ATP-driven Proton Translocation Research Articles

Alanine-scanning Mutagenesis of the ∊ Subunit of the F1-F0 ATP Synthase from Escherichia coli Reveals Two Classes of Mutants

Alanine-scanning mutagenesis was applied to the epsilon subunit of the F1-F0 ATP synthase from E. coli. Nineteen amino acid residues were changed to alanine, either singly or in pairs, between residues 10 and 93. All mutants, when expressed in the epsilon deletion strain XH1, were able to grow on succinate minimal medium. Membranes were prepared from all mutants and assayed for ATP-driven proton translocation, ATP hydrolysis +/- lauryldiethylamine oxide, and sensitivity of ATPase activity to N,N'-dicyclohexylcarbodiimide (DCCD). Most of the mutants fell into 2 distinct classes. The first group had inhibited ATPase activity, with near normal levels of membrane-bound F1, but decreased sensitivity to DCCD. The second group had stimulated ATPase activity, with a reduced level of membrane-bound F1, but normal sensitivity to DCCD. Membranes from all mutants were further characterized by immunoblotting using 2 monoclonal antibodies. A model for the secondary structure of epsilon and its role in the function of the ATP synthase has been developed. Some residues are important for the binding of epsilon to F1 and therefore for inhibition. Other residues, from Glu-59 through Glu-70, are important for the release of inhibition by epsilon that is part of the normal enzyme cycle.

Characterization of mutations in the b subunit of F1F0 ATP synthase in Escherichia coli.

Site-directed mutagenesis was used to investigate the restrictions on Ala-79 of the b subunit in F1F0 adenosine triphosphate synthase. This amino acid had been previously identified as particularly sensitive to mutation (McCormick, K. A., and Cain, B. D. (1991) J. Bacteriol. 173, 7240-7248). Mutant uncF (b) genes were placed under control of the lac promoter and monitored for F1F0 ATP synthase function in an uncF(b) deletion strain. Three deleterious bAla-79 mutations were moved to the unc operon in the chromosome by homologous recombination. Decreases in enzymatic activity in the uncF (b) mutant strains resulted from reduced amounts of enzyme. With the exception of the bAla-79-->Pro mutation, high expression of mutant uncF (b) genes resulted in increases in F1F0 ATP synthase activity which were sufficient to overcome the defects. In addition to the decrease in the amount of enzyme, the bAla-79-->Lys mutation affected ATP synthesis to a much greater extent than ATP-driven proton translocation. The evidence supports our earlier hypothesis, in which bAla-79 was proposed to play an important, but not essential, structural role in F1F0 ATP synthase assembly or stability.

Pyrophosphate-induced acidification of trans cisternal elements of rat liver Golgi apparatus

Trans cisternal elements of the Golgi apparatus from rat liver, identified by thiamin pyrophosphatase cytochemistry, were isolated by preparative free-flow electrophoresis and were found to undergo acidification as measured by a spectral shift in the absorbance of acridine orange. Acidification was supported not only by adenosine triphosphate (ATP) but nearly to the same degree by inorganic pyrophosphate (PPi). The proton gradients generated by either ATP or PPi were collapsed by addition of a neutral H+/K+ exchanger, nigericin, or the protonophore, carbonyl cyanide m-chlorophenylhydrazone, bothat 1.5 μM. Both ATP hydrolysis and ATP-driven proton translocation as well as pyrophosphate hydrolysis and pyrophosphate-driven acidification were stimulated by chloride ions. However, ATP-dependent activities were optimum at pH 6.6, whereas pyrophosphate-dependent activities were optimum at pH 7.6. The Mg2+ optima also were different, being 0.5 mM with ATP and 5 mM with pyrophosphate. With both ATPase and especially pyrophosphatase activity, both by cytochemistry and analysis of free-flow electrophoresis fractions, hydrolysis was more evenly distributed across the Golgi apparatus stack than was either ATP-or PPi-induced inward transport of protons. Proton transport colocalized more closely with thiamin pyrophosphatase activity than did either pyrophosphatase or ATPase activity. ATP- and pyrophosphatate-dependent acidification were maximal in different electrophoretic fractions consistent with the operation of two distinct proton translocation activities, one driven by ATP and one driven by pyrophosphate.

