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.     

Mutant residues suppressing rho(0)-lethality in Kluyveromyces lactis occur at contact sites between subunits of F(1)-ATPase

Characterisation of 35 Kluyveromyces lactis strains lacking mitochondrial DNA has shown that mutations suppressing rho(0)-lethality are limited to the ATP1, 2 and 3 genes coding for the alpha-, beta- and gamma- subunits of mitochondrial F(1)-ATPase. All atp mutations reduce growth on glucose and three alleles, atp1-2, 1-3 and atp3-1, produce a respiratory deficient phenotype that indicates a drop in efficiency of the F(1)F(0)-ATP synthase complex. ATPase activity is needed for suppression as a double mutant containing an atp allele, together with a mutation abolishing catalytic activity, does not suppress rho(0)-lethality. Positioning of the seven amino acids subject to mutation on the bovine F(1)-ATPase structure shows that two residues are found in a membrane proximal region while five amino acids occur at a region suggested to be a molecular bearing. The intriguing juxtaposition of mutable amino acids to other residues subject to change suggests that mutations affect subunit interactions and alter the properties of F(1) in a manner yet to be determined. An explanation for suppressor activity of atp mutations is discussed in the context of a possible role for F(1)-ATPase in the maintenance of mitochondrial inner membrane potential.     

Purification, crystallization, and properties of F1-ATPase complexes from the thermoalkaliphilic Bacillus sp. strain TA2.A1

Recently, we reported the cloning of the atp operon encoding for the F(1)F(0)-ATP synthase from the extremely thermoalkaliphilic bacterium Bacillus sp. strain TA2.A1. In this study, the genes encoding the F(1) moiety of the enzyme complex were cloned from the atp operon into the vector pTrc99A and expressed in Escherichia coli in two variant complexes, F(1)-wt consisting of subunits alpha(3)beta(3)gammadeltaepsilon and F(1)Deltadelta lacking the entire delta-subunit as a prerequisite for overproduction and crystallization trials. Both F(1)-wt and F(1)Deltadelta were successfully overproduced in E. coli and purified in high yield and purity. F(1)Deltadelta was crystallized by micro-batch screening yielding three-dimensional crystals that diffracted to a resolution of 3.1A using a synchrotron radiation source. After establishing cryo and dehydrating conditions, a complete set of diffraction data was collected from a single crystal. No crystals were obtained with F(1)-wt. Data processing of diffraction patterns showed that F(1)Deltadelta crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell parameters of a=121.70, b=174.80, and c=223.50A, alpha, beta, gamma=90.000. The asymmetric unit contained one molecule of bacterial F(1)Deltadelta with a corresponding volume per protein weight (V(M)) of 3.25A(3) Da(-1) and a solvent content of 62.1%. Silver staining of single crystals of F(1)Deltadelta analyzed by SDS-PAGE revealed four bands alpha, beta, gamma, and epsilon with identical M(r)-values as those found in the native F(1)F(0)-ATP synthase isolated from strain TA2.A1 membranes. ATPase assays of F(1)Deltadelta crystals exhibited latent ATP hydrolytic activity that was highly stimulated by lauryldimethylamine oxide, a hallmark of the native enzyme.     

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.     

The typically mitochondrial DNA-encoded ATP6 subunit of the F1F0-ATPase is encoded by a nuclear gene in Chlamydomonas reinhardtii.

The atp6 gene, encoding the ATP6 subunit of F(1)F(0)-ATP synthase, has thus far been found only as an mtDNA-encoded gene. However, atp6 is absent from mtDNAs of some species, including that of Chlamydomonas reinhardtii. Analysis of C. reinhardtii expressed sequence tags revealed three overlapping sequences that encoded a protein with similarity to ATP6 proteins. PCR and 5'- and 3'-RACE were used to obtain the complete cDNA and genomic sequences of C. reinhardtii atp6. The atp6 gene exhibited characteristics of a nucleus-encoded gene: Southern hybridization signals consistent with nuclear localization, the presence of introns, and a codon usage and a polyadenylation signal typical of nuclear genes. The corresponding ATP6 protein was confirmed as a subunit of the mitochondrial F(1)F(0)-ATP synthase from C. reinhardtii by N-terminal sequencing. The predicted ATP6 polypeptide has a 107-amino acid cleavable mitochondrial targeting sequence. The mean hydrophobicity of the protein is decreased in those transmembrane regions that are predicted not to participate directly in proton translocation or in intersubunit contacts with the multimeric ring of c subunits. This is the first example of a mitochondrial protein with more than two transmembrane stretches, directly involved in proton translocation, that is nucleus-encoded.     

