Genes encoding the alpha, gamma, delta, and four F0 subunits of ATP synthase constitute an operon in the cyanobacterium Anabaena sp. strain PCC 7120.

A cluster of genes encoding subunits of ATP synthase of Anabaena sp. strain PCC 7120 was cloned, and the nucleotide sequences of the genes were determined. This cluster, denoted atp1, consists of four F0 genes and three F1 genes encoding the subunits a (atpI), c (atpH), b' (atpG), b (atpF), delta (atpD), alpha (aptA), and gamma (atpC) in that order. Closely linked upstream of the ATP synthase subunit genes is an open reading frame denoted gene 1, which is equivalent to the uncI gene of Escherichia coli. The atp1 gene cluster is at least 10 kilobase pairs distant in the genome from apt2, a cluster of genes encoding the beta (atpB) and epsilon (atpE) subunits of the ATP synthase. This two-clustered ATP synthase gene arrangement is intermediate between those found in chloroplasts and E. coli. A unique feature of the Anabaena atp1 cluster is overlap between the coding regions for atpF and atpD. The atp1 cluster is transcribed as a single 7-kilobase polycistronic mRNA that initiates 140 base pairs upstream of gene 1. The deduced translation products for the Anabaena sp. strain PCC 7120 subunit genes are more similar to chloroplast ATP synthase subunits than to those of E. coli.     

Membrane integration and function of the three F0 subunits of the ATP synthase of Escherichia coli K12

Integration into the cytoplasmic membrane and function of the three F0 subunits, a, b and c, of the membrane-bound ATP synthase of Escherichia coli K12 were analysed in situations where synthesis of only one or two types of subunits was possible. This was achieved by combined use of atp mutations and plasmids carrying and expressing one or two of the atp genes coding for ATP synthase subunits. AU three F0 subunits were found to be required for the establishment of efficient H+ conduction. Subunits a and b individually as well as together were found to bind F1 ATPase to the membrane while subunit c did not. The ATPase activity bound to either of these single subunits, or in pairwise combinations, was not inhibited by N,N'-dicyclohexylcarbodiimide. Also ATP-dependent H+ translocation was not catalysed unless all three F0 subunits were present in the membrane. The integration into the membrane of the subunits a and b was independent of the presence of other ATP synthase subunits.     

Synthesis of a functional F0 sector of the Escherichia coli H+-ATPase does not require synthesis of the alpha or beta subunits of F1.

The uncB, E, F, and H genes of the Escherichia coli unc operon were cloned behind the lac promoter of plasmid pUC9, generating plasmid pBP101. These unc loci code, respectively, for the chi, omega, and psi subunits of the F0 sector and the delta subunit of the F1 sector of the H+-ATP synthase complex. Induction of expression of the four unc genes by the addition of isopropyl-beta-D-thiogalactoside resulted in inhibition of growth. During isopropyl-beta-D-thiogalactoside induction, the three subunits of F0 were integrated into the cytoplasmic membrane with a resultant increase in H+ permeability. A functional F0 was formed from plasmid pBP101 in a genetic background lacking all eight of the unc structural genes coding the F1F0 complex. In the unc deletion background, a reasonable correlation was observed between the amount of F0 incorporated into the membrane and the function measured, i.e., high-affinity binding of F1 and rate of F0-mediated H+ translocation. This correlation indicates that most or all of the F0 assembled in the membrane is active. Although the F0 assembled under these conditions binds F1, only partial restoration of NADH-dependent or ATP-dependent quenching of quinacrine fluorescence was observed with these membranes. Proteolysis of a fraction of the psi subunit may account for this partial deficiency. The experiments described demonstrate that a functional F0 can be assembled in vivo in E. coli strains lacking genes for the alpha, beta, gamma, and epsilon subunits of F1.     

Tobramycin uptake in Escherichia coli is driven by either electrical potential or ATP.

