Asymmetry of the alpha subunit of the chloroplast ATP synthase as probed by the binding of Lucifer Yellow vinyl sulfone

The catalytic portion of the chloroplast ATP synthase (CF1) is structurally asymmetric. Asymmetry of the otherwise symmetrical alpha3beta3 heterohexamer is induced by the presence of tightly bound nucleotides and interactions with the single-copy, smaller subunits. Lucifer Yellow vinyl sulfone (4-amino-N-[3-(vinylsulfonyl)phenyl]naphthalimide-3,6-disulfonic acid) rapidly and covalently binds to lysine 378 on one alpha subunit [Nalin, C. M., Snyder, B., and McCarty, R. E., (1985) Biochemistry 24, 2318-2324] [Shapiro, A. B. (1991) Ph.D. Thesis, Cornell University, Ithaca, NY). The asymmetrical binding of Lucifer Yellow to CF1 provides a method to investigate the cause of asymmetry in the alpha subunits. The reaction of CF1 with Lucifer Yellow was monitored by total fluorescence of bound Lucifer Yellow as well as by quantitative determination of Lucifer Yellow bound to the tryptic peptide that contains lysine 378 of the alpha subunit. The total binding of Lucifer Yellow to CF1 was not affected by the presence of tightly bound nucleotides or nucleotide in the medium. Neither the total binding of Lucifer Yellow to CF1 nor the reaction of alpha-lysine 378 with Lucifer Yellow was changed by the removal of the epsilon subunit, the delta subunit, or both subunits. The extent of incorporation of Lucifer Yellow into lysine 378 of the alpha subunit in (alphabeta)n was about three times that of Lucifer Yellow incorporation into CF1. Reconstitution of (alphabeta)n with gamma restored the binding of one Lucifer Yellow per alpha3beta3gamma. Therefore, the interactions between gamma and the alphabeta heterohexamer are important in conferring asymmetry to the alpha subunits of CF1.     

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.     

The formation or the reduction of a disulfide bridge on the gamma subunit of chloroplast ATP synthase affects the inhibitory effect of the epsilon subunit

We have studied the change of the catalytic activity of chimeric complexes that were formed by chloroplast coupling factor 1 (CF1) -gamma, alpha and beta subunits of thermophilic bacterial F1 after formation or reduction of the disulfide bridge of different gamma subunits modified by oligonucleotide-directed mutagenesis techniques. For this purpose, three mutant gamma subunits were produced: gamma Delta194-230, here 37 amino acids from Pro-194 to Ile-230 are deleted, gammaC199A, Cys-199 is changed to Ala, and gamma Delta200-204, amino acids from Asp-200 to Lys-204 are deleted. All of the chimeric subunit complexes produced from each of these mutant CF1-gamma subunits and alpha and beta subunits from thermophilic bacterial F1 lost the sensitivity against thiol reagents when compared with the complex containing wild-type CF1-gamma. The pH optimum (pH 8.5-9.0) and the concentration of methanol to stimulate ATPase activities were not affected by these mutations. These indicate that the introduction of the mutations did not change the main features of ATPase activity of the chimeric complex. However, the interaction between gamma subunit and epsilon subunit was strongly influenced by the type of gamma subunit itself. Although the ATPase activity of the chimeric complex that contained gamma Delta200-204 or gammaC199A was inhibited by the addition of recombinant epsilon subunit from CF1 similarly to complexes containing the reduced wild-type gamma subunit, the recombinant epsilon subunit did not inhibit the ATPase of the complex, which contained the oxidized form of gamma subunit. Therefore the affinity of the epsilon subunit to the gamma subunit may be dependent on the state of the gamma subunit or the epsilon subunit may bind to the oxidized form of gamma subunit in a mode that does not inhibit the activity. The ATPase activity of the complex that contains gamma Delta194-230 was not efficiently inhibited by epsilon subunit. These results show that the formation or reduction of the disulfide bond on the gamma subunit may induce a conformational change in the region that directly affects the interaction of this subunit with the adjacent epsilon subunit.     

