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.     

Yeast mitochondrial F1F0-ATP synthase exists as a dimer: identification of three dimer-specific subunits.

Using the technique of blue native gel electrophoresis, the oligomeric state of the yeast mitochondrial F1F0-ATP synthase was analysed. Solubilization of mitochondrial membranes with low detergent to protein ratios led to the identification of the dimeric state of the ATP synthase. Analysis of the subunit composition of the dimer, in comparison with the monomer, revealed the presence of three additional small proteins. These dimer-specific subunits of the ATP synthase were identified as the recently described subunit e/Tim11 (Su e/Tim11), the putative subunit g homolog (Su g) and a new component termed subunit k (Su k). Although, as shown here, these three proteins are not required for the formation of enzymatically active ATP synthase, Su e/Tim11 and Su g are essential for the formation of the dimeric state. Su e/Tim11 appears to play a central role in this dimerization process. The dimer-specific subunits are associated with the membrane bound F0-sector. The F0-sector may thereby be involved in the dimerization of two monomeric F1F0-ATP synthase complexes. We speculate that the F1F0-ATP synthase of yeast, like the other complexes of oxidative phosphorylation, form supracomplexes to optimize transduction of energy and to enhance the stability of the complex in the membrane.     

Nucleotide sequence of a cDNA for the alpha subunit of human mitochondrial ATP synthase.

A full length cDNA clone of the alpha subunit of mitochondrial ATP synthase (EC 3.6.1.34) has been isolated from a cDNA library prepared from LX-1 human tumor cells in the lambda-Zap vector. The clone is 1883 base pairs (bp) in length and contains a 1659 bp open reading frame encoding a polypeptide of 553 residues. The deduced amino acid sequence is highly homologous to ATP synthase from several other species.     

Identification of a mitochondrial ATP synthase small subunit gene (RMtATP6) expressed in response to salts and osmotic stresses in rice (Oryza sativa L.)

Large areas of northern China have alkaline soil due to the accumulation of sodium carbonates (NaHCO3, Na2CO3). To understand better how plants can tolerate alkaline soil, a cDNA library was prepared from rice (Oryza sativa L.) roots grown in the presence of NaHCO3 stress. A cDNA clone isolated from this library was identified by a homology search as a mitochondrial ATP synthase 6 kDa subunit gene (RMtATP6; GenBank accession nos AB055076, BAB21526). In transformed yeast and tobacco protoplasts, the RMtATP6 protein was localized in mitochondria using the green fluorescent protein (GFP) marker. Analysis of RMtATP6 mRNA levels suggested that the expression of this gene was induced by stress from sodium carbonates and other sodium salts. Transgenic tobacco overexpressing the RMtATP6 gene had greater tolerance to salt stress at the seedling stage than untransformed tobacco. Among the other genes for F1F0-ATPase of rice, some were found to be up-regulated by some environmental stresses and some were not. These data suggest that the RMtATP6 protein acts as a subunit of ATP synthase, and is expressed in response to stress from several salts, with the other genes coding for the subunits of the same ATP-synthase.     

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.     

