absence of a respiratory Delta mu H+ as well as of isolated Fo reconstituted with F1 or F1-gamma subunit results in direct disulfide cross-linking between cysteine 197 in the carboxy-terminal region of the F0I-PVP(b) subunit and cysteine 91 at the carboxyl end of a small alpha-helix of subunit F1-gamma, both located in the stalk. The F0I-PVP(b) and F1-gamma cross-linking cause dramatic enhancement of oligomycin-sensitive decay of Delta mu H+. In ESMP and MgATP particles the cross-linking is accompanied by decoupling of respiratory ATP synthesis. These effects are consistent with the view that F0I-PVP(b) and F1-gamma are components of the stator and rotor of the proposed rotary motor, respectively. The fact that the carboxy-terminal region of F0I-PVP(b) and the short alpha-helix of F1-gamma can form a direct disulfide bridge shows that these two protein domains are, at least in the resting state of the enzyme, in direct contact. In isolated F0, diamide also induces cross-linking of OSCP with another subunit of F0, but this has no significant effect on proton conduction. When ESMP are treated with diamide in the presence of Delta mu H+ generated by respiration, neither cross-linking between F0I-PVP(b) and F1-gamma subunits nor the associated effects on proton conduction and ATP synthesis is observed. Cross-linking is restored in respiring ESMP by Delta mu H+ collapsing agents as well as by DCCD or oligomycin. These observations indicate that the torque generated by Delta mu H+ decay through Fo induces a relative motion and/or a separation of the F0I-PVP(b) subunit and F1-gamma which places the single cysteine residues, present in each of the two subunits, at a distance at which they cannot be engaged in disulfide bridging.     

Chemical modification of the bovine mitochondrial bc1 complex reveals critical acidic residues involved in the proton pumping activity

Bovine heart ubiquinol-cytochrome c reductase (bc1 complex) was modified with N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ), which is a selective reagent for buried carboxyl groups. EEDQ treatment caused a loss of the proton pumping activity of liposome-reconstituted bc1 complex, without effect on the passive proton conductivity of the proteoliposomes. Although the decoupling effect produced on proton translocation was similar to that elicited by N,N'-dicyclohexylcarbodiimide (DCCD) modification of cytochrome b and subunit IX, EEDQ modified different subunits, namely the Core protein II and the iron-sulfur protein (ISP). A time-dependent increase of the labeling of both subunits was observed which was kinetically comparable with the decrease of the H+/e- ratio. Trypsin treatment of the complex showed that the EEDQ-modified carboxyl group in the ISP belongs to the protruding moiety of the protein, holding the Fe/S cluster. The results obtained show that critical acidic residues, located in different subunits of the bc1 complex, at both sides of the membrane, contribute to its proton pumping activity.     

A triple mutation in the a subunit of the Escherichia coli/Propionigenium modestum F1Fo ATPase hybrid causes a switch from Na+ stimulation to Na+ inhibition

Previously we have shown that the Na+-translocating Escherichia coli (F1-delta)/Propionigenium modestum (Fo+delta) hybrid ATPase acquires a Na+-independent phenotype by the c subunit double mutation F84L, L87V that is reflected by Na+-independent growth of the mutant strain MPC8487 on succinate [Kaim, G., and Dimroth, P. (1995) J. Mol. Biol. 253, 726-738]. Here we describe a new class of mutants that were obtained by random mutagenesis and screening for Na+-independent growth on succinate. All six mutants of the new class contained four mutations in the a subunit (S89P, K220R, V264E, I278N). Results from site-specific mutagenesis revealed that the substitutions K220R, V264E, and I278N were sufficient to create the new phenotype. The resulting E. coli mutant strain MPA762 could only grow in the absence but not in the presence of Na+ ions on succinate minimal medium. This effect of Na+ ions on growth correlated with a Na+-specific inhibition of the mutant ATPase. The Ki for NaCl was 1. 5 mM at pH 6.5, similar to the Km for NaCl in activating the parent hybrid ATPase at this pH. On the other hand, activation by Li+ ions was retained in the new mutant ATPase. In the absence of Na+ or Li+, the mutant enzyme had the same pH optimum at pH 6.5 and twice the specific activity as the parent hybrid ATPase. In accordance with the kinetic data, the reconstituted mutant ATPase catalyzed H+ or Li+ transport but no Na+ transport. These results show for the first time that the coupling ion selectivity of F1Fo ATPases is determined by structural elements not only of the c subunit but also of the a subunit.     