Cloning and functional expression analysis of the alpha subunit of mouse ATP synthase

The alpha subunit of the mitochondrial ATP synthase is part of the F1 enzymatic complex known to bind ADP, phosphate and ATP and is at the heart of the mitochondrial energy-producing mechanism. The mouse embryonal carcinoma variant of the alpha subunit cDNA was cloned and the complete nucleotide sequences of two different lengths of clones were determined. Two distinct polyadenylation sites in the cDNA sequence were detected and two sizes of mRNAs were confirmed by Northern blot hybridization. Two putative ATP-binding motifs - A and B, have been hypothesized for this enzyme based on previous NMR work on another ATP-binding enzyme, adenylate kinase. We have constructed four deletion mutants of the alpha subunit of the mouse F1-ATP synthase to examine the putative role of these domains. The resulting recombinant proteins were expressed and purified. Functional studies with the immobilized mutants proved the significance of both sites for ATP binding.     

A novel Euglena gracilis chloroplast operon encoding four ATP synthase subunits and two ribosomal proteins contains 17 introns

Electromyographic activity of anterior temporal and masseter muscles was measured in 92 young healthy men and women with sound dentitions during rest position, contact in centric occlusion and clench. Male and female mean potentials were similar except in clench, where males had higher electromyographic levels. Mean pooled electromyographic potentials were 1.9 microV (TA) and 1.4 microV (MM) during rest position, 6.5 microV (TA) and 2.8 microV (MM) during contact in centric occlusion. Mean maximum voluntary clench potentials were 181.9 microV (TA) and 216.2 microV (MM) in men, 161.7 microV (TA) and 156.8 microV (MM) in women. Examined muscles were more asymmetric at low electromyographic activity (rest and centric occlusion) with the temporal muscle less asymmetrical than the masseter. In females temporal muscle activity tended to dominate at every contraction level, while in males masseter activity was stronger in clench, and temporal activity in centric occlusion and in rest position.     

Do the bacterial flagellar motor and ATP synthase operate as water turbines?

PURPOSE: Up to 30% of lung cancers (Stage I) with the most favorable outcome recur within 5 years after surgery. This study reviews the pattern of failure after surgical resection in early lung cancers and determines whether flow cytometric DNA variables were prognostic indicators for survival, disease-free survival (DFS), or distant metastasis-free survival (DMFS). METHODS AND MATERIALS: Pathologic specimens from 45 patients at The University of Texas M. D. Anderson Cancer Center who underwent surgical resection and mediastinal nodal dissection for stage I (AJCC) adenocarcinomas of the lung were analyzed by flow cytometry for DNA content. Survival was calculated by the method of Desu and Lee. Chi-square and cross tabulation were used in the analysis. RESULTS: The mean age of the patients was 62 years, and 52.3% were male. All patients were clinical Stage I (T1-2 N0), Karnofsky performance status > or = 70, and had a weight loss <10 lbs. Median overall survival (OS) and DFS were 50 months and 33 months, respectively. OS, DFS, and DMFS at 1, 3 and 5 years were 73%, 57%, and 35%; 63%, 53%, and 45%; and 67%, 56%, and 48%, respectively. Analysis of all 45 patients revealed 86% of patients developing brain metastasis had an abnormal DNA content > or = 30%, whereas 4% of patients with brain metastasis had abnormal DNA content < 30% (p = 0.01). This correlation maintained significance when only pT1/2 lesions were analyzed. There was a significant statistical correlation between abnormal DNA and 5-year OS, with 74% OS for those with abnormal DNA < 30% vs. 42% for > or = 30% (p = 0.036). The 5-year DFS for pT1/2 patients was significantly correlated with abnormal DNA content: 53% for patients with abnormal DNA < 30% vs. 17% for patients with abnormal DNA > or = 30%, respectively (p = 0.03). Of those with %S fraction (%S) < 2, 13% failed locally compared to 41% of those with %S > or = 2. There was a highly significant correlation between DNA index (DNAI) and aneuploid %S: 68% of patients with a DNAI > or = 1.7 had > or = 2.6 aneuploid %S, whereas only 13% of patients with DNAI > or = 1.7 had aneuploid %S < 2.6. (p < 0.001). Grouping the percent of abnormal DNA and overall %S according to low vs. mixed vs. high values correlated with DFS (p = 0.02). CONCLUSIONS: This study confirms significant correlation between a high DNA index and a higher frequency of brain metastasis, as well as worse OS. Although DNA content variables were not predictive of recurrence at other sites, brain metastasis represents the worst outcome from distant metastasis. Further studies are needed, as well as prospective trials, for evaluating adjuvant therapy in patients with adverse DNA variables following complete surgical resection for early disease. If high-risk patients could be identified after resection, adjuvant therapy (chemotherapy or elective brain irradiation) could be administered.     

