A simple method of purification of a soluble oligomycin-insensitive mitochondrial ATPase.

A simple method for the purification of the soluble oligomycin-insensitive mitochondrial ATPase from heart is described. It consists of adsorption of the of the enzyme to Sepharose hexylammonium followed by elution with KCl and a precipitation step with (NH₄)₂SO₄. In sodium dodec'yl sulfate gels, the enzyme shows the A, B, C, D, and E subunits; however, the D and E subunits appear only when the gels are loaded with a high concentration of protein. The K_m for Mg-ATP is approximately 0.6 mm and is inhibited by ADP.     

Aurovertin binds to the beta subunit of yeast mitochondrial ATPase.

Isolated beta subunit of ATPase (F1) from yeast mitochondria does not catalyze an ATPase reaction but still binds the specific F1 inhibitor aurovertin. Binding was measured by enhancement of aurovertin fluorescence; it was as tight as that to F1-ATPase. No binding was observed with F1 or with isolated beta subunit from a single-gene nuclear yeast mutant whose F1-ATPase was resistant to aurovertin.     

Biosynthetic pathway of mitochondrial ATPase subunit 9 in Neurospora crassa

Subunit 9 of mitochondrial ATPase (Su9) is synthesized in reticulocyte lysates programmed with Neurospora poly A-RNA, and in a Neurospora cell free system as a precursor with a higher apparent molecular weight than the mature protein (Mr 16,400 vs. 10,500). The RNA which directs the synthesis of Su9 precursor is associated with free polysomes. The precursor occurs as a high molecular weight aggregate in the postribosomal supernatant of reticulocyte lysates. Transfer in vitro of the precursor into isolated mitochondria is demonstrated. This process includes the correct proteolytic cleavage of the precursor to the mature form. After transfer, the protein acquires the following properties of the assembled subunit: it is resistant to added protease, it is soluble in chloroform/methanol, and it can be immunoprecipitated with antibodies to F1-ATPase. The precursor to Su9 is also detected in intact cells after pulse labeling. Processing in vivo takes place posttranslationally. It is inhibited by the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). A hypothetical mechanism is discussed for the intracellular transfer of Su9. It entails synthesis on free polysomes, release of the precursor into the cytosol, recognition by a receptor on the mitochondrial surface, and transfer into the inner mitochondrial membrane, which is accompanied by proteolytic cleavage and which depends on an electrical potential across the inner mitochondrial membrane.     

The mitochondrial ATPase. Selective modification of a nitrogen residue in the beta subunit.

1. When mitochondrial ATPase, which has been modified on a single tyrosine residue by 4-chloro-7-nitrobenzofurazan, is incubated at pH 9.0, the 7-nitrobenzofurazan group undergoes an intramolecular transfer to a nitrogen residue. The rate of this transfer is sensitive to the binding of adenine nucleotides to the enzyme. The resulting N-nitrobenzofurazan ATPase has little or no activity. 2. The fluorescence of the N-nitrobenzofurazan group in the modified ATPase is quenched on binding of ADP. 3. Electrophoresis of the modified enzyme in sodium dodecyl sulphate on a 10% polyacrylamide gel shows that the fluorescence of the N-nitrobenzofurazan chromophore is exclusively in the beta subunit. 4. The rate of transfer of the nitrobenzofurazan group from tyrosyl oxygen to nitrogen on the enzyme is compared with the rate of transfer between model compounds. 5. The interaction of the N-nitrobenzofurazan ATPase with aurovertin is reported.     

Human liver cDNA clones encoding proteolipid subunit 9 of the mitochondrial ATPase complex

Clones encoding the proteolipid subunit 9 of the mitochondrial ATPase complex have been isolated from a lambda gt10 library of human liver cDNA sequences, using a probe of Neurospora crassa cDNA encoding subunit 9. From nucleotide sequence analysis it is concluded that the amino acid sequence of mature human subunit 9 is identical to that of its bovine counterpart. By comparing the sequence of two cDNA clones (denoted human 1 and 2) to those of two bovine cDNA clones (denoted P1 and P2) recently described by Gay and Walker (EMBO J. 4, 3519-3524, 1985) it is evident that there are close sequence relationships between human 1 and bovine P1, and between human 2 and bovine P2, although both human clones are truncated at their 5'-ends. Thus, as in bovine cells there appears to be at least two human genes encoding subunit 9.     

The effect of altered membrane sterol composition on the temperature dependence of yeast mitochondrial ATPase

The sterol content of cells of $\text{Saccharomyces cerevisiae}$ was manipulated by growing the organism anaerobically in a medium containing excess supplements of unsaturated fatty acids and a range of supplements of ergosterol. Anaerobic mitochondrial precursor structures were isolated whose membrane lipids contain the same fatty acid composition but whose sterol content varies from 7 to 105 mg/g mitochondrial protein. Arrhenius plots of the mitochondrial ATPase activity of the different preparations show a discontinuity with Arrhenius activation energies of about +40 and +80 KJ/mole, respectively, above and below the transition temperature. However, the temperature of the transition is markedly dependent on sterol composition, and increases by up to 17° as the sterol content of the mitochondria is progressively decreased. These results support the concepts that membrane lipid composition influences the activity of membrane-bound enzymes, and that sterols promote the gel to liquid phase transition in biological membranes.     