Characterization of the 46,000-dalton subunit of eIF-4F

Three protein synthesis initiation factors, eukaryotic initiation factor (eIF)-4A, -4B, and -4F are required for the ATP-dependent binding of mRNA to the ribosome. To extend the characterization of the eIF-4A-like subunit of eIF-4F, a cDNA clone encoding eIF-4A has been isolated from a rabbit liver cDNA library and sequenced. The clone is almost full length for the coding region and complete for the 3' noncoding region. The sequence of the rabbit cDNA has been compared to the sequence of the two similar, but not identical, genes and cDNAs encoding mouse eIF-4A (termed eIF-4AI and eIF-4AII). The rabbit cDNA sequence is very similar to the mouse eIF-4AI genomic and liver cDNA sequence with 100% identity at the amino acid level and 90% identity at the nucleotide level within the protein coding region; however, there is very little similarity in the 3' noncoding region. Amino acid sequencing of purified rabbit reticulocyte eIF-4A protein indicates that it is eIF-4AI (encoded by the eIF-4AI gene and cDNA) and none of the amino acid residues sequenced are in disagreement with those predicted from the mouse liver or rabbit liver cDNA sequences. Subsequently, we have analyzed the p46 subunit of eIF-4F, a three subunit protein whose molecular weights have been estimated by sodium dodecyl sulfate gel electrophoresis to be 220,000, 46,000 and 24,000. The p46 subunit has physical properties similar to eIF-4A. This subunit was isolated from rabbit reticulocyte eIF-4F and sequenced chemically. Our results indicate that this peptide is a mixture of eIF-4AI and eIF-4AII in an approximate ratio of 4 to 1, respectively. No eIF-4AII was observed in our rabbit reticulocyte eIF-4A preparation. Therefore we have concluded that either the eIF-4AI and the eIF-4AII proteins were resolved from each other in the purification of rabbit reticulocyte eIF-4A or that eIF-4AII preferentially associates with the p220 and p24 subunits of eIF-4F. Evidence favoring the latter possibility is discussed.     

Isolation and mapping of a putative b subunit of human ATP synthase (ATP-BL) from human leukocytes.

From a human-leukocyte cDNA library, we cloned cDNA encoding a novel protein, which has a significant homology with the b subunit of ATP synthase (proton-transporting ATPase, F1F0-ATPase; EC3.6.1.34) derived from Anabaena sp. strain PCC 7120. The cDNA has an open reading frame of 1314 nucleotides corresponding to 438 amino acids. The coding sequence was 37.9% identical over 57 amino acid with b subunit of ATP synthase. The 34-amino-acid region of the predicted peptide sequence displays a coiled-coil motif that could form a complex with some other protein(s). We designated this novel gene as ATP-BL because of its homology to the b subunit of ATP synthase. The ATP-BL locus was mapped by fluorescence in situ hybridization (FISH) and radiation hybrid mapping to the q24 region of chromosome 16.     

Isolated epsilon subunit of thermophilic F1-ATPase binds ATP

F1-ATPase, a soluble part of the F0F1-ATP synthase, has subunit structure alpha3beta3gammadeltaepsilon in which nucleotide-binding sites are located in the alpha and beta subunits and, as believed, in none of the other subunits. However, we report here that the isolated epsilon subunit of F1-ATPase from thermophilic Bacillus strain PS3 can bind ATP. The binding was directly demonstrated by isolating the epsilon subunit-ATP complex with gel filtration chromatography. The binding was not dependent on Mg2+ but was highly specific for ATP; however, ADP, GTP, UTP, and CTP failed to bind. The epsilon subunit lacking the C-terminal helical hairpin was unable to bind ATP. Although ATP binding to the isolated epsilon subunits from other organisms has not been detected under the same conditions, a possibility emerges that the epsilon subunit acts as a built in cellular ATP level sensor of F0F1-ATP synthase.     

Each yeast mitochondrial F1F0-ATP synthase complex contains a single copy of subunit 8

The stoichiometry of subunit 8 in yeast mitochondrial F(1)F(0)-ATP synthase (mtATPase) has been evaluated using an immunoprecipitation approach. Single HA or FLAG epitopes were introduced at the N-terminus of subunit 8. Expression of each tagged subunit 8 variant in yeast cells lacking endogenous subunit 8 restored a respiratory phenotype and had little measurable effect on ATP hydrolase activity of the isolated enzyme. Moreover, the two epitope-tagged subunit 8 variants could be stably co-expressed in the same host cells and both of HA-Y8 and FLAG-Y8 could be detected in ATP synthase complexes isolated by native gel electrophoresis. Mitochondria isolated from each yeast strain were solubilized to release ATP synthase complexes in either the monomeric or dimeric forms. In each case, monoclonal antibodies directed against either the FLAG or HA epitope could immunoprecipitate intact ATP synthase complexes. When both HA-Y8 and FLAG-Y8 were co-expressed in cells, monomeric ATP synthases contained only a single subunit 8 variant after immunoprecipitation, corresponding to the particular antibody used (HA or FLAG). By contrast, both subunit 8 variants were recovered in samples of immunoprecipitated dimeric ATP synthase complexes, irrespective of the antibody used. We conclude that each monomeric yeast mitochondrial ATP synthase complex contains a single copy of subunit 8.     