A human homolog of bacterial acetolactate synthase genes maps within the CADASIL critical region

CADASIL, a recently identified autosomal dominant condition characterized by the recurrence of subcortical infarcts leading to dementia, was previously mapped to chromosome 19p13.1 within a 2-cM interval, D19S226-D19S199. No recombination event was observed with D19S841, a highly polymorphic microsatellite marker isolated from a cosmid mapped to this region. We recently identified within this cosmid a conserved sequence that we used to screen a fetal brain cDNA library and isolated an ubiquitous and abundantly transcribed gene. We did not detect any mutation of this gene in CADASIL patients, suggesting that it is not implicated in this disorder. Interestingly, this gene encodes a putative protein homologous to several thiamine pyrophosphate-binding proteins previously identified in bacteria, yeast, and plants. The proteins with the highest degree of similarity were the acetolactate synthase enzymes which, in prokaryotes, are involved in the branched chain amino acid biosynthetic pathway, raising fascinating questions on the yet unknown function of this gene in mammals.     

Isolation of supernumerary yeast ATP synthase subunits e and i. Characterization of subunit i and disruption of its structural gene ATP18

Two subunits of the yeast ATP synthase have been isolated. Subunit e was found loosely associated to the complex. Triton X-100 at a 1% concentration removed this subunit from the ATP synthase. The N-terminal sequencing of subunit i has been performed. The data are in agreement with the sequence of the predicted product of a DNA fragment of Saccharomyces cerevisiae chromosome XIII. The ATP18 gene encodes subunit i, which is 59 amino acids long and corresponds to a calculated mass of 6687 Da. Its pI is 9.73. It is an amphiphilic protein having a hydrophobic N-terminal part and a hydrophilic C-terminal part. It is not apparently related to any subunit described in other ATP synthases. The null mutant showed low growth on nonfermentable medium. Mutant mitochondria display a low ADP/O ratio and a decrease with time in proton pumping after ATP addition. Subunit i is associated with the complex; it is not a structural component of the enzyme but rather is involved in the oxidative phosphorylations. Similar amounts of ATP synthase were measured for wild-type and null mutant mitochondria. Because 2-fold less specific ATPase activity was measured for the null mutant than for the wild-type mitochondria, we make the hypothesis that the observed decrease in the turnover of the mutant enzyme could be linked to a proton translocation defect through F0.     

Regulation of cytochrome c oxidase by interaction of ATP at two binding sites, one on subunit VIa

Cytochrome c oxidase isolated from a wild-type yeast strain and a mutant in which the gene for subunit VIa had been disrupted were used to study the interaction of adenine nucleotides with the enzyme complex. At low ionic strength (25 mM potassium phosphate), in the absence of nucleotides, the cytochrome c oxidase activity of the mutant enzyme lacking subunit VIa was higher than that of the wild-type enzyme. Increasing concentrations of ATP, in the physiological range, enhanced the cytochrome c oxidase activity of the mutant much more than the activity of the wild-type strain, whereas ADP, in the same concentration range, had no significant effect on the activity of the cytochrome c oxidase of either strain. These results indicate an interaction of ATP with subunit VIa in the wild-type enzyme that prevents the stimulation of the activity observed in the mutant enzyme. The stimulation of the mutant enzyme implies the presence of a second ATP binding site on the enzyme. Quantitative titrations with the fluorescent adenine nucleotide analogues 2'(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) and 2'(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) confirmed the presence of two binding sites for adenine nucleotides per monomer of wild-type cytochrome c oxidase and one binding site per monomer of mutant enzyme. Covalent photolabeling of yeast cytochrome c oxidase with radioactive 2-azido-ATP further confirmed the presence of an ATP binding site on subunit VIa.(ABSTRACT TRUNCATED AT 250 WORDS)     

The alpha/beta subunit interaction in H(+)-ATPase (ATP synthase). An Escherichia coli alpha subunit mutation (Arg-alpha 296-->Cys) restores coupling efficiency to the deleterious beta subunit mutant (Ser-beta 174-->Phe)

