Bovine Mitochondrial ATP Synthase Research Articles

A Novel Multiplex Polymerase Chain Reaction Method for the Identification of Brachyspina Syndrome Carriers in Chinese Holstein Cattle

A Novel Multiplex Polymerase Chain Reaction Method for the Identification of Brachyspina Syndrome Carriers in Chinese Holstein Cattle Brachyspina syndrome (BS) is a monogenic autosomal recessive hereditary defect in Holstein dairy cattle. It is caused by a 3.3-kb deletion in the Fanconi anemia complementation group I (FANCI) gene on BTA21, which removes exons 25 to 27 and leads to a frame-shift and premature stop codon in exon 28. A consequence of BS is reduced reproduction efficiency and milk production in the dams due to loss of homozygous fetuses. In this study, we successfully developed a novel single-tube multiplex PCR assay to identify the recessive allele of BS with the bovine mitochondrial ATP synthase F0 subunit 8 (ATP8) genes as an internal positive control. We used this technique to detect BS alleles in a total of 63 Holstein bulls and 500 Chinese Holstein cows born between 2013 and 2015. Among them, 42 carriers were found (two sires and 40 cows). Combined with our previous study that assayed 206 Holstein bulls and 136 cows (born between 1996 and 2012) and identified 10 bull carriers and three cow carriers, the overall observed carrier frequency is 4.5% in bulls (269 assayed), and 6.8% in cows (636 assayed). Our method provides not only a reliable, economic, and practical diagnostic technique for BS carrier detection, but also promotes BS detection and monitoring system for the genetic improvement project of Chinese Holstein cattle.

CHARACTERIZATION OF ATP GENE IN Calotropis procera MITOCHONDRIAL GENOME

The drought-tolerant wild plant C. procera is important in medicine, industry and ornamental fields. Generally, its bark and leaves are used for many Folk medicine treatments. ATP4 (mitochondrion ATPase subunit 4), one of ATP gene family provides instructions for making transporter proteins called ATPases, which use energy from ATP molecule to move substances, such as fats, sugars, charged atoms or molecules (ions), and drugs, across the cell membranes. In this study, we uncovered and characterized ATP4 (ATP4, NCBI accession no. KP171515) gene in this medicinal plant from the de novo assembled transcriptome contigs of the high-throughput sequencing dataset. A number of GenBank accessions for ATP4 sequences were blasted with the recovered de novo assembled contigs. Homology modeling of the deduced amino acids was further carried out using Swiss-Model, accessible via the EXPASY. Superimposition of C. procera ATP4 full sequence model on Chain A, Subcomplex of the Stator of Bovine Mitochondrial ATP Synthase (PDB accession no. 2CLY_A) was constructed using RasMol and Deep-View programs. The functional domains of the novel ATP4 amino acids sequence were identified from the NCBI conserved domain data-base (CDD, accession no. cl21478) that provide insights into sequence structure/function relationships, as well as domain models imported from a number of external source databases (Pfam, SMART, COG, PRK, TIGRFAM).

Dietary macronutrients modulate the fatty acyl composition of rat liver mitochondrial cardiolipins

The interaction of dietary fats and carbohydrates on liver mitochondria were examined in male FBNF1 rats fed 20 different low-fat isocaloric diets. Animal growth rates and mitochondrial respiratory parameters were essentially unaffected, but mass spectrometry-based mitochondrial lipidomics profiling revealed increased levels of cardiolipins (CLs), a family of phospholipids essential for mitochondrial structure and function, in rats fed saturated or trans fat-based diets with a high glycemic index. These mitochondria showed elevated monolysocardiolipins (a CL precursor/product of CL degradation), elevated ratio of trans-phosphocholine (PC) (18:1/18:1) to cis-PC (18:1/18:1) (a marker of thiyl radical stress), and decreased ubiquinone Q9; the latter two of which imply a low-grade mitochondrial redox abnormality. Extended analysis demonstrated: i) dietary fats and, to a lesser extent, carbohydrates induce changes in the relative abundance of specific CL species; ii) fatty acid (FA) incorporation into mature CLs undergoes both positive (>400-fold) and negative (2.5-fold) regulation; and iii) dietary lipid abundance and incorporation of FAs into both the CL pool and specific mature tetra-acyl CLs are inversely related, suggesting previously unobserved compensatory regulation. This study reveals previously unobserved complexity/regulation of the central lipid in mitochondrial metabolism.

