Mutational analysis of the glycine-rich region of the c subunit of the Escherichia coli F0F1 ATPase.

Eight strains carrying amino acid substitutions within the c subunit of the F0F1 ATPase of Escherichia coli have been constructed by using site-directed mutagenesis. Three strains carrying the substitutions Gly-23----Leu, Ala-24----Leu, and Gly-38----Leu, which reside in or near the highly conserved glycine-rich region of the c subunit, are unable to carry out oxidative phosphorylation. Membranes prepared from these strains possess basal levels of ATPase activity. In contrast, strains carrying the substitutions Ile-30----Phe, Gly-33----Leu, Gly-58----Leu, and Lys-34----Val and the Lys-34----Val, Glu-37----Gln double substitution were found to possess a coupled phenotype similar to that of the wild type.     

Mutations in the conserved proline 43 residue of the uncE protein (subunit c) of Escherichia coli F1F0-ATPase alter the coupling of F1 to F0

The conserved Pro43 residue of the uncE protein (subunit c) of the Escherichia coli F1F0-ATPase was changed to Ser or Ala by oligonucleotide-directed mutagenesis, and the mutations were incorporated into the chromosome. The resultant mutant strains were capable of oxidative phosphorylation as indicated by their ability to grow on succinate and had growth yields on glucose that were 80-90% of wild type. Membrane vesicles from the mutants were slightly less efficient than wild type vesicles in ATP-driven proton pumping as indicated by ATP-dependent quenching of quinacrine fluorescence. The decreased quenching response was not due to increased H+ leakiness of the mutant membranes or to loss of F1-ATPase activity from the membrane. These results indicate that the mutant F1F0-ATPases are defective in coupling ATP hydrolysis to H+ translocation. The membrane ATPase activity of the mutants was inhibited less by dicyclohexylcarbodiimide than that of wild type. The decrease in sensitivity to inhibition by dicyclohexylcarbodiimide was caused primarily by dissociation of the F1-ATPase from the mutant F0 in the ATPase assay mixture. These results support the idea that Pro43, and neighboring conserved polar residues play an important role in the binding and functional coupling of F1 to F0. Although a Pro residue is found at position 43 in all species of subunit c studied, surprisingly, it is not absolutely essential to function.     

Effects of mutations of conserved Lys-155 and Thr-156 residues in the phosphate-binding glycine-rich sequence of the F1-ATPase beta subunit of Escherichia coli.

beta Lys-155 in the glycine-rich sequence of the beta subunit of Escherichia coli F1-ATPase has been shown to be near the gamma-phosphate moiety of ATP by affinity labeling (Ida, K., Noumi, T., Maeda, M., Fukui, T., and Futai, M. (1991) J. Biol. Chem. 266, 5424-5429). For examination of the roles of beta Lys-155 and beta Thr-156, mutants (beta Lys-155-->Ala, Ser, or Thr; beta Thr-156-->Ala, Cys, Asp, or Ser; beta Lys-155/beta Thr-156-->beta Thr-155/beta Lys-156; and beta Thr-156/beta Val-157-->beta Ala-156/beta Thr-157) were constructed, and their properties were studied extensively. The beta Ser-156 mutant was active in ATP synthesis and had approximately 1.5-fold higher membrane ATPase activity than the wild type. Other mutants were defective in ATP synthesis, had < 0.1% of the membrane ATPase activity of the wild type, and showed no ATP-dependent formation of an electrochemical proton gradient. The mutants had essentially the same amounts of F1 in their membranes as the wild type. Purified mutant enzymes (beta Ala-155, beta Ser-155, beta Ala-156, and beta Cys-156) showed low rates of multisite (< 0.02% of the wild type) and unisite (< 1.5% of the wild type) catalyses. The k1 values of the mutant enzymes for unisite catalysis were lower than that of the wild type: not detectable with the beta Ala-156 and beta Cys-156 enzymes and 10(2)-fold lower with the beta Ala-155 and beta Ser-155 enzymes. The beta Thr-156-->Ala or Cys enzyme showed an altered response to Mg2+, suggesting that beta Thr-156 may be closely related to Mg2+ binding. These results suggest that beta Lys-155 and beta Thr-156 are essential for catalysis and are possibly located in the catalytic site, although beta Thr-156 could be replaced by a serine residue.     