Functional and idling rotatory motion within F1-ATPase

ATP synthase mediates proton flow through its membrane portion, F0, which drives the synthesis of ATP in its headpiece, F1. The F1-portion contains a hexagonal array of three subunits alpha and three beta encircling a central subunit gamma, that in turn interacts with a smaller epsilon and with F0. Recently we reported that the application of polarized absorption recovery after photobleaching showed the ATP-driven rotation of gamma over at least two, if not three, beta. Here we extend probes of such rotation aided by a new theory for assessing continuous versus stepped, Brownian versus unidirectional molecular motion. The observed relaxation of the absorption anisotropy is fully compatible with a unidirectional and stepping rotation of gamma over three equidistantly spaced angular positions in the hexagon formed by the alternating subunits alpha and beta. The results strongly support a rotational catalysis with equal participation of all three catalytic sites. In addition we report a limited rotation of gamma without added nucleotides, perhaps idling and of Brownian nature, that covers only a narrow angular domain.     

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.     

Molecular basis for the coupling ion selectivity of F1F0 ATP synthases: probing the liganding groups for Na+ and Li+ in the c subunit of the ATP synthase from Propionigenium modestum

The conserved glutamate residue at position 65 of the Propionigenium modestum c subunit is directly involved in binding and translocation of Na+ across the membrane. The site-specific introduction of the cQ32I and cS66A substitutions in the putative vicinity to cE65 inhibited growth of the single-site mutants on succinate minimal agar, indicating that both amino acid residues are important for proper function of the oxidative phosphorylation system. This growth inhibition was abolished, however, if the cF84L/cL87V double mutation was additionally present in the P. modestum c subunit. The newly constructed Escherichia coli strain MPC848732I, harboring the cQ32I/cF84L/cL87V triple mutation, revealed a change in the coupling ion specificity from Na+ to H+. ATP hydrolysis by this enzyme was therefore not activated by NaCl, and ATP-driven H+ transport was not affected by this alkali salt. Both activities were influenced, however, by LiCl. These data demonstrate the loss of the Na+ binding site and retention of Li+ and H+ binding sites within this mutant ATPase. In the E. coli strain MPC848766A (cS66A/cF84L/cL87V), the specificity of the ATPase was further restricted to H+ as the exclusive coupling ion. Therefore, neither Na+ nor Li+ stimulated the ATPase activity, and no ATP-driven Li+ transport was observed. The ATPase of the E. coli mutant MPC32N (cQ32N) was activated by NaCl and LiCl. The mutant ATPase exhibited a 5-fold higher Km for NaCl but no change in the Km for LiCl in comparison to that of the parent strain. These results demonstrate that the binding of Na+ to the c subunit of P. modestum requires liganding groups provided by Q32, E65, and S66. For the coordination of Li+, two liganding partners, E65 and S66, are sufficient, and H+ translocation was mediated by E65 alone.     

The second stalk composed of the b- and delta-subunits connects F0 to F1 via an alpha-subunit in the Escherichia coli ATP synthase

The b- and delta-subunits of the Escherichia coli ATP synthase are critical for binding ECF1 to the F0 part, and appear to constitute the stator necessary for holding the alpha3beta3 hexamer as the c-epsilon-gamma domain rotates during catalysis. Previous studies have determined that the b-subunits are dimeric for a large part of their length, and interact with the F1 part through the delta-subunit (Rodgers, A. J. W., Wilkens, S., Aggeler, R., Morris, M. B., Howitt, S. M., and Capaldi, R. A. (1997) J. Biol. Chem. 272, 31058-31064). To further study b-subunit interactions, three mutants were constructed in which Ser-84, Ala-144, and Leu-156, respectively, were replaced by Cys. Treatment of purified ECF1F0 from all three mutants with CuCl2 induced disulfide formation resulting in b-subunit dimer cross-link products. In addition, the mutant bL156C formed a cross-link from a b-subunit to an alpha-subunit via alphaCys90. Neither b-b nor b-alpha cross-linking had significant effect on ATPase activities in any of the mutants. Proton pumping activities were measured in inner membranes from the three mutants. Dimerization of the b-subunit did not effect proton pumping in mutants bS84C or bA144C. In the mutant bL156C, CuCl2 treatment reduced proton pumping markedly, probably because of uncoupling caused by the b-alpha cross-link formation. The results show that the alpha-subunit forms part of the binding site on ECF1 for the b2delta domain and that the b-subunit extends all the way from the membrane to the top of the F1 structure. Some conformational flexibility in the connection between the second stalk and F1 appears to be required for coupled catalysis.     