Arrangement of subunits in intact mammalian mitochondrial ATP synthase determined by cryo-EM

Mitochondrial ATP synthase is responsible for the synthesis of ATP, a universal energy currency in cells. Whereas X-ray crystallography has revealed the structure of the soluble region of the complex and the membrane-intrinsic c-subunits, little is known about the structure of the six other proteins (a, b, f, A6L, e, and g) that comprise the membrane-bound region of the complex in animal mitochondria. Here, we present the structure of intact bovine mitochondrial ATP synthase at ∼18 Å resolution by electron cryomicroscopy of single particles in amorphous ice. The map reveals that the a-subunit and c(8)-ring of the complex interact with a small contact area and that the b-subunit spans the membrane without contacting the c(8)-ring. The e- and g-subunits extend from the a-subunit density distal to the c(8)-ring. The map was calculated from images of a preparation of the enzyme solubilized with the detergent dodecyl maltoside, which is visible in electron cryomicroscopy maps. The structure shows that the micelle surrounding the complex is curved. The observed bend in the micelle of the detergent-solubilized complex is consistent with previous electron tomography experiments and suggests that monomers of ATP synthase are sufficient to produce curvature in lipid bilayers.

Inhibition of ATPase activity of Escherichia coli ATP synthase by polyphenols

We have studied the inhibitory effect of five polyphenols namely, resveratrol, piceatannol, quercetin, quercetrin, and quercetin-3-β- d glucoside on Escherichia coli ATP synthase. Recently published X-ray crystal structures of bovine mitochondrial ATP synthase inhibited by resveratrol, piceatannol, and quercetin, suggest that these compounds bind in a hydrophobic pocket between the γ-subunit C-terminal tip and the hydrophobic inside of the surrounding annulus in a region critical for rotation of the γ-subunit. Herein, we show that resveratrol, piceatannol, quercetin, quercetrin, or quercetin-3-β- d glucoside all inhibit E. coli ATP synthase but to different degrees. Whereas piceatannol inhibited ATPase essentially completely (∼0 residual activity), inhibition by other compounds was partial with ∼20% residual activity by quercetin, ∼50% residual activity by quercetin-3-β- d glucoside, and ∼60% residual activity by quercetrin or resveratrol. Piceatannol was the most potent inhibitor (IC 50 ∼14 μM) followed by quercetin (IC 50 ∼33 μM), quercetin-3-β- d glucoside (IC 50 ∼71 μM), resveratrol (IC 50 ∼94 μM), quercitrin (IC 50 ∼120 μM). Inhibition was identical in both F 1F o membrane preparations as well as in isolated purified F 1. In all cases inhibition was reversible. Interestingly, resveratrol and piceatannol inhibited both ATPase and ATP synthesis whereas quercetin, quercetrin or quercetin-3-β- d glucoside inhibited only ATPase activity and not ATP synthesis.

Cryo-EM Structure of the Yeast ATP Synthase

We have used electron cryomicroscopy of single particles to determine the structure of the ATP synthase from Saccharomyces cerevisiae. The resulting map at 24 Å resolution can accommodate atomic models of the F 1–c 10 subcomplex, the peripheral stalk subcomplex, and the N-terminal domain of the oligomycin sensitivity conferral protein. The map is similar to an earlier electron cryomicroscopy structure of bovine mitochondrial ATP synthase but with important differences. It resolves the internal structure of the membrane region of the complex, especially the membrane embedded subunits b, c, and a. Comparison of the yeast ATP synthase map, which lacks density from the dimer-specific subunits e and g, with a map of the bovine enzyme that included e and g indicates where these subunits are located in the intact complex. This new map has allowed construction of a model of subunit arrangement in the F O motor of ATP synthase that dictates how dimerization of the complex via subunits e and g might occur.