Mutations in three of the putative transmembrane helices of subunit a of the Escherichia coli F 1F 0-ATPase disrupt ATP-driven proton translocation

Three missense mutants in subunit a of the Escherichia coli F 1F 0-ATPase were isolated and characterized after hydroxylamine mutagenesis of a plasmid carrying the uncB (subunit a) gene. The mutations resulted in Asp 119 → His, Ser 152 → Phe, or Gly 197 → Arg substitutions in subunit a Function was not completely abolished by any of the mutations. The F 0 membrane sector was assembled in all three cases as judged by restoration of dicyclohexylcarbodiimide sensitivity to the F 1F 0-ATPase. The H + translocation capacity of F 0 was reduced in all three mutants. ATP-driven H +-translocation was also reduced, with the response in the Gly 197 → Arg mutant being almost nil and that in the Asp 119 → His and Ser 152 → Phe mutants less severely affected. The substituted residues are predicted to lie in the second, third, and fourth transmembrane helices suggested in most models for subunit a. The Gly 197 → Arg mutation lies in a very conserved region of the protein and the substitution may disrupt a structure that is critical to function. The Asp 119 → His and Ser 152 → Phe mutations also lie in areas with sequence conservation. A further analysis of randomly generated mutants may provide more information on regions of the protein that are crucial to function. Heterodiploid transformants, carrying plasmids with either the wild-type uncB gene or mutant uncB genes in an uncB (Trp 231 → stop) background, were characterized biochemically. The truncated subunit a was not detected in membranes of the background strain by Western blotting, and the uncB + plasmid complemented strain showed normal biochemistry. The uncB mutant genes were shown to cause equivalent defects in either the heterodiploid background configuration, or after incorporation into an otherwise wild-type unc operon. The subunit a (Trp 231 → stop) background strain was shown to bind F 1-ATPase nearly normally despite lacking subunit a in its membrane.

Mutagenesis of the alpha subunit of the F1Fo-ATPase from Escherichia coli. Mutations at Glu-196, Pro-190, and Ser-199.

In an attempt to identify amino acid residues involved in proton translocation by the Fo sector of the Escherichia coli F1Fo-ATPase, 16 mutations at the carboxyl-terminal third of the a subunit have been isolated, and their phenotypes have been partially characterized. Thirteen mutations were constructed by "cassette" mutagenesis at two highly conserved residues, aglu196 and apro190. Two mutations were products of oligonucleotide-directed mutagenesis of a portion of of oligonucleotide-directed mutagenesis of a portion of the uncB gene cloned into an M13 vector. One mutation was isolated after in vitro mutagenesis of the entire uncB gene in a plasmid vector with hydroxylamine. Amino acid substitutions for aglu196 (Asp, Gln, His, Asn, Lys, Ala, Ser, Pro) affect ATP-driven proton translocation and passive proton permeability by Fo to varying extents, but do not prevent growth on minimal succinate media. Amino acid substitutions of glutamine or arginine for apro190 affect F1Fo-ATPase assembly and eliminate ATP-driven proton translocation, while the substitution of asparagine at this position does not significantly affect either assembly or proton translocation. The substitution of amino acids threonine or alanine for aser199 causes no detectable phenotypic change from wild type. These and other mutations are discussed in terms of the assembly, structure, and function of the a subunit. It is concluded that aglu196 and apro190 are not obligate components of the proton channel, but that they affect proton translocation indirectly.

Reconstitution of the lysosomal proton pump.

Lysosomal membrane proteins solubilized with octyl beta-D-glucopyranoside were reconstituted into proteoliposomes using acetone/ether-washed phospholipids from Escherichia coli. Assays of the quenching of acridine orange fluorescence showed that addition of both ATP and valinomycin to K+-loaded proteoliposomes led to the formation of a pH gradient that was acidic inside. ATP-driven acidification took place in the absence of permeant anions and was inhibited by the "protonophore", carbonylcyanide p-trifluoromethoxyphenylhydrazone, indicating that only H+ was transported actively. Proton translocation was readily blocked by N-ethylmaleimide (10 microM gave 50% inhibition of fluorescence quenching) but was unaffected by oligomycin (50 nM), orthovanadate (50 microM), or ouabain (0.5 mM); similarly, only N-ethylmaleimide affected ATP hydrolysis by proteoliposomes (88% inhibition). Other work showed that reconstitution of ATP-driven proton translocation required the presence of glycerol during protein solubilization and that optimal recovery depended on the use of both glycerol and phospholipid at this stage. We conclude that acidification of the lysosome is mediated by an ATPase capable of electrogenic H+ translocation without molecular coupling to other ionic species.