The Ser-beta 174 residue of the Escherichia coli H(+)-ATPase beta subunit has been shown to be near the catalytic site together with Gly-beta 149, Gly-beta 172, Glu-beta 192, and Val-beta 198 (Iwamoto, A., Park, M.-Y., Maeda, M., and Futai, M. (1993) J. Biol. Chem. 268, 3156-3160). In this study, we introduced various residues at position 174 and found that the larger the side chain volume of the residue introduced, the lower the enzyme activity became. The Phe-beta 174 mutant was defective in energy coupling between catalysis and transport, whereas the Leu-beta 174 mutant could couple efficiently, although both mutants had essentially the same ATPase activities (approximately 10% of the wild type). The defective energy coupling of the Phe-beta 174 mutant was suppressed by the second mutation (Arg-alpha 296-->Cys) in the alpha subunit. The Cys-alpha 296/Phe-beta 174 mutant had essentially the same membrane ATPase activity as the Phe-beta 174 single mutant when assayed under the conditions that stabilize the double mutant enzyme. These results indicate the importance of the alpha/beta interaction, especially that between the regions near Arg-alpha 296 and Ser-beta 174, for energy coupling in the H(+)-ATPase. The 2 residues (Ser-beta 174 and Arg-alpha 296) may be located nearby at the interface of the two subunits. About 1 mol of N-[14C]ethylmaleimide could bind to 1 mol of the alpha subunit of Cys-alpha 296/Phe-beta 174 or Cys-alpha 296 mutant ATPase, but could not inhibit the enzyme activity. This is the first intersubunit mutation/suppression approach to ATPase catalysis and its energy coupling.     

Characterization of the import pathway of the F(A)d subunit of mitochondrial ATP synthase into isolated plant mitochondria

The synthetic precursor of the F(A)d subunit of mitochondrial ATP synthase was imported into isolated soybean cotyledon mitochondria. Import of the F(A)d precursor was accompanied by processing to a lower molecular weight mature form. The F(A)d precursor displayed the following import characteristics not seen before with plant mitochondria: efficient import in the absence of external ATP and import of wheat germ-translated precursor. Pretreatment of the F(A)d precursor with NEM did not inhibit import. Taken together with the lack of a requirement for external ATP, this indicates that this precursor does not require extramitochondrial ATP-dependent factors for import. Binding studies indicated that the F(A)d precursor bound to a proteinaceous component of the mitochondrial outer membrane. Inhibitor studies indicated that processing was most likely via the general mitochondrial processing peptidase. The results suggest that import of this subunit occurs via a pathway different from the general import pathway described for the majority of precursor proteins.     

Rate of accumulation of Luxol Fast Blue staining material and mitochondrial ATP synthase subunit 9 in motor neuron degeneration mice

The rate of accumulation of Luxol Fast Blue staining material in the hippocampus of motor neuron degeneration (mnd/mnd) mice, a model of Batten Disease, was quantitated. Stained material increased linearly up to 8 months of age. A quantitative immunoassay was used to measure levels of mitochondrial ATP synthase subunit 9 in brain and liver of mnd/mnd mice. Levels of subunit 9 increased progressively throughout the lifespan of mnd/mnd mice reaching levels approximately 5-fold higher than in control animals. The rate of accumulation of subunit 9 is not consistent with any simple complete or partial degradation defect that is constant throughout the animal's life. Two more complicated models are discussed which are consistent with the observed accumulation rate of subunit 9.     

ATP synthesis by the F0F1 ATP synthase from thermophilic Bacillus PS3 reconstituted into liposomes with bacteriorhodopsin. 2. Relationships between proton motive force and ATP synthesis