Angle determination for side views in single particle electron microscopy

In order to build a first model in single particle electron microscopy the relative angular orientation of each image of a protein complex must be determined. These orientations can be described by three Eulerian angles. Images of complexes that present the same view can be aligned in two-dimensions and averaged in order to increase their signal-to-noise ratio. Based on these averaged images, several standard approaches exist for determining Euler angles for randomly oriented projection images. The common lines and angular reconstitution methods work well for particles with symmetry while the random conical tilting and related orthogonal tilt reconstruction methods work in most cases but require the acquisition of tilt pairs of images. For the situation where views of particles can be identified that are rotations about a single axis parallel to the grid, an alternative algorithm to determine the orientations of class averages without the need to acquire tilt pairs can be applied. This type of view of a complex is usually called a side view. This paper describes the detailed workings and characterization of an algorithm, named rotational analysis, which uses real-space fiducial markers derived from the averages themselves to determine the Euler angles for side views. We demonstrate how this algorithm works in practice by applying it to a data set of images of affinity-purified bovine mitochondrial ATP synthase.

Identification of Two Proteins Associated with Mammalian ATP Synthase

Bovine mitochondrial ATP synthase commonly is isolated as a monomeric complex that contains 16 protein subunits and the natural IF(1) inhibitor protein in substoichiometric amounts. Alternatively ATP synthase can be isolated in dimeric and higher oligomeric states using digitonin for membrane solubilization and blue native or clear native electrophoresis for separation of the native mitochondrial complexes. Using blue native electrophoresis we could identify two ATP synthase-associated membrane proteins with masses smaller than 7 kDa and isoelectric points close to 10 that previously had been removed during purification. We show that in the mitochondrial membrane both proteins are almost quantitatively bound to ATP synthase. Both proteins had been identified earlier in a different context, but their association with ATP synthase was unknown. The first one had been named 6.8-kDa mitochondrial proteolipid because it can be isolated by chloroform/methanol extraction from mitochondrial membranes. The second one had been denoted as diabetes-associated protein in insulin-sensitive tissue (DAPIT), which may provide a clue for further functional and clinical investigations.

Lysine 43 Is Trimethylated in Subunit c from Bovine Mitochondrial ATP Synthase and in Storage Bodies Associated with Batten Disease

The hydrophobic membrane protein, subunit c, has been isolated from ATP synthase purified from bovine heart mitochondria. It has also been obtained from lysosomal storage bodies associated with ceroid lipofuscinosis from ovine liver and from human brain tissue of a victim of Batten disease. It is likely that the lysosomal protein has originated from the mitochondrion. These samples have been characterized by mass spectrometric methods. Irrespective of its source, subunit c has an intact molecular mass of 7650 Da, 42 Da greater than the value calculated from the amino acid sequence, and the protein has been modified post-translationally. In all three samples, the modification is associated with lysine 43, which lies in a polar loop region linking the two transmembrane alpha-helices of the protein. This residue is conserved throughout vertebrate sequences. The additional mass arises from trimethylation and not acetylation at the epsilon-N-position of the residue. These experiments show that the post-translational modification of subunit c is not, as has been suggested, an abnormal phenomenon associated with the etiology of Batten disease and ceroid lipofucinoses. Evidently, it occurs either before or during import of the protein into mitochondria or at a mitochondrial location after completion of the import process. The function of the trimethyllysine residue in the assembled ATP synthase complex is obscure. The residue and the modification are not conserved in all ATP synthases, and their role in the assembly and (or) functioning of the enzyme appear to be confined to higher organisms.

Cross-linking of the endogenous inhibitor protein (IF1) with rotor (gamma, epsilon) and stator (alpha) subunits of the mitochondrial ATP synthase.

The location of the endogenous inhibitor protein (IF1) in the rotor/stator architecture of the bovine mitochondrial ATP synthase was studied by reversible cross-linking with dithiobis(succinimidylpropionate) in soluble F1I and intact F1F0I complexes of submitochondrial particles. Reducing two-dimensional electrophoresis, Western blotting, and fluorescent cysteine labeling showed formation of alpha-IF1, IF1-IF1, gamma-IF1, and epsilon-IF1 cross-linkages in soluble F1I and in native F1F0I complexes. Cross-linking blocked the release of IF1 from its inhibitory site and therefore the activation of F1I and F1F0I complexes in a dithiothreitol-sensitive process. These results show that the endogenous IF1 is at a distance < or = 12 angstroms to gamma and epsilon subunits of the central rotor of the native mitochondrial ATP synthase. This finding strongly suggests that, without excluding the classical assumption that IF1 inhibits conformational changes of the catalytic beta subunits, the inhibitory mechanism of IF1 may involve the interference with rotation of the central stalk.