Met23Lys mutation in subunit gamma of F(O)F(1)-ATP synthase from Rhodobacter capsulatus impairs the activation of ATP hydrolysis by protonmotive force

H(+)-F(O)F(1)-ATP synthase couples proton flow through its membrane portion, F(O), to the synthesis of ATP in its headpiece, F(1). Upon reversal of the reaction the enzyme functions as a proton pumping ATPase. Even in the simplest bacterial enzyme the ATPase activity is regulated by several mechanisms, involving inhibition by MgADP, conformational transitions of the epsilon subunit, and activation by protonmotive force. Here we report that the Met23Lys mutation in the gamma subunit of the Rhodobacter capsulatus ATP synthase significantly impaired the activation of ATP hydrolysis by protonmotive force. The impairment in the mutant was due to faster enzyme deactivation that was particularly evident at low ATP/ADP ratio. We suggest that the electrostatic interaction of the introduced gammaLys23 with the DELSEED region of subunit beta stabilized the ADP-inhibited state of the enzyme by hindering the rotation of subunit gamma rotation which is necessary for the activation.     

Escherichia coli H(+)-ATPase: role of the delta subunit in binding Fl to the Fo sector.

The roles of the Escherichia coli H(+)-ATPase (FoFl) delta subunit (177 amino acid residues) was studied by analyzing mutants. The membranes of nonsense (Gln-23----end, Gln-29----end, Gln-74----end) and missense (Gly-150----Asp) mutants had very low ATPase activities, indicating that the delta subunit is essential for the binding of the Fl portion to Fo. The Gln-176----end mutant had essentially the same membrane-bound activity as the wild type, whereas in the Val-174----end mutant most of the ATPase activity was in the cytoplasm. Thus Val-174 (and possibly Leu-175 also) was essential for maintaining the structure of the subunit, whereas the two carboxyl terminal residues Gln-176 and Ser-177 were dispensable. Substitutions were introduced at various residues (Thr-11, Glu-26, Asp-30, Glu-42, Glu-82, Arg-85, Asp-144, Arg-154, Asp-161, Ser-163), including apparently conserved hydrophilic ones. The resulting mutants had essentially the same phenotypes as the wild type, indicating that these residues do not have any significant functional role(s). Analysis of mutations (Gly-150----Asp, Pro, or Ala) indicated that Gly-150 itself was not essential, but that the mutations might affect the structure of the subunit. These results suggest that the overall structure of the delta subunit is necessary, but that individual residues may not have strict functional roles.     

The role of the epsilon subunit in the Escherichia coli ATP synthase. The C-terminal domain is required for efficient energy coupling

The role of the C-domain of the epsilon subunit of ATP synthase was investigated by fusing either the 20-kDa flavodoxin (Fd) or the 5-kDa chitin binding domain (CBD) to the N termini of both full-length epsilon and a truncation mutant epsilon(88-stop). All mutant epsilon proteins were stable in cells and supported F1F0 assembly. Cells expressing the Fd-epsilon or Fd-epsilon(88-stop) mutants were unable to grow on acetate minimal medium, indicating their inability to carry out oxidative phosphorylation because of steric blockage of rotation. The other forms of epsilon supported growth on acetate. Membrane vesicles containing Fd-epsilon showed 23% of the wild type ATPase activity but no proton pumping, suggesting that the ATP synthase is intrinsically partially uncoupled. Vesicles containing CBD-epsilon were indistinguishable from the wild type in ATPase activity and proton pumping, indicating that the N-terminal fusions alone do not promote uncoupling. Fd-epsilon(88-stop) caused higher rates of uncoupled ATP hydrolysis than Fd-epsilon, and epsilon(88-stop) showed an increased rate of membrane-bound ATP hydrolysis but decreased proton pumping relative to the wild type. Both results demonstrate the role of the C-domain in coupling. Analysis of the wild type and epsilon(88-stop) mutant membrane ATPase activities at concentrations of ATP from 50 mum to 8 mm showed no significant dependence of the ratio of bound/released ATPase activity on ATP concentration. These results support the hypothesis that the main function of the C-domain in the Escherichia coli epsilon subunit is to reduce uncoupled ATPase activity, rather than to regulate coupled activity.     