Mycochromone decreases ATP-driven proton translocation in plant plasma membrane- and tonoplast-enriched vesicles

Mycochromone decreases ATP-driven proton translocation in plant plasma membrane- and tonoplast-enriched vesicles

Topology and function of "stalk" proteins in the bovine mitochondrial H+-ATPase.

Proton translocating ATPases comprise a hydrophilic sector F1, a membrane sector F0, and, in the case of bovine mitochondria, a connecting "stalk" which is believed to contain the oligomycin sensitivity-conferring protein (OSCP) and coupling factor 6 (F6). The present study was undertaken to verify the accessibility of F6 and OSCP to trypsin and to examine the functional consequences of such treatment. Our data show that F1 binds equally to trypsin-treated F0 and untreated F0, but the former complexes exhibit cold lability and only partial sensitivity to oligomycin. Furthermore, these complexes fail to exhibit ATP-driven proton translocation or ATP-32Pi exchange activity. Trypsinization of F0 does not, however, inhibit passive proton conductance through the membrane sector but actually enhances it. Immunological data indicate extensive degradation of OSCP under conditions where F6 proteolysis is insignificant. Intact H+-ATPase complexes are relatively resistant to both the structural and functional effects of trypsin. We conclude that OSCP is predominantly an extrinsic protein which is shielded by F1 in the native membrane. F6 may also be an extrinsic protein but is shielded from trypsinization by OSCP and/or other F0 polypeptides. The exposed, trypsin-sensitive segments of OSCP are not required for passive proton conductance through F0 but may be required for ATP-driven reactions. We propose that bovine mitochondrial OSCP is a functional analogue of subunit b in the Escherichia coli H+-ATPase.

A chemically explicit model for the molecular mechanism of the F1F0 H+-ATPase/ATP synthases.

A general hypothesis for the molecular mechanism of membrane transport based on current knowledge of protein structure and the nature of ligand-induced protein conformational changes has recently been proposed [Scarborough, G. A. (1985) Microbiol. Rev. 49, 214-231]. According to this hypothesis, the essential reaction undergone by all proteinaceous transport catalysts is a ligand-induced hinge-bending-type conformational change that results in the transposition of binding-site residues from access on one side of the membrane to access on the other side. Subsequent release and/or alteration of the ligand or ligands that induce the conformational change facilitates the converse conformational change, which returns the binding-site residues to their original position. With this simple cyclic ligand-dependent gating process as a central feature, biochemically orthodox mechanisms for virtually all known transporters are readily conceived. In this article, a chemically explicit model for the molecular mechanism of the F1F0 H+-ATPase/ATP synthases of mitochondria, bacteria, and chloroplasts, formulated within the guidelines of this general transport paradigm, is presented. At least three points of potential interest arise from this exercise. First, with the aid of the model, it is possible to visualize how energy transduction catalyzed by these enzymes might proceed, with no major events left unspecified. Second, explicit possibilities as to the molecular nature of electric field effects on the transport process are raised. And finally, it is shown that enzyme conformational changes, energy-dependent binding-affinity changes, and several other related phenomena as well, need not be taken as evidence of "action at a distance" or indirect energy coupling mechanisms, as is sometimes assumed, because such events are also integral features of the mechanism presented, even though all of the key reactions proposed for both ATP-driven proton translocation and proton translocation-driven ATP synthesis occur at the enzyme active site.

Electrogeneicity of the lysosomal proton pump

Using acridine orange as a pH gradient probe, the effects of valinomycin and FCCP on the pH gradient across lysosomal membranes in an ATP-free medium as well as on the rate of the inward ATP-driven proton translocation were investigated. Both lysosome-enriched and highly purified lysosomal preparations from rat liver were used with identical results. Ionophore effects were found to be different depending upon whether passive ion fluxes or ATP-driven H + translocation were involved and supported the existence of a membrane potential in the latter case. Anions stimulated the rate of ATP-driven proton translocation and stimulation increased with increasing anion lipophilicity. These results strongly support the electrogenic nature of the lysosomal proton pump.

Determinants of clathrin-coated vesicle acidification.

The proton-translocating ATPase of clathrin-coated vesicles of bovine brain is characterized by ATP specificity. Chloride or bromide serve as co-ions balancing electrogenic proton pumping. ATP-driven proton translocation can be observed in the absence of chloride, provided the membrane potential is collapsed by K+ moving out in the presence of valinomycin. Chloride transport can be observed independent of proton movements in the absence of ATP, provided an inside positive membrane potential is generated by K+ moving from the outside to the inside in the presence of valinomycin.