Phosphate exchange and ATP synthesis by DMSO-pretreated purified bovine mitochondrial ATP synthase

Purified soluble bovine mitochondrial F1Fo-ATP synthase contained 2mol of ATP, 2mol of ADP and 6mol of Pi/mol. Incubation of this enzyme with 1mM [32P]Pi caused the exchange of 2mol of Pi/mol of F1Fo-ATP synthase. The labelled phosphates were not displaced by ATP. Transfer of F1Fo-ATP synthase to a buffer containing 30% (v/v) DMSO and 1mM [32P]Pi resulted in the loss of bound nucleotides with the retention of 1mol of ATP/mol of F1Fo-ATP synthase. Six molecules of [32P]Pi were incorporated by exchange with the existing bound phosphate. Removal of the DMSO by passage of the enzyme through a centrifuged column of Sephadex G-50 resulted in the exchange of one molecule of bound [32P]Pi into the bound ATP. Azide did not prevent this [32P]Pi ↔ ATP exchange reaction. The bound labelled ATP could be displaced from the enzyme by exogenous ATP. Addition of ADP to the DMSO-pretreated F1Fo-ATP synthase in the original DMSO-free buffer resulted in the formation of an additional molecule of bound ATP. It was concluded that following pretreatment with and subsequent removal of DMSO the F1Fo-ATP synthase contained one molecule of ATP at a catalytic site which was competent to carry out a phosphateŐATP exchange reaction using enzyme-bound inorganic radiolabelled phosphate. In the presence of ADP an additional molecule of labelled ATP was formed from enzyme-bound Pi at a second catalytic site. The bound phosphateŐATP exchange reaction is not readily accommodated by current mechanisms for the ATP synthase.

Phosphate exchange and ATP synthesis by DMSO-pretreated purified bovine mitochondrial ATP synthase.

Purified soluble bovine mitochondrial F(1)F(o)-ATP synthase contained 2 mol of ATP, 2 mol of ADP and 6 mol of P(i)/mol. Incubation of this enzyme with 1 mM [(32)P]P(i) caused the exchange of 2 mol of P(i)/mol of F(1)F(o)-ATP synthase. The labelled phosphates were not displaced by ATP. Transfer of F(1)F(o)-ATP synthase to a buffer containing 30% (v/v) DMSO and 1 mM [(32)P]P(i) resulted in the loss of bound nucleotides with the retention of 1 mol of ATP/mol of F(1)F(o)-ATP synthase. Six molecules of [(32)P]P(i) were incorporated by exchange with the existing bound phosphate. Removal of the DMSO by passage of the enzyme through a centrifuged column of Sephadex G-50 resulted in the exchange of one molecule of bound [(32)P]P(i) into the bound ATP. Azide did not prevent this [(32)P]P(i)<-->ATP exchange reaction. The bound labelled ATP could be displaced from the enzyme by exogenous ATP. Addition of ADP to the DMSO-pretreated F(1)F(o)-ATP synthase in the original DMSO-free buffer resulted in the formation of an additional molecule of bound ATP. It was concluded that following pretreatment with and subsequent removal of DMSO the F(1)F(o)-ATP synthase contained one molecule of ATP at a catalytic site which was competent to carry out a phosphate-ATP exchange reaction using enzyme-bound inorganic radiolabelled phosphate. In the presence of ADP an additional molecule of labelled ATP was formed from enzyme-bound P(i) at a second catalytic site. The bound phosphate-ATP exchange reaction is not readily accommodated by current mechanisms for the ATP synthase.

F1 and F0 connections in the bovine mitochondrial ATP synthase: the role of the of alpha subunit N-terminus, oligomycin-sensitivity conferring protein (OCSP) and subunit d.

We have studied the functional effect of limited proteolysis by trypsin of the constituent subunits in the native and reconstituted F1F0 complex and isolated F1 of the bovine heart mitochondrial ATP synthase (EC 3.6.1.34). Chemical cross-linking of oligomycin-sensitivity conferring protein (OSCP) with other subunits of the ATP synthase and the consequent functional effects were also investigated. The results obtained show that the alpha subunit N-terminus is essential for the correct, functional connection of F1 to F0. The alpha-subunit N-terminus contacts OSCP which, in turn, contacts the F0I-PVP(b) and the F0-d subunits. The N-terminus of subunit alpha, OSCP, a segment of subunit d and the C-terminal and central region of F0I-PVP(b) subunits are peripherally located with respect to subunits gamma and delta which are completely shielded in the F1F0 complex against trypsin digestion. This qualifies the N-terminus of subunit alpha, OSCP, subunit d and F0I-PVP(b) as components of the lateral element of the stalk. These subunits, rather than being confined at one side of the complex which would leave most of the central part of the gamma subunit uncovered, surround the gamma and the delta subunits located in the central stalk.