Halotolerant cyanobacterium Aphanothece halophytica contains an Na+-dependent F1F0-ATP synthase with a potential role in salt-stress tolerance

Aphanothece halophytica is a halotolerant alkaliphilic cyanobacterium that can grow in media of up to 3.0 m NaCl and pH 11. Here, we show that in addition to a typical H(+)-ATP synthase, Aphanothece halophytica contains a putative F(1)F(0)-type Na(+)-ATP synthase (ApNa(+)-ATPase) operon (ApNa(+)-atp). The operon consists of nine genes organized in the order of putative subunits β, ε, I, hypothetical protein, a, c, b, α, and γ. Homologous operons could also be found in some cyanobacteria such as Synechococcus sp. PCC 7002 and Acaryochloris marina MBIC11017. The ApNa(+)-atp operon was isolated from the A. halophytica genome and transferred into an Escherichia coli mutant DK8 (Δatp) deficient in ATP synthase. The inverted membrane vesicles of E. coli DK8 expressing ApNa(+)-ATPase exhibited Na(+)-dependent ATP hydrolysis activity, which was inhibited by monensin and tributyltin chloride, but not by the protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The Na(+) ion protected the inhibition of ApNa(+)-ATPase by N,N'-dicyclohexylcarbodiimide. The ATP synthesis activity was also observed using the Na(+)-loaded inverted membrane vesicles. Expression of the ApNa(+)-atp operon in the heterologous cyanobacterium Synechococcus sp. PCC 7942 showed its localization in the cytoplasmic membrane fractions and increased tolerance to salt stress. These results indicate that A. halophytica has additional Na(+)-dependent F(1)F(0)-ATPase in the cytoplasmic membrane playing a potential role in salt-stress tolerance.     

A topological analysis of subunit alpha from Escherichia coli F1F0-ATP synthase predicts eight transmembrane segments

The membrane topology of subunit alpha from the Escherichia coli F1F0-ATP synthase was studied using a gene fusion technique. Fusion proteins linking different amino-terminal fragments of the alpha subunit with an enzymatically active fragment of alkaline phosphatase were constructed by both random transposition of TnphoA and site-directed mutagenesis. Those proteins with high levels of alkaline phosphatase activity are predicted to define periplasmic domains of alpha, and this was confirmed by testing for cell growth in minimal medium supplemented with polyphosphate (P greater than 75) as the sole source of phosphate. The enzymatic activity of some fusion proteins was shown to be sensitive to glucose present in the growth medium. Results from subcellular fractionation experiments suggest that these fusion proteins may be inactive even though they have a periplasmic alkaline phosphatase. The enzymatic activity appears dependent upon proteolytic release of the alkaline phosphatase moiety from its alpha subunit membrane anchor and suggests the target of glucose repression may be a protease present in the periplasm. For the topological analysis of the alpha subunit, a total of 28 unique fusion proteins were studied and the results were consistent with a model of alpha containing eight transmembrane segments, including periplasmic amino and carboxyl termini. Surprisingly, separate periplasmic domains were identified near amino acids 200, 233, and 270. These results suggest the flanking membrane spans are only 10-15 amino acids in length and not able to span a standard 30 A bilayer in an alpha-helical conformation. These short spans may have interesting mechanistic implications for the function of F0, because they contain several amino acids which appear critical for proton translocation. Finally, a fusion of alkaline phosphatase at amino acid 271, the carboxyl-terminal residue, but not at amino acid 260, was able to complement the strain RH305 (uncB-) for growth on succinate and suggests the last 11 amino acids of the alpha subunit are critical to the function of F1F0-ATP synthase.     