The stalk region of the Escherichia coli ATP synthase. Tyrosine 205 of the gamma subunit is in the interface between the F1 and F0 parts and can interact with both the epsilon and c oligomer

The soluble portion of the Escherichia coli F1F0 ATP synthase (ECF1) and E. coli F1F0 ATP synthase (ECF1F0) have been isolated from a novel mutant gammaY205C. ECF1 isolated from this mutant had an ATPase activity 3.5-fold higher than that of wild-type enzyme and could be activated further by maleimide modification of the introduced cysteine. This effect was not seen in ECF1F0. The mutation partly disrupts the F1 to F0 interaction, as indicated by a reduced efficiency of proton pumping. ECF1 containing the mutation gammaY205C was bound to the membrane-bound portion of the E. coli F1F0 ATP synthase (ECF0) isolated from mutants cA39C, cQ42C, cP43C, and cD44C to reconstitute hybrid enzymes. Cu2+ treatment or reaction with 5,5'-dithio-bis(2-nitro-benzoic acid) induced disulfide bond formation between the Cys at gamma position 205 and a Cys residue at positions 42, 43, or 44 in the c subunit but not at position 39. Using Cu2+ treatment, this covalent cross-linking was obtained in yields as high as 95% in the hybrid ECF1 gammaY205C/cQ42C and in ECF1F0 isolated from the double mutant of the same composition. The covalent linkage of the gamma to a c subunit had little effect on ATPase activity. However, ATP hydrolysis-linked proton translocation was lost, by modification of both gamma Cys-205 and c Cys-42 by bulky reagents such as 5,5'-dithio-bis (2-nitro-benzoic acid) or benzophenone-4-maleimide. In both ECF1 and ECF1F0 containing a Cys at gamma 205 and a Cys in the epsilon subunit (at position 38 or 43), cross-linking of the gamma to the epsilon subunit was induced in high yield by Cu2+. No cross-linking was observed in hybrid enzymes in which the Cys was at position 10, 65, or 108 of the epsilon subunit. Cross-linking of gamma to epsilon had only a minimal effect on ATP hydrolysis. The reactivity of the Cys at gamma 205 showed a nucleotide dependence of reactivity to maleimides in both ECF1 and ECF1F0, which was lost in ECF1 when the epsilon subunit was removed. Our results show that there is close interaction of the gamma and epsilon subunits for the full-length of the stalk region in ECF1F0. We argue that this interaction controls the coupling between nucleotide binding sites and the proton channel in ECF1F0.     

Direct observation of the rotation of epsilon subunit in F1-ATPase

Rotation of the epsilon subunit in F1-ATPase from thermophilic Bacillus strain PS3 (TF1) was observed under a fluorescence microscope by the method used for observation of the gamma subunit rotation (Noji, H., Yasuda, R., Yoshida, M., and Kinosita, K., Jr. (1997) Nature 386, 299-302). The alpha3 beta3 gamma epsilon complex of TF1 was fixed to a solid surface, and fluorescently labeled actin filament was attached to the epsilon subunit through biotin-streptavidin. In the presence of ATP, the filament attached to epsilon subunit rotated in a unidirection. The direction of the rotation was the same as that observed for the gamma subunit. The rotational velocity was slightly slower than the filament attached to the gamma subunit, probably due to the experimental setup used. Thus, as suggested from biochemical studies (Aggeler, R., Ogilvie, I. , and Capaldi, R. A. (1997) J. Biol. Chem. 272, 19621-19624), the epsilon subunit rotates with the gamma subunit in F1-ATPase during catalysis.     