Acylation of monolysocardiolipin in rat heart

Cardiolipin is a major mitochondrial membrane glycerophospholipid in the mammalian heart. In this study, the ability of the isolated intact rat heart to remodel cardiolipin and the mitochondrial enzyme activities that reacylate monolysocardiolipin to cardiolipin in vitro were characterized. Adult rat heart cardiolipin was found to contain primarily linoleic and oleic acids. Perfusion of the isolated intact rat heart in the Langendorff mode with various radioactive fatty acids, followed by analysis of radioactivity incorporated into cardiolipin and its immediate precursor phosphatidylglycerol, indicated that unsaturated fatty acids entered into cardiolipin mainly by deacylation followed by reacylation. The in vitro mitochondrial acylation of monolysocardiolipin to cardiolipin was coenzyme A-dependent with a pH optimum in the alkaline range. Significant activity was also present at physiological pH. With oleoyl-coenzyme A as substrate, the apparent K(m) for oleoyl-coenzyme A and monolysocardiolipin were 12.5 microm and 138.9 microm, respectively. With linoleoyl-coenzyme A as substrate, the apparent K(m) for linoleoyl-coenzyme A and monolysocardiolipin were 6.7 microm and 59.9 microm, respectively. Pre-incubation at 50 degrees C resulted in different profiles of enzyme inactivation for the two activities. Both activities were affected similarly by phospholipids, triacsin C, and various lipid binding proteins but were affected differently by various detergents and myristoyl-coenzyme A. [(3)H]cardiolipin was not formed from monolyso[(3)H]cardiolipin in the absence of acyl-coenzyme A. Monolysocardiolipin acyltransferase activities were observed in mitochondria prepared from various other rat tissues. In summary, the data suggest that the isolated intact rat heart has the ability to rapidly remodel cardiolipin and that rat heart mitochondria contain coenzyme A-dependent acyltransferase(s) for the acylation of monolysocardiolipin to cardiolipin. A simple and reproducible in vitro assay for the determination of acyl-coenzyme A- dependent monolysocardiolipin acyltransferase activity in mammalian tissues with exogenous monolysocardiolipin substrate is also presented.

Oligomycin sensitivity conferring protein (OSCP) of bovine heart mitochondrial ATP synthase: high-affinity OSCP-Fo interactions require a local alpha-helix at the C-terminal end of the subunit.

Earlier studies on oligomycin sensitivity conferring protein (OSCP) of bovine mitochondrial ATP synthase (F1Fo) indicated that a deletion mutant form (CD-10), lacking the last 10 amino acid residues (K181-L190), was unable to bind to the Fo segment, or reconstitute energy-linked reactions in OSCP-depleted F1Fo complexes [Joshi et al. (1996) Biochemistry 35, 12094-12103]. So far as known, the K181-L190 region of all mammalian species of OSCP harbors four charged residues at positions 181, 184, 187, and 188, while secondary structure predictions suggest that the K178-M186 region has a high propensity to form a helix [Ovchinnikov et al. (1984) FEBS Lett. 166, 19-22; Higuti et al. (1993) Biochim. Biophys. Acta 1172, 311-314; Grinkevich et al. ( 1994) Biol. Membr. 11, 310-323; Engelbrecht et al. (1991) Z. Naturforsch., C: Biochem., Biophys., Biol.,Virol. 46, 759-764]. Present studies were undertaken to clarify the role of individual amino acids in the K181-L190 region in OSCP-stimulated energy coupling. Our data show that simultaneous replacements of all four charged residues by uncharged but polar glutamines, or of K181-R184 by apolar alanines, had no significant influence either on the total alpha-helix content of the mutant forms or on the ability of mutant OSCPs to couple energy-linked reactions. However, a substitution of the K181-M186 region by six proline residues led to complete loss in the coupling activity of the resultant mutant. A detailed analysis of the 6-proline mutant form revealed that the variant was indistinguishable from WT OSCP with respect to expression characteristics, affinity for S-Sepharose, and ability to interact with F1, but was unable to complex with the Fo segment. These studies suggest that the global protein structure was not destabilized. The helix potential prediction analyses showed that the 6-proline OSCP displayed a marked decrease in the helix-forming propensity in the region corresponding to residues 175-190. Quantitative CD analyses to measure helical content demonstrated that both of the mutant forms 6-proline-OSCP and CD-10 had a somewhat lower alpha-helical content compared to WT protein, while synthetic peptides corresponding in sequence to the K178-L190 region displayed a high propensity to form a helix. Taken together, these results suggest that the C-terminal end of OSCP encompasses an alpha-helix which is crucial for high-affinity interactions of the C-terminal end of this subunit with Fo in the F1Fo enzyme.