Role of the delta subunit in enhancing proton conduction through the F0 of the Escherichia coli F1F0 ATPase.

We studied the effect of the delta subunit of the Escherichia coli F1 ATPase on the proton permeability of the F0 proton channel synthesized and assembled in vivo. Membranes isolated from an unc deletion strain carrying a plasmid containing the genes for the F0 subunits and the delta subunit were significantly more permeable to protons than membranes isolated from the same strain carrying a plasmid containing the genes for the F0 subunits alone. This increased proton permeability could be blocked by treatment with either dicyclohexyl-carbodiimide or purified F1, both of which block proton conduction through the F0. After reconstitution with purified F1 in vitro, both membrane preparations could couple proton pumping to ATP hydrolysis. These results demonstrate that an interaction between the delta subunit and the F0 during synthesis and assembly produces a significant change in the proton permeability of the F0 proton channel.     

Functional role of charged residues in the transmembrane segments of the yeast plasma membrane H+-ATPase

As defined by hydropathy analysis, the membrane-spanning segments of the yeast plasma membrane H(+)-ATPase contain seven negatively charged amino acids (Asp and Glu) and four positively charged amino acids (Arg and His). To explore the functional role of these residues, site-directed mutants at all 11 positions and at Glu-288, located near the cytoplasmic end of M3, have been constructed and expressed in yeast secretory vesicles. Substitutions at four of the positions (Glu-129, Glu-288, Asp-833, and Arg-857) had no significant effect on ATP hydrolysis or ATP-dependent proton pumping, substitutions at five additional positions (Arg-695, His-701, Asp-730, Asp-739, and Arg-811) led to misfolding of the ATPase and blockage at an early stage of biogenesis, and substitutions of Asp-143 allowed measurable biogenesis but nearly abolished ATP hydrolysis and proton transport. Of greatest interest were mutations of Glu-703 in M5 and Glu-803 in M8, which altered the apparent coupling between hydrolysis and transport. Three Glu-703 mutants (E703Q, E703L, E703D) showed significantly reduced pumping over a wide range of hydrolysis values and thus appeared to be partially uncoupled. At Glu-803, by contrast, one mutant (E803N) was almost completely uncoupled, while another (E803Q) pumped protons at an enhanced rate relative to the rate of ATP hydrolysis. Both Glu-703 and Glu-803 occupy positions at which amino acid substitutions have been shown to affect transport by mammalian P-ATPases. Taken together, the results provide growing evidence that residues in membrane segments 5 and 8 of the P-ATPases contribute to the cation transport pathway and that the fundamental mechanism of transport has been conserved throughout the group.     

Investigation of the catalytic site within the ATP-grasp domain of Clostridium symbiosum pyruvate phosphate dikinase