A1Ao-ATP synthase of Methanobrevibacter ruminantium couples sodium ions for ATP synthesis under physiological conditions

An unresolved question in the bioenergetics of methanogenic archaea is how the generation of proton-motive and sodium-motive forces during methane production is used to synthesize ATP by the membrane-bound A(1)A(o)-ATP synthase, with both proton- and sodium-coupled enzymes being reported in methanogens. To address this question, we investigated the biochemical characteristics of the A(1)A(o)-ATP synthase (MbbrA(1)A(o)) of Methanobrevibacter ruminantium M1, a predominant methanogen in the rumen. Growth of M. ruminantium M1 was inhibited by protonophores and sodium ionophores, demonstrating that both ion gradients were essential for growth. To study the role of these ions in ATP synthesis, the ahaHIKECFABD operon encoding the MbbrA(1)A(o) was expressed in Escherichia coli strain DK8 (Δatp) and purified yielding a 9-subunit protein with an SDS-stable c oligomer. Analysis of the c subunit amino acid sequence revealed that it consisted of four transmembrane helices, and each hairpin displayed a complete Na(+)-binding signature made up of identical amino acid residues. The purified MbbrA(1)A(o) was stimulated by sodium ions, and Na(+) provided pH-dependent protection against inhibition by dicyclohexylcarbodiimide but not tributyltin chloride. ATP synthesis in inverted membrane vesicles lacking sodium ions was driven by a membrane potential that was sensitive to cyanide m-chlorophenylhydrazone but not to monensin. ATP synthesis could not be driven by a chemical gradient of sodium ions unless a membrane potential was imposed. ATP synthesis under these conditions was sensitive to monensin but not cyanide m-chlorophenylhydrazone. These data suggest that the M. ruminantium M1 A(1)A(o)-ATP synthase exhibits all the properties of a sodium-coupled enzyme, but it is also able to use protons to drive ATP synthesis under conditions that favor proton coupling, such as low pH and low levels of sodium ions.     

Manipulations in the peripheral stalk of the Saccharomyces cerevisiae F1F0-ATP synthase

The Saccharomyces cerevisiae F(1)F(0)-ATP synthase peripheral stalk is composed of the OSCP, h, d, and b subunits. The b subunit has two membrane-spanning domains and a large hydrophilic domain that extends along one side of the enzyme to the top of F(1). In contrast, the Escherichia coli peripheral stalk has two identical b subunits, and subunits with substantially altered lengths can be incorporated into a functional F(1)F(0)-ATP synthase. The differences in subunit structure between the eukaryotic and prokaryotic peripheral stalks raised a question about whether the two stalks have similar physical and functional properties. In the present work, the length of the S. cerevisiae b subunit has been manipulated to determine whether the F(1)F(0)-ATP synthase exhibited the same tolerances as in the bacterial enzyme. Plasmid shuffling was used for ectopic expression of altered b subunits in a strain carrying a chromosomal disruption of the ATP4 gene. Wild type growth phenotypes were observed for insertions of up to 11 and a deletion of four amino acids on a nonfermentable carbon source. In mitochondria-enriched fractions, abundant ATP hydrolysis activity was seen for the insertion mutants. ATPase activity was largely oligomycin-insensitive in these mitochondrial fractions. In addition, very poor complementation was seen in a mutant with an insertion of 14 amino acids. Lengthier deletions yielded a defective enzyme. The results suggest that although the eukaryotic peripheral stalk is near its minimum length, the b subunit can be extended a considerable distance.     

The atp operon: nucleotide sequence of the region encoding the alpha-subunit of Escherichia coli ATP-synthase

Part of the atp (or unc) operon encoding the alpha, beta, gamma, delta, and epsilon subunits of Escherichia coli ATP-synthase has been cloned into the plasmid pACYC 184. The DNA coding for the largest of these proteins, the alphas subunit, has been sequenced by cloning into the bacteriophage M13 and sequencing with dideoxy nucleotide chain terminators. It comprises 1539 nucleotides corresponding to a protein of 513 amino acids.     