The bound adenine nucleotides of purified bovine mitochondrial ATP synthase.

The experiments in this study were directed towards defining the nucleotide content of purified beef-heart mitochondrial F1F0 ATP synthase during binding and hydrolysis of ATP. The purified, soluble synthase as prepared contained 2 mol ATP and 2 mol ADP/mol enzyme. Three of these four nucleotides were exchangeable on incubation with radiolabelled MgATP. Passage of the ATP synthase through a column of Sephadex G-50 readily removed 1 mol ADP/mol. The remaining bound nucleotides were not displaced by incubation with 1 mM GTP or 5 mM sodium sulfite, the latter an activator of the ATPase activity of the synthase. Incubation of the synthase with 250 microM MgATP in the presence of 3 mM sodium azide, an inhibitor of the ATPase, resulted in the transitory formation of a form of the enzyme in which 5-6 nucleotide-binding sites were loaded with ATP and/or ADP, thus showing that the ATP synthase, like the soluble F1 ATPase, contained a minimum of six nucleotide-binding sites. The presence of an ATP-regenerating system during incubation with MgATP resulted in the loading of 5-6 sites to yield a form of the enzyme containing 3-4 mol ATP and 2 mol ADP/mol synthase even after passage through a centrifuged column. Following hydrolysis of the medium MgATP, the enzyme reached a stable form containing 2 mol ATP and 2 mol ADP/mol synthase. Like the form of the enzyme originally prepared, 1 mol ADP/mol synthase was readily released. However, this ADP remained bound to the synthase in the presence of GTP if azide was present. These results are discussed in the context of current ideas about nucleotide-binding sites on the F1 ATPase portion of the F1F0 ATP synthase. It is concluded that the properties of the sites on the F1F0 synthase show some differences from those on the F1 ATPase.

Oligomycin sensitivity conferring protein of mitochondrial ATP synthase: deletions in the N-terminal end cause defects in interactions with F1, while deletions in the C-terminal end cause defects in interactions with F0.

The structure/function relationships of oligomycin sensitivity conferring protein (OSCP) of bovine mitochondrial ATP synthase were studied by nested deletion mutagenesis, followed by analyses of the resultant OSCPs for their ability to restore partial reactions of ATP synthesis in OSCP-depleted F1-F0 complexes. Our results indicate that, from the N-terminus of OSCP, up to 13 amino acid residues could be deleted without any effect on OSCP coupling activity. However, deletion of 16 or more residues led to a slow decline in the ability of resultant mutant forms to restore ATP synthesis. Compared to the wild-type form of OSCP, deletion mutant ND-28 (deletion of residues 1-28) is 50% as active in its ability to reconstitute ATP-Pi exchange activity. Detailed analyses of mutant ND-28 revealed that it was able to bind to the membrane segment (F0) of ATP synthase and restore oligomycin-sensitive ATPase activity in OSCP-depleted F1-F0 complexes. However, it did not bind to soluble segment F1, nor did it confer cold stability to either soluble F1 or reconstituted F1-F0 complex. On the other hand, studies on nested deletions on the C-terminal end indicate that three residues could be deleted without compromising the energy-coupling activity of OSCP. However, truncations of five or more residues caused an impairment in the ability of resultant mutant forms to restore ATP-Pi exchange activity in OSCP-depleted complexes. Mutant CD-10 (deletion of amino acids 181-190) was completely ineffective as a coupling factor. Detailed analyses of this mutant revealed that the subunit was able to bind to soluble F1 segment and confer cold stability to the enzyme but was neither able to associate with the membrane segment (F0) nor able to reconstitute high oligomycin sensitivity in depleted F1-F0 complexes. We take these data to suggest that the N-terminal end of OSCP corresponding to residues G16-N28 is essential for binding of the coupling factor to soluble F1 but not for coupling the energy of proton translocation to the synthesis of ATP; on the other hand, the carboxyl-terminal end of OSCP containing amino acids K181-M186 is important for F0-OSCP interactions as well as for the coupling of the energy of delta microH+ during the synthesis of ATP. These results suggest a model for OSCP in which the N-terminus is associated with the F1 segment and the C-terminus is associated with the F0 segment, while the central part of the polypeptide forms three or more helices constituting the stalk in the intact F1F0 enzyme.