Pyruvate phosphate dikinase (PPDK) catalyzes the interconversion of ATP, P(i), and pyruvate with AMP, PP(i), and phosphoenolpyruvate (PEP) in three partial reactions as follows: 1) E-His + ATP --> E-His-PP.AMP; 2) E-His-PP.AMP + P(i) --> E-His-P.AMP.PP(i); and 3) E-His-P + pyruvate --> E.PEP using His-455 as the carrier of the transferred phosphoryl groups. The crystal structure of the Clostridium symbiosum PPDK (in the unbound state) reveals a three-domain structure consisting of consecutive N-terminal, central His-455, and C-terminal domains. The N-terminal and central His-455 domains catalyze partial reactions 1 and 2, whereas the C-terminal and central His-455 domains catalyze partial reaction 3. Attempts to obtain a crystal structure of the enzyme with substrate ligands bound at the nucleotide binding domain have been unsuccessful. The object of the present study is to demonstrate Mg(II) activation of catalysis at the ATP/P(i) active site, to identify the residues at the ATP/P(i) active site that contribute to catalysis, and to identify roles for these residues based on their positions within the active site scaffold. First, Mg(II) activation studies of catalysis of E + ATP + P(i) --> E-P + AMP + PP(i) partial reaction were carried out using a truncation mutant (Tem533) in which the C-terminal domain is absent. The kinetics show that a minimum of 2 Mg(II) per active site is required for the reaction. The active site residues used for substrate/cofactor binding/activation were identified by site-directed mutagenesis. Lys-22, Arg-92, Asp-321, Glu-323, and Gln-335 mutants were found to be inactive; Arg-337, Glu-279, Asp-280, and Arg-135 mutants were partially active; and Thr-253 and Gln-240 mutants were almost fully active. The participation of the nucleotide ribose 2'-OH and alpha-P in enzyme binding is indicated by the loss of productive binding seen with substrate analogs modified at these positions. The ATP, P(i), and Mg(II) ions were docked into the PPDK N-terminal domain crevice, in an orientation consistent with substrate/cofactor binding modes observed for other members of the ATP-Grasp fold enzyme superfamily and consistent with the structure-function data. On the basis of this docking model, the ATP polyphosphate moiety is oriented/activated for pyrophosphoryl transfer through interaction with Lys-22 (gamma-P), Arg-92 (alpha-P), and the Gly-101 to Met-103 loop (gamma-P) as well as with the Mg(II) cofactors. The P(i) is oriented/activated for partial reaction 2 through interaction with Arg-337 and a Mg(II) cofactor. The Mg(II) ions are bound through interaction with Asp-321, Glu-323, and Gln-335 and substrate. Residues Glu-279, Asp-280, and Arg-135 are suggested to function in the closure of an active site loop, over the nucleotide ribose-binding site.     

F1ATPase of Escherichia coli: a mutation (uncA401) located in the middle of the alpha subunit affects the conformation essential for F1 activity

F1ATPase from the Escherichia coli mutant of H+-ATPase, AN120 (uncA401), has less than 1% of the wild type activity and has been shown to be defective in the alpha subunit by in vitro reconstitution experiments. In the present study, the mutation site was located within a domain of the subunit by recombinant DNA technology. For this, a series of recombinant plasmids carrying various portions of the alpha subunit gene were constructed and used for genetic recombination with AN120. Analysis of the recombinants indicated that the mutation site could be located between amino acid residues 370 and 387. The biochemical properties of the mutant F1 were analyzed further using the fluorescent ATP analog DNS-ATP (2'-(5-dimethylaminonaphthalene-1-sulfonyl)-amino-2'-deoxy ATP). The single turnover process of E. coli F1ATPase proposed by Matsuoka et al. [(1982) J. Biochem. 92, 1383-1398.] was compared in the mutant and wild type F1's. Mutant F1 bound DNS-ATP and hydrolyzed it as efficiently as wild type F1. Results showed that binding of ATP to a low affinity site, possibly in the beta subunit, caused decrease of fluorescence of DNS-ATP in the wild type F1 and that this effect of ATP binding was inhibited by DCCD (dicyclohexyl carbodiimide). However, this effect was not inhibited by DCCD in the mutant F1, suggesting that in the proposed process some step(s) after ATP binding to the low affinity site differed in the mutant and wild F1's. When Pi was added to F1 bound to DNS-ATP or to aurovertin, a fluorescent probe capable of binding to the beta subunit, the opposite changes of fluorescence of these probes in the mutant and wild type F1's were observed, suggesting that the conformational change induced by phosphate binding was altered in the mutant F1. On the basis of the estimated mutation site and the biochemical properties of the mutant F1, the correlation of the domain of this site in the alpha subunit with the function of F1 ATPase is discussed.     