Aminoglycoside antibiotics such as streptomycin and tobramycin must traverse the bacterial cytoplasmic membrane prior to initiating lethal effects. Previous data on Escherichia coli, Staphylococcus aureus, and Bacillus subtilis have demonstrated that transport of aminoglycosides is regulated by delta psi, the electrical component of the proton motive force. However, several laboratories have observed that growth of bacterial cells can occur in the apparent absence of delta psi, and we wished to confirm these studies with E. coli and further investigate whether transport of aminoglycosides could occur in the absence of a membrane potential. Treatment of acrA strain CL2 with the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) dissipated delta psi, decreased intracellular ATP levels, and resulted in cessation of growth; after a variable period of time (3 to 7 h), growth resumed, ultimately achieving growth rates comparable to those of untreated cells. Absence of delta psi in these cells was confirmed by absence of [3H]tetraphenyl phosphonium+ uptake as measured by membrane filtration, lack of flagellar motion, and inability of these cells to transport proline (but not methionine). Regrowth was associated with restoration of normal intracellular ATP as measured by luciferin-luciferase bioluminescence assay. Unlike unacclimatized CL2 cells treated with CCCP, these cells transported [3H]tobramycin similarly to untreated cells; aminoglycoside-induced killing was seen in association with transport. These studies suggest that under certain circumstances aminoglycoside transport can be driven by ATP (or other high-energy activated phosphate donors) alone, in the absence of a measurable delta psi. delta uncBC mutants of CL2 incapable of interconverting delta psi and ATP were treated with CCCP, resulting in dissipation of delta psi but no alteration in ATP content. Despite maintenance of normal ATP, there was no transport of [3H] bramycin, confirming that under normal growth conditions ATP has no role in the transport of aminoglycosides.     

ATP synthase: subunit-subunit interactions in the stator stalk

In ATP synthase, proton translocation through the Fo subcomplex and ATP synthesis/hydrolysis in the F1 subcomplex are coupled by subunit rotation. The static, non-rotating portions of F1 and Fo are attached to each other via the peripheral "stator stalk", which has to withstand elastic strain during subunit rotation. In Escherichia coli, the stator stalk consists of subunits b2delta; in other organisms, it has three or four different subunits. Recent advances in this area include affinity measurements between individual components of the stator stalk as well as a detailed analysis of the interaction between subunit delta (or its mitochondrial counterpart, the oligomycin-sensitivity conferring protein, OSCP) and F1. The current status of our knowledge of the structure of the stator stalk and of the interactions between its subunits will be discussed in this review.     

Respiratory-driven Na+ electrical potential in the bacterium Vitreoscilla

Vitreoscilla is a Gram-negative bacterium with unique respiratory physiology in which Na+ was implicated as a coupling cation for the generation of a transmembrane electrical gradient (delta psi). Thus, cells respiring in the presence of 110 mM Na+ generated a delta psi of -142 mV compared to only -42 and -56 mV for Li+ and choline, respectively, and even the -42 and -56 mV were insensitive to the protonophore 3,5-di-tert-butyl-4-hydroxybenzaldehyde (DTHB). The kinetics of delta psi formation and collapse correlated well with the kinetics of Na+ fluxes but not with those of H+ fluxes. Cyanide inhibited respiration, Na+ extrusion, and delta psi formation 81% or more, indicating that delta psi formation and Na+ extrusion were coupled to respiration. Experiments were performed to distinguish among three possible transport systems for this coupling: (1) a Na(+)-transporting ATPase; (2) an electrogenic Na+/H+ antiport system; (3) a primary Na+ pump directly driven by the free energy of electron transport. DCCD and arsenate decreased cellular ATP up to 86% but had no effect on delta psi, evidence against a Na(+)-transporting ATPase. Low concentrations of DTHB had no effect on delta psi; high concentrations transiently collapsed delta psi, but led to a stimulation of Na+ extrusion, the opposite of that expected for a Na+/H+ antiport system. Potassium ion, which collapses delta psi, also stimulated Na+ extrusion. The experimental evidence is against Na+ extrusion by mechanisms 1 and 2 and supports the existence of a respiratory-driven primary Na+ pump for generating delta psi in Vitreoscilla.     

Biochemical and molecular characterization of a Na+-translocating F1Fo-ATPase from the thermoalkaliphilic bacterium Clostridium paradoxum

Clostridium paradoxum is an anaerobic thermoalkaliphilic bacterium that grows rapidly at pH 9.8 and 56 degrees C. Under these conditions, growth is sensitive to the F-type ATP synthase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD), suggesting an important role for this enzyme in the physiology of C. paradoxum. The ATP synthase was characterized at the biochemical and molecular levels. The purified enzyme (30-fold purification) displayed the typical subunit pattern for an F1Fo-ATP synthase but also included the presence of a stable oligomeric c-ring that could be dissociated by trichloroacetic acid treatment into its monomeric c subunits. The purified ATPase was stimulated by sodium ions, and sodium provided protection against inhibition by DCCD that was pH dependent. ATP synthesis in inverted membrane vesicles was driven by an artificially imposed chemical gradient of sodium ions in the presence of a transmembrane electrical potential that was sensitive to monensin. Cloning and sequencing of the atp operon revealed the presence of a sodium-binding motif in the membrane-bound c subunit (viz., Q28, E61, and S62). On the basis of these properties, the F1Fo-ATP synthase of C. paradoxum is a sodium-translocating ATPase that is used to generate an electrochemical gradient of + that could be used to drive other membrane-bound bioenergetic processes (e.g., solute transport or flagellar rotation). In support of this proposal are the low rates of ATP synthesis catalyzed by the enzyme and the lack of the C-terminal region of the epsilon subunit that has been shown to be essential for coupled ATP synthesis.     