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.     

Subunit gamma-green fluorescent protein fusions are functionally incorporated into mitochondrial F1F0-ATP synthase,arguing against a rigid cap structure at the top of F1

We have investigated the question of the presence of a cap structure located at the top of the F(1) alpha(3)beta(3) hexamer of the yeast mitochondrial F(1)F(0)-ATP synthase complex. Specifically, we sought to determine whether the putative cap has a rigid structure and occludes the central shaft space formed by the alpha(3)beta(3) hexamer or alternatively whether the cap is more flexible permitting access to the central shaft space under certain conditions. Thus, we sought to establish whether subunit gamma, an essential component of the F(1) central stalk housed within the central shaft space and whose N and C termini would both lie beneath a putative cap, could be fused at its C terminus to green fluorescent protein (GFP) without loss of enzyme function. The GFP moiety serves to report on the integrity and location of fusion proteins containing different length polypeptide linkers between GFP and subunit gamma, as well as being a potential occluding structure in itself. Functional incorporation of subunit gamma-GFP fusions into ATP synthase of yeast cells lacking native subunit gamma was demonstrated by the ability of intact complexes to hydrolyze ATP and retain sensitivity to oligomycin. Our conclusion is that the putative cap structure cannot be an inflexible structure, but must be of a more flexible nature consistent with the accommodation of subunit gamma-GFP fusions within functional ATP synthase complexes.     

Topological and functional study of subunit h of the F1Fo ATP synthase complex in yeast Saccharomyces cerevisiae

Subunit h, a 92-residue-long, hydrophilic, acidic protein, is a component of the yeast mitochondrial F1Fo ATP synthase. This subunit, homologous to the mammalian factor F6, is essential for the correct assembly and/or functioning of this enzyme since yeast cells lacking it are not able to grow on nonfermentable carbon sources. Chemical cross-links between subunit h and subunit 4 have previously been shown, suggesting that subunit h is a component of the peripheral stalk of the F1Fo ATP synthase. The construction of cysteine-containing subunit h mutants and the use of bismaleimide reagents provided insights into its environment. Cross-links were obtained between subunit h and subunits alpha, f, d, and 4. These results and secondary structure predictions allowed us to build a structural model and to propose that this subunit occupies a central place in the peripheral stalk between the F1 sector and the membrane. In addition, subunit h was found to have a stoichiometry of one in the F1Fo ATP synthase complex and to be in close proximity to another subunit h belonging to another F1Fo ATP synthase in the inner mitochondrial membrane. Finally, functional characterization of mitochondria from mutants expressing different C-terminal shortened subunit h suggested that its C-terminal part is not essential for the assembly of a functional F1Fo ATP synthase.     

Environmental study of subunit i, a F(o) component of the yeast ATP synthase

The topology of subunit i, a component of the yeast F(o)F(1)-ATP synthase, was determined by the use of cysteine-substituted mutants. The N(in)-C(out) orientation of this intrinsic subunit was confirmed by chemical modification of unique cysteine residues with 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid. Near-neighbor relationships between subunit i and subunits 6, f, g, and d were demonstrated by cross-link formation following sulfhydryl oxidation or reaction with homobifunctional and heterobifunctional reagents. Our data suggest interactions between the unique membrane-spanning segment of subunit i and the first transmembranous alpha-helix of subunit 6 and a stoichiometry of 1 subunit i per complex. Cross-linked products between mutant subunits i and proteins loosely bound to the F(o)F(1)-ATP synthase suggest that subunit i is located at the periphery of the enzyme and interacts with proteins of the inner mitochondrial membrane that are not involved in the structure of the yeast ATP synthase.     