The correlation between the rate of ATP synthesis and light-induced proton flux was investigated in proteoliposomes reconstituted with bacteriorhodopsin and ATP synthase from thermophilic Bacillus PS3. By variation of the actinic light intensity it was found that ATP synthase activity depended in a sigmoidal manner on the amplitude of the transmembrane light-induced pH gradient. Maximal rates of ATP synthesis (up to to 200 nmol ATP x min(-1) x mg protein (-1) were obtained at saturating light intensities under a steady-state pH gradient of about pH 1.25. It was demonstrated that this was the maximal deltapH attainable at 40 degrees C in reconstituted proteoliposomes, due to the feedback inhibition of bacteriorhodopsin by the proton gradient it generates. In the absence of valinomycin, a small but significant transmembrane electrical potential could develop at 40 degrees C, contributing to an increase in the rate of ATP synthesis. The H+/ATP stoichiometry was measured at the static-head (equilibrium) conditions from the ratio of the phosphate potential to the size of the light-induced pH gradient and a value of about four was obtained under the maximal electrochemical proton gradient. Increasing the amount of bacteriorhodopsin in the proteoliposomes at a constant F0F1 concentration led to a large increase in the rate of ATP synthesis whereas the magnitude of delta pH remained the same or, at very high bacteriorhodopsin levels, decreased. Consequently the H+/ATP stoichiometry was found to increase significantly with increasing bacteriorhodopsin content. Reconstitutions with mixtures of native and impaired bacteriorhodopsin (Asp96-->Asn mutated bacteriorhodopsin) further demonstrated that this increase in the coupling efficiency could not be related to protein-protein interactions but rather to bacteriorhodopsin donating H+ to the ATP synthase. Increasing the amount of negatively charged phospholipids in the proteoliposomes also increased the coupling efficiency between bacteriorhodopsin and ATP synthase at a constant transmembrane pH gradient. Similar results were obtained with chloroplast ATP synthase. Furthermore, ATP synthase activities induced by delta pH/delta psi transitions were independent of bacteriorhodopsin or anionic lipid levels. These observations were interpreted as indicating that, in bacteriorhodopsin/ATP synthase, proteoliposomes, a localized pathway for coupling light-driven H+ transport by bacteriorhodopsin to ATP synthesis by F0F1 might exist under specific experimental conditions.     

Isolation and characterization of a cDNA encoding Arabidopsis thaliana mevalonate kinase by genetic complementation in yeast

A 1.64-kb cDNA encoding an Arabidopsis thaliana mevalonate kinase (MK) was cloned by complementation of the erg 12-1 mutation affecting MK in the yeast Saccharomyces cerevisiae, and the nucleotide sequence was determined. The longest open reading frame encodes a protein of 378 amino acids (aa) with a predicted molecular mass of 40,650 Da. A striking feature of the cDNA sequence is a long 5' untranslated region (322 bp). The deduced aa sequence reveals that the plant enzyme shows strong similarities to the yeast and mammalian enzymes, especially the strong hydrophobicity percentage and several conserved regions. Southern analysis suggests that probably only one locus exists in the A. thaliana genome.     

Genetic fusions of subunit c in the F0 sector of H+-transporting ATP synthase. Functional dimers and trimers and determination of stoichiometry by cross-linking analysis

The multicopy c subunit of the H+-transporting ATP synthase of Escherichia coli folds through the transmembrane F0 sector as a hairpin of two hydrophobic alpha-helices with the proton-translocating aspartyl-61 side chain centered in the second transmembrane helix. The number of subunits c in the F0 complex, which is thought to determine the H+-pumping/ATP stoichiometry, was previously not determined with exactness but thought to range from 9-12. The studies described here indicate that the exact number is 12. Based upon the precedent of the subunit c in vacuolar-type ATPases, which are composed of four transmembrane helices and seem to have evolved by gene duplication of an F0-type progenitor gene, we constructed genetically fused dimers and trimers of E. coli subunit c. Both the dimeric and trimeric forms proved to be functional. These results indicate that the total number of subunit c in F0 should be a multiple of 2 and 3. Based upon a previous study in which the oligomeric organization of c subunits in F0 was determined by cross-linking of Cys-substituted subunits (Jones, P. C. , Jiang, W., and Fillingame, R. H. (1998) J. Biol. Chem. 273, 17178-17185), we introduced Cys into the first and last transmembrane helices of subunit c monomers, dimers, and trimers and attempted to generate cross-linked products by oxidation with Cu(II)-(1,10-phenanthroline)2. Double Cys substitutions at two sets of positions gave rise to extensive cross-linked multimers. Multimers of the monomer that extended up to the position of c12 were correlated and calibrated with distinct cross-linked species of the appropriate doubly Cys-substituted dimers (i.e. c2, c4, . c12) and doubly Cys-substituted trimers (i.e. c3, c6, c9, c12). The results show that there are 12 copies of subunit c per F0 in E. coli, the exact number having both mechanistic and structural significance.