Yeast mitochondrial F1F0-ATP synthase exists as a dimer: identification of three dimer-specific subunits

Using the technique of blue native gel electrophoresis, the oligomeric state of the yeast mitochondrial F1F0-ATP synthase was analysed. Solubilization of mitochondrial membranes with low detergent to protein ratios led to the identification of the dimeric state of the ATP synthase. Analysis of the subunit composition of the dimer, in comparison with the monomer, revealed the presence of three additional small proteins. These dimer-specific subunits of the ATP synthase were identified as the recently described subunit e/Tim11 (Su e/Tim11), the putative subunit g homolog (Su g) and a new component termed subunit k (Su k). Although, as shown here, these three proteins are not required for the formation of enzymatically active ATP synthase, Su e/Tim11 and Su g are essential for the formation of the dimeric state. Su e/Tim11 appears to play a central role in this dimerization process. The dimer-specific subunits are associated with the membrane bound F0-sector. The F0-sector may thereby be involved in the dimerization of two monomeric F1F0-ATP synthase complexes. We speculate that the F1F0-ATP synthase of yeast, like the other complexes of oxidative phosphorylation, form supracomplexes to optimize transduction of energy and to enhance the stability of the complex in the membrane.     

Purification and properties of the F1F0 ATPase of Ilyobacter tartaricus, a sodium ion pump

The ATPase of Ilyobacter tartaricus was solubilized from the bacterial membranes and purified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed the usual subunit pattern of a bacterial F1F0 ATPase. The polypeptides with apparent molecular masses of 56, 52, 35, 16.5, and 6.5 kDa were identified as the alpha, beta, gamma, epsilon, and c subunits, respectively, by N-terminal protein sequencing and comparison with the sequences of the corresponding subunits from the Na(+)-translocating ATPase of Propionigenium modestum. Two overlapping sequences were obtained for the polypeptides moving with an apparent molecular mass of 22 kDa (tentatively assigned as b and delta subunits). No sequence could be determined for the putative a subunit (apparent molecular mass, 25 kDa). The c subunits formed a strong aggregate with the apparent molecular mass of 50 kDa which required treatment with trichloroacetic acid for dissociation. The ATPase was inhibited by dicyclohexyl carbodiimide, and Na+ ions protected the enzyme from this inhibition. The ATPase was specifically activated by Na+ or Li+ ions, markedly at high pH. After reconstitution into proteoliposomes, the enzyme catalyzed the ATP-dependent transport of Na+, Li+, or Hi+. Proton transport was specifically inhibited by Na+ or Li+ ions, indicating a competition between these alkali ions and protons for binding and translocation across the membrane. These experiments characterize the I. tartaricus ATPase as a new member of the family of FS-ATPases, which use Na+ as the physiological coupling ion for ATP synthesis.     

The oligomycin sensitivity conferring protein of rat liver mitochondrial ATP synthase: arginine 94 is important for the binding of OSCP to F1

The oligomycin sensitivity conferring protein (OSCP) is an essential subunit of the mitochondrial ATP synthase (F0F1) long regarded as being directly involved in the energetic coupling of proton transport to ATP synthesis. To gain insight into the function of OSCP, mutations were made in a highly conserved central region of the subunit, and the recombinant proteins were studied using several biochemical assays. Rat liver OSCP was expressed to high levels in Escherichia coli, solubilized from inclusion bodies, renatured, and purified to homogeneity. The recombinant protein was able to reconstitute oligomycin-sensitive ATPase activity to inner membrane vesicles depleted of F1 and OSCP, and bound to F1 with a stoichiometry of 1:1. A novel fluorescence anisotropy assay was developed to study the affinity of binding of F1 to OSCP, providing a Kd value of 51 +/- 11 nM. Two highly conserved, charged residues (E91 and R94) which lie within the central region of OSCP were mutated, and the recombinant proteins (E91Q, R94Q, and R94A) were purified to homogeneity and judged by CD spectroscopy to have structures similar to that of the wild-type protein. Both R94 mutants demonstrated little or no binding to F1, while the E91Q bound in a manner identical to that of wild-type OSCP. Significantly, all three mutant proteins were able to reconstitute F1 with membranes and to confer oligomycin sensitivity to the same extent as wild-type OSCP. These results demonstrate that a single tight binding site exists on isolated rat liver F1 for OSCP, and implicate arginine 94 as playing a critical role in this site. In addition, these results indicate that this tight binding site is not required for conferral of oligomycin sensitivity to the reconstituted F0F1 complex.