Cross-linking and labeling of the Escherichia coli F1F0-ATP synthase reveal a compact hydrophilic portion of F0 close to an F1 catalytic subunit

The subunit arrangement of the F0 sector of the Escherichia coli ATP synthase is examined using hydrophilic and hydrophobic (cleavable) cross-linking reagents and the water-soluble labeling reagent [35S] diazoniumbenzenesulfonate ( [35S]DABS). Cross-linking is performed on purified ATP synthase and inverted minicell membranes. ATP synthase incorporated into liposomes is labeled with [35S]DABS. Three cross-linked products involving the F0 subunits (a, b, and c) are observed with the purified ATP synthase in solution: a-b, b2, and c2 dimers. A cross-link between the F0 and F1 is detected and occurs between the a and beta subunits. A cross-linker independent association between the b and beta subunits is also evident, suggesting that the two subunits are close enough to form a disulfide bridge. A cross-linking reagent stable to reducing agents produces a b-beta dimer, as detected by immunoblotting with anti-beta serum. The c subunit does not cross-link with any F1 polypeptide. Minicell membranes containing ATP synthase polypeptides radioactively labeled in vivo similarly show b2 and c2 dimers after cross-linking. [35S]DABS labels the a and b, but not c, subunits, showing that the a and b, but not c, subunits possess hydrophilic domains. Thus, certain domains of subunits a and b extend from the membrane and are in close proximity to one another and the F1 catalytic subunit beta.     

Mutagenic analysis of the a subunit of the F1F0 ATP synthase in Escherichia coli: Gln-252 through Tyr-263.

The a subunit of F1F0 ATP synthase contains a highly conserved region near its carboxyl terminus which is thought to be important in proton translocation. Cassette site-directed mutagenesis was used to study the roles of four conserved amino acids Gln-252, Phe-256, Leu-259, and Tyr-263. Substitution of basic amino acids at each of these four sites resulted in marked decreases in enzyme function. Cells carrying a subunit mutations Gln-252-->Lys, Phe-256-->Arg, Leu-259-->Arg, and Tyr-263-->Arg all displayed growth characteristics suggesting substantial loss of ATP synthase function. Studies of both ATP-driven proton pumping and proton permeability of stripped membranes indicated that proton translocation through F0 was affected by the mutations. Other mutations, such as the Phe-256-->Asp mutation, also resulted in reduced enzyme activity. However, more conservative amino acid substitutions generated at these same four positions produced minimal losses of F1F0 ATP synthase. The effects of mutations and, hence, the relative importance of the amino acids for enzyme function appeared to decrease with proximity to the carboxyl terminus of the a subunit. The data are most consistent with the hypothesis that the region between Gln-252 and Tyr-263 of the a subunit has an important structural role in F1F0 ATP synthase.     

Defective gamma subunit of ATP synthase (F1F0) from Escherichia coli leads to resistance to aminoglycoside antibiotics.

A strain of Escherichia coli which was derived from a gentamicin-resistant clinical isolate was found to be cross-resistant to neomycin and streptomycin. The molecular nature of the genetic defect was found to be an insertion of two GC base pairs in the uncG gene of the mutant. The insertion led to the production of a truncated gamma subunit of 247 amino acids in length instead of the 286 amino acids that are present in the normal gamma subunit. A plasmid which carried the ATP synthase genes from the mutant produced resistance to aminoglycoside antibiotics when it was introduced into a strain with a chromosomal deletion of the ATP synthase genes. Removal of the genes coding for the beta and epsilon subunits abolished antibiotic resistance coded by the mutant plasmid. The relationship between antibiotic resistance and the gamma subunit was investigated by testing the antibiotic resistance of plasmids carrying various combinations of unc genes. The presence of genes for the F0 portion of the ATP synthase in the presence or absence of genes for the gamma subunit was not sufficient to cause antibiotic resistance. alpha, beta, and truncated gamma subunits were detected on washed membranes of the mutant by immunoblotting. The first 247 amino acid residues of the gamma subunit may be sufficient to allow its association with other F1 subunits in such a way that the proton gate of F0 is held open by the mutant F1.     