ATPF1 binding site, a positive cis-acting regulatory element of the mammalian ATP synthase alpha-subunit gene.

An analysis of the promoter of a bovine and human nuclear-encoded mitochondrial ATP synthase alpha-subunit gene (ATPA) revealed the presence of a positive control element. DNase I footprinting and electrophoretic mobility shift assays demonstrated that this cis-acting regulatory element contains a binding site for a protein present in human HeLa nuclei, termed ATP factor 1 (ATPF1). The ATPF1 binding site contains the sequence, CANNTG, a sequence identical to the recognition site for a family of transcription factors containing a basic region adjacent to a helix-loop-helix domain. Site-directed point mutations of the basic helix-loop-helix binding site demonstrated the critical role of this element for both binding of ATPF1 and also for transcriptional activation of the ATPA gene.

Structural organization of a nuclear gene for the α-subunit of the bovine mitochondrial ATP synthase complex

The structural organization of an expressed bovine gene ( ATPA1) that encodes an isoform of the α-subunit of the mitochondrial F 0F 1 ATP synthase was determined. The gene extends over 10 kilobasepairs and is divided into 12 exons. The first exon encodes the 5′ untranslated region and approximately one-half of the presequence that targets this protein to the mitochondrion. The remainder of the presequence, together with three amino acids of the mature protein, are encoded by exon 2. Primer extension and nuclease protection analyses revealed multiple sites of transcription initiation. The 5′ flanking region of the ATPA1 gene can drive the transcription of a reporter gene in an orientation-dependent manner. This promoter region contains several sequence elements which might play an important role in regulating the expression of this gene, including possible TATA and CCAAT boxes, putative Sp1-binding sites, and sequences resembling AP-1, AP-2, AP-4 and cAMP-responsive elements. The ATPA1 gene also contains sequences homologous to several motifs that are shared among some nuclear genes encoding mitochondrial proteins. These include Mt1, Mt3, Mt4, a respiratory enhancer, and NRF-2 sites. Tissue-specific differences in the ATPA1 mRNA levels were observed with high levels found in skeletal muscle and heart, and lower levels in other tissues.

The F 0 subunits of bovine mitochondrial ATP synthase complex: Purification, antibody production, and interspecies cross-immunoreactivity

The known subunits of the membrane sector F 0 of the bovine mitochondrial ATP synthase complex are subunits b, d, 6, F 6, OSCP (oligomycin sensitivity-conferring protein), the DCCD (dicyclohexylcarbodiimide) binding proteolipid, and A6L. The first six subunits were purified from SMP or preparations of the ATP synthase complex, and monospecific antibodies were raised against each. The antisera were shown to be competent for immunoblotting, and each antiserum recognized a single polypeptide of the expected M r in preparations of the ATP synthase complex. Immunoblots utilizing antibodies to OSCP and subunits d and 6, which exhibit the same M r on dodecyl sulfate—polyacrylamide gels, showed clearly that these polypeptides are immunologically distinct. Immunological cross-reactivity was demonstrated between bovine, human, rat, Saccharomyces cerevisiae, Paracoccus denitrificans, and Escherichia coli for subunit 6; between bovine, human, and rat for subunits b, d, OSCP, and F 6; and between bovine and rat for the DCCD binding proteolipid. Anti-subunit 6 antiserum, before or after immunopurification against the ATP synthase complex, recognized a single polypeptide in the bovine ATP synthase complex and S. cerevisiae mitochondria, but two polypeptides of different M r in bovine SMP, human, and rat mitochondria, and Paracoccus and E. coli membranes.