A specific adaptation in the a subunit of thermoalkaliphilic F1FO-ATP synthase enables ATP synthesis at high pH but not at neutral pH values

Analysis of the atp operon from the thermoalkaliphilic Bacillus sp. TA2.A1 and comparison with other atp operons from alkaliphilic bacteria reveals the presence of a conserved lysine residue at position 180 (Bacillus sp. TA2.A1 numbering) within the a subunit of these F(1)F(o)-ATP synthases. We hypothesize that the basic nature of this residue is ideally suited to capture protons from the bulk phase at high pH. To test this hypothesis, a heterologous expression system for the ATP synthase from Bacillus sp. TA2.A1 (TA2F(1)F(o)) was developed in Escherichia coli DK8 (Deltaatp). Amino acid substitutions were made in the a subunit of TA2F(1)F(o) at position 180. Lysine (aK180) was substituted for the basic residues histidine (aK180H) or arginine (aK180R), and the uncharged residue glycine (aK180G). ATP synthesis experiments were performed in ADP plus P(i)-loaded right-side-out membrane vesicles energized by ascorbate-phenazine methosulfate. When these enzyme complexes were examined for their ability to perform ATP synthesis over the pH range from 7.0 to 10.0, TA2F(1)F(o) and aK180R showed a similar pH profile having optimum ATP synthesis rates at pH 9.0-9.5 with no measurable ATP synthesis at pH 7.5. Conversely, aK180H and aK180G showed maximal ATP synthesis at pH values 8.0 and 7.5, respectively. ATP synthesis under these conditions for all enzyme forms was sensitive to DCCD. These data strongly imply that amino acid residue Lys(180) is a specific adaptation within the a subunit of TA2F(1)F(o) to facilitate proton capture at high pH. At pH values near the pK(a) of Lys(180), the trapped protons readily dissociate to reach the subunit c binding sites, but this dissociation is impeded at neutral pH values causing either a blocking of the proposed H(+) channel and/or mechanism of proton translocation, and hence ATP synthesis is inhibited.     

Identification and analysis of the rnc gene for RNase III in Rhodobacter capsulatus.

The large subunit ribosomal RNA of the purple bacterium Rhodobacter capsulatus shows fragmentation into pieces of 14 and 16S, both fragments forming the functional equivalent of intact 23S rRNA. An RNA-processing step removes an extra stem-loop structure from the 23S rRNA [Kordes, E., Jock, S., Fritsch, J., Bosch, F. and Klug, G. (1994) J. Bacteriol., 176, 1121-1127]. Taking advantage of the fragmentation deficient mutant strain Fm65, we used genetic complementation to find the mutated gene responsible for this aberration. It was identified as the Rhodobacter homologue to mc from Escherichia coli encoding endoribonuclease III (RNase III). The predicted protein has 226 amino acids with a molecular weight of 25.5 kDa. It shares high homology with other known RNase III enzymes over the full length. In particular it shows the double-stranded RNA-binding domain (dsRBD) motif essential for binding of dsRNA substrates. The Fm65 mutant has a frame shift mutation resulting in complete loss of the dsRBD rendering the enzyme inactive. The cloned Rhodobacter enzyme can substitute RNase III activity in an RNase III deficient E. coli strain. Contrary to E. coli, the Rhodobacter mc is in one operon together with the lep gene encoding the leader peptidase.     

Formation of the yeast F1F0-ATP synthase dimeric complex does not require the ATPase inhibitor protein,Inh1

The yeast F1F0-ATP synthase forms dimeric complexes in the mitochondrial inner membrane and in a manner that is supported by the F0-sector subunits, Su e and Su g. Furthermore, it has recently been demonstrated that the binding of the F1F0-ATPase natural inhibitor protein to purified bovine F1-sectors can promote their dimerization in solution (Cabezon, E., Arechaga, I., Jonathan P., Butler, G., and Walker J. E. (2000) J. Biol. Chem. 275, 28353-28355). It was unclear until now whether the binding of the inhibitor protein to the F1 domains contributes to the process of F1F0-ATP synthase dimerization in intact mitochondria. Here we have directly addressed the involvement of the yeast inhibitor protein, Inh1, and its known accessory proteins, Stf1 and Stf2, in the formation of the yeast F1F0-ATP synthase dimer. Using mitochondria isolated from null mutants deficient in Inh1, Stf1, and Stf2, we demonstrate that formation of the F(1)F(0)-ATP synthase dimers is not adversely affected by the absence of these proteins. Furthermore, we demonstrate that the F1F0-ATPase monomers present in su e null mutant mitochondria can be as effectively inhibited by Inh1, as its dimeric counterpart in wild-type mitochondria. We conclude that dimerization of the F1F0-ATP synthase complexes involves a physical interaction of the membrane-embedded F0 sectors from two monomeric complexes and in a manner that is independent of inhibitory activity of the Inh1 and accessory proteins.