A homologous sequence between H+-ATPase (F0F1) and cation-transporting ATPases. Thr-285----Asp replacement in the beta subunit of Escherichia coli F1 changes its catalytic properties

A sequence of 10 amino acids (I-C-S-D-K-T-G-T-L-T) of ion motive ATPases such as Na+/K+-ATPase is similar to the sequence of the beta subunit of H+-ATPases, including that of Escherichia coli (I-T-S-T-K-T-G-S-I-T) (residues 282-291). The Asp (D) residue phosphorylated in ion motive ATPase corresponds to Thr (T) of the beta subunit. This substitution may be reasonable because there is no phosphoenzyme intermediate in the catalytic cycle of F1-ATPase. We replaced Thr-285 of the beta subunit by an Asp residue by in vitro mutagenesis and reconstituted the alpha beta gamma complex from the mutant (or wild-type) beta and wild-type alpha and gamma subunits. The uni- and multisite ATPase activities of the alpha beta gamma complex with mutant beta subunits were about 20 and 30% of those with the wild-type subunit. The rate of ATP binding (k1) of the mutant complex under uni-site conditions was about 10-fold less than that of the wild-type complex. These results suggest that Thr-285, or the region in its vicinity, is essential for normal catalysis of the H+-ATPase. The mutant complex could not form a phosphoenzyme under the conditions where the H+/K+-ATPase is phosphorylated, suggesting that another residue(s) may also be involved in formation of the intermediate in ion motive ATPase. The wild-type alpha beta gamma complex had slightly different kinetic properties from the wild-type F1, possibly because it did not contain the epsilon subunit.     

Lysine 480 is not an essential residue for ATP binding or hydrolysis by Na,K-ATPase.

Lysine 480 has been suggested to be essential for ATP binding and hydrolysis by Na,K-ATPase because it is labeled by reagents that are thought to react with the ATPase from within the ATP binding site. In order to test this hypothesis, Lys-480 was changed to Ala, Arg, or Glu by site-directed mutagenesis, and the resultant Na,K-ATPase molecules were expressed in yeast cells. The ATPase activity of each of the mutants was similar to the activity of the wild type enzyme indicating that Lys-480 is not essential for ATP hydrolysis. The binding of [3H]ouabain in both ATP-dependent and inorganic phosphate-dependent reactions was used to determine the apparent affinity of each mutant for ATP or Pi. The K0.5(ATP) for ouabain binding to phosphoenzyme formed from ATP was 1-3 microM for Lys-480, Arg-480, and Ala-480, whereas for Glu-480 the K0.5(ATP) was 18 microM. The K0.5(Pi) for ouabain binding to phosphoenzyme formed from inorganic phosphate was 16-28 microM for Lys-480, Arg-480, and Ala-480, but was 74 microM for Glu-480. The Kd for ouabain binding was similar for both the wild type and mutant Na,K-ATPase molecules (3-6 nM). These data indicate that the substitution of an acidic amino acid for lysine at position 480 appears to reduce the affinity of the Na,K-ATPase for both ATP and phosphate. It is concluded that Lys-480 is not essential for ATP binding or hydrolysis or for phosphate binding by Na,K-ATPase but is likely to be located within the ATP binding site of the Na,K-ATPase.     

The proton pore in the Escherichia coli F0F1-ATPase: a requirement for arginine at position 210 of the a-subunit