Translational initiation frequency of atp genes from Escherichia coli: identification of an intercistronic sequence that enhances translation.

The c, b and delta subunit genes of the Escherichia coli atp operon were cloned individually in an expression vector between the tac fusion promoter and the galK gene. The relative rates of subunit synthesis directed by the cloned genes were similar in vitro and in vivo and compared favourably with the subunit stoichiometry of the assembled proton-translocating ATP synthase of E. coli in vivo. The rate of synthesis of subunit c was at least six times that of subunit b and 18 times that of subunit delta. Progressive shortening of the long intercistronic sequence lying upstream of the subunit c gene showed that maximal expression of this gene is dependent upon the presence of a sequence stretching greater than 20 bp upstream of the Shine-Dalgarno site. This sequence thus acts to enhance the rate of translational initiation. The possibility that similar sequences might perform the same function in other operons of E. coli and bacteriophage lambda is also discussed. Translation of the subunit b cistron is partially coupled to translation of the preceding subunit c cistron. In conclusion, the expression of all the atp operon genes could be adjusted to accommodate the subunit requirements of ATP synthase assembly primarily by means of mechanisms which control the efficiency of translational initiation and re-initiation at the respective cistron start codons.     

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.     

Complete DNA sequence of the atp operon of the sodium-dependent F1Fo ATP synthase from Ilyobacter tartaricus and identification of the encoded subunits

The atp operon of Ilyobacter tartaricus, strain DSM 2382, was completely sequenced using conventional and inverse polymerase chain reaction (i-PCR) techniques. It contains nine open reading frames that were attributed to eight structural genes of the F(1)F(o) ATP synthase and the atpI gene, which is not part of the enzyme complex. The initiation codons of all atp genes, except that of atpB coding for the a subunit, were identified by the corresponding N-terminal amino acid sequence. The hydrophobic a subunit was identified by MALDI mass spectrometry. The atp genes of I. tartaricus are arranged in one operon with the sequence atpIBEFHAGDC comprising 6,992 base pairs with a GC content of 38.1%. The F(1)F(o) ATP synthase of I. tartaricus has a calculated molecular mass of 510 kDa and includes 4,810 amino acids. The gene sequences and products reveal significant identities to atp genes of other Na(+)-translocating F(1)F(o) ATP synthases, especially in the F(o) subunits a and c which are directly involved in ion translocation.     

Introduction of a carboxyl group in the first transmembrane helix of Escherichia coli F1Fo ATPase subunit c and cytoplasmic pH regulation

The multicopy subunit c of the H(+)-transporting F1Fo ATP synthase of Escherichia coli folds across the membrane as a hairpin of two hydrophobic alpha helices. The subunits interact in a front-to-back fashion, forming an oligomeric ring with helix 1 packing in the interior and helix 2 at the periphery. A conserved carboxyl, Asp(61) in E. coli, centered in the second transmembrane helix is essential for H+ transport. A second carboxylic acid in the first transmembrane helix is found at a position equivalent to Ile28 in several bacteria, some the cause of serious infectious disease. This side chain has been predicted to pack proximal to the essential carboxyl in helix 2. It appears that in some of these bacteria the primary function of the enzyme is H+ pumping for cytoplasmic pH regulation. In this study, Ile28 was changed to Asp and Glu. Both mutants were functional. However, unlike the wild type, the mutants showed pH-dependent ATPase-coupled H+ pumping and passive H+ transport through Fo. The results indicate that the presence of a second carboxylate enables regulation of enzyme function in response to cytoplasmic pH and that the ion binding pocket is aqueous accessible. The presence of a single carboxyl at position 28, in mutants I28D/D61G and I28E/D61G, did not support growth on a succinate carbon source. However, I28E/D61G was functional in ATPase-coupled H+ transport. This result indicates that the side chain at position 28 is part of the ion binding pocket.     