An ATP-driven proton pump in clathrin-coated vesicles.

Clathrin containing coated vesicles prepared from bovine brain catalyzed ATP-driven proton translocation and a 32Pi-ATP exchange reaction. Both activities were measured in the presence of 5 micrograms of oligomycin/mg of protein which completely inhibited these reactions catalyzed by submitochondrial particles. Analyses performed during the purification procedure demonstrated that the oligomycin-resistant pump was concentrated and highly purified in the fractions containing coated vesicles. Moreover, vesicles precipitated by either monoclonal or polyclonal antibodies against clathrin contained the H+ pump activity. Dicyclohexylcarbodiimide (0.5 mM) and N-ethylmaleimide (1 mM) added to the assay mixture inhibited the pump completely, whereas neither vanadate, sodium azide, efrapeptin, or mitochondrial ATPase inhibitor had an effect.

Lack of involvement of lipoic acid in membrane-associated energy transduction in Escherichia coli

Oxidative phosphorylation, active transport of proline, aerobic- and ATP-driven proton translocation and transhydrogenation of NADP + by NADH, occurred in lipoic acid-deficient cells or vesicles of a lipoic acid auxotroph of E. coli , W1485 lip 2. Addition of lipoic acid had little effect on these processes. Tributyltin chloride, which has been proposed to inhibit oxidative phosphorylation by reaction with lipoic acid (Cain et al. , Biochem. J. (1977) 166 , 593), was an effective inhibitor of aerobic and ATP-dependent proton translocation and transhydrogenation in lipoic acid-deficient vesicles from this organism. Our results do not support the proposal of Partis et al. (FEBS Lett. (1977) 75 , 47) that lipoic acid is involved in the energy transducing processes associated with the membrane of E. coli .

Stoichiometry of Adenosine Triphosphate-driven Proton Translocation in Bovine Heart Submitochondrial Particles

Abstract Inward-directed proton translocation driven by ATP hydrolysis was investigated in bovine heart submitochondrial particles under conditions where no net pH change occurred upon ATP hydrolysis. A biphasic time course of ATP hydrolysis, due to the presence of adenylate kinase activity, was observed by following the release of 32Pi from [β, γ-32P]ATP under comparable conditions. An equation was derived for calculation of the H+:ATP ratio from the time course of ATP hydrolysis and the transient pH change of the medium due to proton translocation. H+:ATP ratios as high as 1.7 were observed, although a seasonal variation was noted. However, oligomycin raised the H+:O ratios for respiration-driven proton translocation, suggesting that the experimentally observed H+:ATP ratios were suboptimal. The results indicate that the true stoichiometry of ATP-driven proton translocation, characteristic of the ATPase mechanism is 2H+:ATP. The same stoichiometry has been reported previously for outward-directed proton translocation in intact mitochondria.

Partial Resolution of the Enzymes Catalyzing Oxidative Phosphorylation: XXVI. SPECIFICITY OF PHOSPHOLIPIDS REQUIRED FOR ENERGY TRANSFER REACTIONS

Abstract 1. Vesicles catalyzing 32Pi-ATP exchange and ATP-driven proton translocation were reconstituted with chemically defined phospholipids and mitochondrial membrane proteins which were virtually free of electron transport carriers. 2. Phosphatidylcholine and phosphatidylethanolamine were both required for the reconstitution of vesicles with high exchange activity. The optimal ratio of the two phospholipids was variable depending on the source and composition of the phospholipids. With highly purified preparations a 3- to 4-fold excess of phosphatidylethanolamine over phosphatidylcholine yielded optimal rates. An equal molar ratio gave low exchange activity, which was markedly accelerated by low amounts of cardiolipin or another acidic phospholipid. Coenzyme Q10 was not required. 3. Synthetic preparations substituted for natural phospholipids. The presence of unsaturated fatty acyl groups in the phospholipid appeared to be essential for the reconstitution of active vesicles. Phospholipids with fully saturated acyl groups were actually inhibitory. On the other hand, phospholipids with unnatural side chains were active provided they contained unsaturated groups. 4. The morphology of the reconstituted vesicles as seen in electron micrographs varied with the different phospholipids used in reconstitution. 5. Optimal conditions of reconstitution of the components which were solubilized with cholate were studied with [carboxy-14C]cholate. Rapid removal of cholate by passage of the components through a Sephadex column resulted in inactive particles. Removal of cholate by dialysis over several hours was optimal. The reconstituted vesicles had a higher phospholipid content and lower proton permeability than sub-mitochondrial particles.