Site-directed mutagenesis was used to generate three mutations in the uncB gene encoding the a-subunit of the F0 portion of the F0F1-ATPase of Escherichia coli. These mutations directed the substitution of Arg-210 by Gln, or of His-245 by Leu, or of both Lys-167 and Lys-169 by Gln. The mutations were incorporated into plasmids carrying all the structural genes encoding the F0F1-ATPase complex and these plasmids were used to transform strain AN727 (uncB402). Strains carrying either the Arg-210 or His-245 substitutions were unable to grow on succinate as sole carbon source and had uncoupled growth yields. The substitution of Lys-167 and Lys-169 by Gln resulted in a strain with growth characteristics indistinguishable from a normal strain. The properties of the membranes from the Arg-210 or His-245 mutants were essentially identical, both being proton impermeable and both having ATPase activities resistant to the inhibitor DCCD. Furthermore, in both mutants, the F1-ATPase activities were inhibited by about 50% when bound to the membranes. The membrane activities of the mutant with the double lysine change were the same as for a normal strain. The results are discussed in relation to a previously proposed model for the F0 (Cox, G.B., Fimmel, A.L., Gibson, F. and Hatch, L. (1986) Biochim. Biophys. Acta 849, 62-69).     

Coupling factor F1 ATPase with defective beta subunit from a mutant of Escherichia coli

The defective coupling factor F1 ATPase from a mutant strain (KF11) of Escherichia coli was purified to a practically homogeneous form. The final specific activity of Mg2+-ATPase was 6-9 units/mg protein, which is about 10-15 times lower than that of F1 ATPase from the wild-type strain. The mutant F1 had a ratio of Ca2+-ATPase to Mg2+-ATPase of about 3.5, whereas the wild-type F1 had ratio of about 0.8. The mutant F1 was more unstable than wild-type F1: on storage at -80 degrees C for 2 weeks, about 80% of its activity (dependent on Ca2+ or Mg2+) was lost, whereas none of the activity of the wild-type F1 was lost. The following results indicate that the mutation is in the beta subunit. (i) High Mg2+-ATPase activity (about 20 units/mg protein) was reconstituted when the beta subunit from wild type F1 was added to dissociated mutant F1 and the mixture was dialyzed against buffer containing ATP and Mg2+. (ii) Low ATPase activity having the same ratio of Ca2+-ATPase to Mg2+-ATPase as the mutant F1 was reconstituted when a mixture of the beta subunit from the mutant F1 and the alpha and gamma subunits from wild-type F1 was dialyzed against the same buffer. (iii) Tryptic peptide analysis of the beta subunit of the mutant showed a difference in a single peptide compared with the wild-type strain.     

ATP binding to the ϵ subunit of thermophilic ATP synthase is crucial for efficient coupling of ATPase and H+ pump activities

ATP binding to the ? subunit of F1-ATPase, a soluble subcomplex of TFoF1 (FoF1-ATPase synthase from the thermophilic Bacillus strain PS3), affects the regulation of F1-ATPase activity by stabilizing the compact, ATPase-active, form of the ? subunit [Kato, S., Yoshida, M. and Kato-Yamada, Y. (2007) J. Biol. Chem. 282, 37618-37623]. In the present study, we report how ATP binding to the ? subunit affects ATPase and H+ pumping activities in the holoenzyme TFoF1. Wild-type TFoF1 showed significant H+ pumping activity when ATP was used as the substrate. However, GTP, which bound poorly to the ? subunit, did not support efficient H+ pumping. Addition of small amounts of ATP to the GTP substrate restored coupling between GTPase and H+ pumping activities. Similar uncoupling was observed when TFoF1 contained an ATP-binding-deficient ? subunit, even with ATP as a substrate. Further analysis suggested that the compact conformation of the ? subunit induced by ATP binding was required to couple ATPase and H+ pumping activities in TFoF1 unless the ? subunit was in its extended-state conformation. The present study reveals a novel role of the ? subunit as an ATP-sensitive regulator of the coupling of ATPase and H+ pumping activities of TFoF1.     