A nuclear gene encoding the beta subunit of the mitochondrial ATP synthase in Nicotiana plumbaginifolia.

The beta subunit of the mitochondrial ATP synthase in Nicotiana plumbaginifolia is encoded by two nuclear genes, atp2-1 and atp2-2, which are both expressed. The complete nucleotide sequence of atp2-1 has been determined. It contains eight introns ranging from 88 to 1453 bp. The last intron contains a putative insertion element (Inp), of 812 bp bordered by 35-bp inverted repeats which share an 11-bp homology with Agrobacterium tumefaciens T-DNA borders. Sequences homologous to Inp are present in multiple copies in the N. plumbaginifolia and the N. tabacum genome but not in more distant species. The atp2-1 encoded polypeptide is highly homologous to beta subunits from other ATP synthases but it contains an extension at the N terminus which is probably involved in mitochondrial targeting. A sequence homology between exon 4 of atp2-1 and exon 1 of the human ras genes suggests a common ancestral origin for these exons.     

Characterization of a H+-ATPase in rat brain synaptic vesicles. Coupling to L-glutamate transport

Synaptic vesicles contain a H+-ATPase that generates a proton electrochemical gradient (delta mu H+) required for the uptake of neurotransmitters into the organelles. In this study, the synaptic vesicle H+-ATPase was examined for structural and functional similarities with other identified ATPases that generate a delta mu H+ across membranes. The synaptic vesicle H+-ATPase displayed immunological similarity with the 115-, 72-, and 39-kDa subunits of a vacuolar-type H+-ATPase purified from chromaffin granules. Functionally, the ATP-dependent H+ pumping across synaptic vesicles and ATP hydrolysis were sensitive to the sulfhydryl-modifying reagents, N-ethylmaleimide and 4-chloro-7-nitrobenz-2-oxa-1,3-diazole, at concentrations known to affect vacuolar-type H+-ATPases. In addition, as with vacuolar-type H+-ATPases, the presence of NO3-, SO4(2-), or F- inhibited the generation of a delta mu H+, but addition of vanadate or oligomycin had no effect. The delta mu H+ is a function of the pH gradient (delta pH) and membrane potential (delta psi sv) across the synaptic vesicle. Acidification (delta pH) of the synaptic vesicle interior was enhanced in the presence of permeant anions, such as Cl-, or the K+ ionophore, valinomycin. In the absence of permeant anions, the H+-ATPase generated a delta psi sv that effected the transport of L-glutamate into the synaptic vesicles. Dissipation of delta psi sv by incubation with increased external Cl- or nigericin resulted in the abolition of glutamate uptake, despite the continued maintenance of a delta mu H+ across the synaptic vesicle as a substantial delta pH. The results suggest that the synaptic vesicle H+-ATPase is of a vacuolar type and energizes the uptake of anionic glutamate by virtue of the delta psi sv component of the delta mu H+ it generates.     

Identification of subunit g of yeast mitochondrial F1F0-ATP synthase, a protein required for maximal activity of cytochrome c oxidase.

By means of a yeast genome database search, we have identified an open reading frame located on chromosome XVI of Saccharomyces cerevisiae that encodes a protein with 53% amino acid similarity to the 11.3-kDa subunit g of bovine mitochondrial F1F0-ATP synthase. We have designated this ORF ATP20, and its product subunit g. A null mutant strain, constructed by insertion of the HIS3 gene into the coding region of ATP20, retained oxidative phosphorylation function. Assembly of F1F0-ATP synthase in the atp20-null strain was not affected in the absence of subunit g and levels of oligomycin-sensitive ATP hydrolase activity in mitochondria were normal. Immunoprecipitation of F1F0-ATP synthase from mitochondrial lysates prepared from atp20-null cells expressing a variant of subunit g with a hexahistidine motif indicated that this polypeptide was associated with other well-characterized subunits of the yeast complex. Whilst mitochondria isolated from the atp20-null strain had the same oxidative phosphorylation efficiency (ATP : O) as that of the control strain, the atp20-null strain displayed approximately a 30% reduction in both respiratory capacity and ATP synthetic rate. The absence of subunit g also reduced the activity of cytochrome c oxidase, and altered the kinetic control of this complex as demonstrated by experiments titrating ATP synthetic activity with cyanide. These results indicate that subunit g is associated with F1F0-ATP synthase and is required for maximal levels of respiration, ATP synthesis and cytochrome c oxidase activity in yeast.