Remarkable stability of the proton translocating F1FO-ATP synthase from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1

For functional characterization, we isolated the F1FO-ATP synthase of the thermophilic cyanobacterium Thermosynechococcus elongatus. Because of the high content of phycobilisomes, a combination of dye-ligand chromatography and anion exchange chromatography was necessary to yield highly pure ATP synthase. All nine single F1FO subunits were identified by mass spectrometry. Western blotting revealed the SDS stable oligomer of subunits c in T. elongatus. In contrast to the mass archived in the database (10,141 Da), MALDI-TOF-MS revealed a mass of the subunit c monomer of only 8238 Da. A notable feature of the ATP synthase was its ability to synthesize ATP in a wide temperature range and its stability against chaotropic reagents. After reconstitution of F1FO into liposomes, ATP synthesis energized by an applied electrochemical proton gradient demonstrated functional integrity. The highest ATP synthesis rate was determined at the natural growth temperature of 55 degrees C, but even at 95 degrees C ATP production occurred. In contrast to other prokaryotic and eukaryotic ATP synthases which can be disassembled with Coomassie dye into the membrane integral and the hydrophilic part, the F1FO-ATP synthase possessed a particular stability. Also with the chaotropic reagents sodium bromide and guanidine thiocyanate, significantly harsher conditions were required for disassembly of the thermophilic ATP synthase.     

Coupled rotation within single F0F1 enzyme complexes during ATP synthesis or hydrolysis

F0F1 ATP synthases are the smallest rotary motors in nature and work as ATP factories in bacteria, plants and animals. Here we report on the first observation of intersubunit rotation in fully coupled single F0F1 molecules during ATP synthesis or hydrolysis. We investigate the Na+-translocating ATP synthase of Propionigenium modestum specifically labeled by a single fluorophore at one c subunit using polarization-resolved confocal microscopy. Rotation during ATP synthesis was observed with the immobilized enzyme reconstituted into proteoliposomes after applying a diffusion potential, but not with a Na+ concentration gradient alone. During ATP hydrolysis, stepwise rotation of the labeled c subunit was found in the presence of 2 mM NaCl, but not without the addition of Na+ ions. Moreover, upon the incubation with the F0-specific inhibitor dicyclohexylcarbodiimide the rotation was severely inhibited.     

Membrane topography of the coupling ion binding site in Na+-translocating F1F0 ATP synthase.

A carbodiimide with a photoactivatable diazirine substituent was synthesized and incubated with the Na(+)-translocating F(1)F(0) ATP synthase from both Propionigenium modestum and Ilyobacter tartaricus. This caused severe inhibition of ATP hydrolysis activity in the absence of Na(+) ions but not in its presence, indicating the specific reaction with the Na(+) binding c-Glu(65) residue. Photocross-linking was investigated with the substituted ATP synthase from both bacteria in reconstituted 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC)-containing proteoliposomes. A subunit c/POPC conjugate was found in the illuminated samples but no a-c cross-links were observed, not even after ATP-induced rotation of the c-ring. Our substituted diazirine moiety on c-Glu(65) was therefore in close contact with phospholipid but does not contact subunit a. Na(+)in/(22)Na(+)out exchange activity of the ATP synthase was not affected by modifying the c-Glu(65) sites with the carbodiimide, but upon photoinduced cross-linking, this activity was abolished. Cross-linking the rotor to lipids apparently arrested rotational mobility required for moving Na(+) ions back and forth across the membrane. The site of cross-linking was analyzed by digestions of the substituted POPC using phospholipases C and A(2) and by mass spectroscopy. The substitutions were found exclusively at the fatty acid side chains, which indicates that c-Glu(65) is located within the core of the membrane.