The mechanism of ATP synthase: a reassessment of the functions of the b and a subunits

A model for the mechanism of ATP synthase was proposed previously (Cox, G.B., Jans, D.A., Fimmel, A.L., Gibson, F. and Hatch, L. (1984) Biochim. Biophys. Acta 768, 201-208) in which the b subunit of the Fo of Escherichia coli rotated. The driving force was proposed to be an interaction between two charged residues in the membrane, namely, Lys-23 of the b subunit and Asp-61 of the c subunit. To test this proposal the Lys-23 of the b subunit was replaced by threonine using site-directed mutagenesis. The resulting mutant, although it had an impairment in the assembly of the F1F0-ATPase, was normal with respect to oxidative phosphorylation. The role of the a subunit, which had been previously proposed to be a structural one, was reassessed by examination of the possible secondary and tertiary structure of the analogous proteins from several sources. Not only did these subunits appear to have very similar structures, but in each there was a highly conserved helical arm on one of the transmembrane helices which could form a proton channel if it interacted with the Asp-61 of the c subunit. A revised model is therefore presented in which five transmembrane helices from the a subunit and two from the b subunit are surrounded by a ring of c subunits. The highly conserved nature of the structures of the a, b and c subunits from various organisms suggests that the model may have relevance for ATP synthases from bacterial plasma membranes, mitochondria and chloroplasts.     

H(+)-ATPase gamma subunit of Escherichia coli. Role of the conserved carboxyl-terminal region

Cloned uncG genes (wild-type or in vitro mutagenized) for the Escherichia coli gamma subunit were introduced into the uncG mutant Gln-14----end), and the functions of the mutant subunits were studied. The F1's with Ala-283----end and Thr-277----end mutant gamma subunits had 63 and 14% of the wild-type ATPase activity, respectively, and mutants with these subunits showed reduced growth by oxidative phosphorylation, indicating that the 10 residues at the carboxyl terminus (286th residue) are important, but dispensable, for catalysis. On the other hand, F1 with a Gln-269----end gamma subunit was inactive. Replacement of conserved residues (Gln-269, Thr-273, or Glu-275) between Gln-269 and Leu-276 gave enzymes with significantly reduced ATPase activity (2-41% of that of the wild-type) and lower ATP-driven proton conduction. Thus these residues are required for the normal catalytic activity of F1, although they are not absolutely essential. Membranes with amino acid replacements (Thr-277----end, Gln-269----Leu, or Glu-275----Lys) and the frameshift mutation (downstream of Thr-277) had about 15% of the wild-type ATPase activity, but showed different degrees of ATP-dependent H+ translocation and growth yield by oxidative phosphorylation, suggesting that the gamma subunit, especially its carboxyl-terminal region, functions in coupling between catalysis and H+ translocation.     

Genetic complementation between mutant b subunits in F1F0 ATP synthase

In Escherichia coli, a parallel homodimer of identical b subunits constitutes the peripheral stalk of F(1)F(0) ATP synthase. Although the two b subunits have long been viewed as a single functional unit, the asymmetric nature of the enzyme complex suggested that the functional roles of each b subunit should not necessarily be considered equivalent. Previous mutagenesis studies of the peripheral stalk suffered from the fact that mutations in the uncF(b) gene affected both of the b subunits. We developed a system to express and study F(1)F(0) ATP synthase complexes containing two different b subunits. Two mutations already known to inactivate the F(1)F(0) ATP synthase complex have been studied using this experimental system. An evolutionarily conserved arginine, b(Arg-36), was known to be crucial for F(1)F(0) ATP synthase function, and the last four C-terminal amino acids had been shown to be important for enzyme assembly. Experiments expressing one of the mutants with a wild type b subunit demonstrated the presence of heterodimers in F(1)F(0) ATP synthase complexes. Activity assays suggested that the heterodimeric F(1)F(0) complexes were functional. When the two defective b subunits were expressed together and in the absence of any wild type b subunit, an active F(1)F(0) ATP synthase complex was assembled. This mutual complementation between fully defective b subunits indicated that each of the two b subunits makes a unique contribution to the functions of the peripheral stalk, such that one mutant b subunit is making up for what the other is lacking.     

Conserved polar loop region of Escherichia coli subunit c of the F1F0 H+-ATPase. Glutamine 42 is not absolutely essential, but substitutions alter binding and coupling of F1 to F0

The uncE114 mutation (Gln42----Glu) in subunit c of the Escherichia coli H+ ATP synthetase causes uncoupling of proton translocation from ATP hydrolysis (Mosher, M. E., White, L. K., Hermolin, J., and Fillingame, R. H. (1985) J. Biol. Chem. 260, 4807-4814). In the background of strain ER, the mutation led to dissociation of F1 from the membrane. Ten revertants to the uncE114 mutation were isolated, and the uncE gene was cloned and sequenced. Six of the revertants were intragenic and had substitutions of glycine, alanine, or valine for the mutant glutamate residue at position 42. The intragenic, revertant uncE genes were incorporated into an otherwise wild type chromosome of strain ER. Membrane vesicles prepared from each of the revertants showed a restoration of F1 binding to F0. The Val42 revertant differed from the other two revertants in that the ATPase activity of F1 was inhibited when membrane bound. This was shown by the stimulation of ATPase activity when F1 was released from the membrane. The Gly42 and Ala42 revertants demonstrated membrane ATPase activity that was resistant to dicyclohexylcarbodiimide treatment. Resistance was shown to be due to the increased dissociation of F1 from the membrane under ATPase assay conditions. The Ala42 revertant showed a significant reduction in ATP-dependent quenching of quinacrine fluorescence that was attributed to less efficient coupling of ATP hydrolysis to H+ translocation, whereas the other revertants showed responses very near to that of wild type. Minor changes in the F1-F0 interaction in all three revertants were indicated by an increase in H+ leakiness, as judged by reduced NADH-dependent quenching of quinacrine fluorescence. The minor defects in the revertants support the idea that residue 42 is involved in the binding and coupling of F1 to F0 but also show that the conserved glutamine (or asparagine) is not absolutely necessary in this function.     

Arginine 328 of the beta-subunit of the mitochondrial ATPase in yeast is essential for protein stability

The mitochondrial ATPase is rapidly inactivated by the arginine selective reagent phenylglyoxal. Recently, the purported major reacting residue has been reported for the chloroplast enzyme (Viale, A. M., and Vallejos, R. H. (1985) J. Biol. Chem. 260, 4958-4962) corresponding to Arg-328 in the beta-subunit of the yeast Saccharomyces cerevisiae mitochondrial ATPase, a highly conserved residue in the ATPase. This arginine residue was concluded to be in the active site of the ATPase and possibly involved in the binding of nucleotides. To test this hypothesis, site-directed mutagenesis of the yeast enzyme has been used to replace Arg-328 with alanine and lysine. The modified genes were transformed into a yeast strain, DMY111, which contained a null mutation in the gene coding for the beta-subunit of the ATPase. Both of the substitutions were functional in vivo as demonstrated by the ability of yeast transformants to grow on a nonfermentable carbon source. The water soluble F1-ATPase with Ala-328 and Lys-328 were extremely unstable, but could be stabilized with glycerol. The rate of enzymatic decay followed first order kinetics with half-lives of 1.1 and 4.0 min for the mutants with Ala-328 and Lys-328 in 10% and 5% glycerol, respectively, while the wild type enzyme was stable even in the absence of glycerol. Kinetic analysis of both ATPase and GTPase has been determined. The wild type enzyme had two observable apparent Km and Vmax values for ATPase which were 0.056 mM-1 and 67 units/min/mg and 0.140 mM-1 and 100 units/min/mg. The mutant enzyme containing Lys-328 showed similar kinetic values of 0.066 mM-1 and 23 units/min/mg and 0.300 mM-1 and 43 units/min/mg. The mutant enzyme containing Ala-328, however, only demonstrated a single site with values of 0.121 mM-1 and 45 units/min/mg. In contrast to ATPase activity, kinetic values for GTPase were nearly identical for the wild type and mutant enzymes. Opposite to predicted results, the mutant enzymes were more sensitive to the reagent phenylglyoxal. These results indicate that Arg-328 is important for protein stability, but not involved in catalysis.