The Saccharomyces cerevisiae ATP22 gene codes for the mitochondrial ATPase subunit 6-specific translation factor

Mutations in the Saccharomyces cerevisiae ATP22 gene were previously shown to block assembly of the F0 component of the mitochondrial proton-translocating ATPase. Further inquiries into the function of Atp22p have revealed that it is essential for translation of subunit 6 of the mitochondrial ATPase. The mutant phenotype can be partially rescued by the presence in the same cell of wild-type mitochondrial DNA and a rho- deletion genome in which the 5'-UTR, first exon, and first intron of COX1 are fused to the fourth codon of ATP6. The COX1/ATP6 gene is transcribed and processed to the mature mRNA by splicing of the COX1 intron from the precursor. The hybrid protein translated from the novel mRNA is proteolytically cleaved at the normal site between residues 10 and 11 of the subunit 6 precursor, causing the release of the polypeptide encoded by the COX1 exon. The ability of the rho- suppressor genome to express subunit 6 in an atp22 null mutant constitutes strong evidence that translation of subunit 6 depends on the interaction of Atp22p with the 5'-UTR of the ATP6 mRNA.     

The biogenesis of rat liver mitochondrial ATPase. Subunit composition of the normal ATPase complex and of the deficient complex formed when mitochondrial protein synthesis is blocked.

1. An ATPase complex containing 12 subunits was isoalted from rat liver mitochondria. 2. In vivo inhibition of mitochondrial protein synthesis by the chloramphenicol analogue thiamphenicol leads to the formation of an oligomycin-insensitive membrane-bound ATPase complex in mitochondria of regenerating rat liver. 3. This oligomycin-insensitive, membrane-bound ATPase was isolated by the same procedure as the ATPase complex from regenerating livers of untreated animals. 4. SDS-polyacrylamide gel electrophoresis of in vivo labelled ATPase complexes from control and from thiamphenicol-treated rats reveals that three subunits out of the 12 are not synthesized or assembled when the mitochondrial translation activity is blocked. 5. From the subunits synthesized and assembled when mitochondrial pror (Fo) of the ATPase complex (subunit 5). 6. The oligomycin sensitivity-conferring protein seems absent in the ATPase complex formed in the presence of thiamphenicol.     

Shotgun proteomics for the characterization of subunit composition of mitochondrial complex I

Here we propose shotgun proteomics as an alternative method to gel-based bottom-up proteomic platform for the structural characterization of mitochondrial NADH:ubiquinone oxidoreductase (complex I). The approach is based on simultaneous identification of subunits after global digestion of the intact complex. Resulting mixture of tryptic peptides is purified, concentrated, separated and online analyzed using nano-scale reverse-phase nano-ESI-MS/MS in a single information dependent acquisition mode. The usefulness of the method is demonstrated in our work on the well described model system of complex I from bovine heart mitochondria. The shotgun method led to the identification and partial sequence characterization of 42 subunits representing more than 95% coverage of the complex. In particular, almost all nuclear (except MLRQ) and 5 mitochondria DNA encoded subunits (except ND4L and ND6) were identified. Furthermore, it was possible to identify 30 co-purified proteins of the inner mitochondrial membrane structurally not belonging to complex I. The method's efficiency is shown by comparing it to two classical 1 D gel-based strategies. Shotgun proteomics is less laborious, significantly faster and requires less sample material with minimal treatment, facilitating the screening for post-translational modifications and quantitative and qualitative differences of complex I subunits in genetic disorders.     

Archaebacterial ATPase: studies on subunit composition and quaternary structure of the F1-analogous ATPase from Sulfolobus acidocaldarius

A modified procedure for the purification of soluble ATPase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius is described. In addition to (alpha) 65 and (beta) 51 kDa polypeptides, further subunits gamma * (20 kDa) and delta * (12 kDa) are demonstrated to be components of the enzyme, exhibiting a total molecular mass of 380 kDa. Molecular electron microscopic images of the native enzyme indicate a quaternary structure probably formed by the gamma *, delta *-complex as a central mass surrounded by a pseudohexagon of the peripherally arranged larger alpha and beta subunits. As can be derived from both molecular mass and electron microscopy data, the archaebacterial Sulfolobus-ATPase emerges to exist as an alpha 3 beta 3-quaternary structure with respect to the larger subunits. This is normally found in typical F1-ATPases of eubacterial and eukaryotic organisms. Therefore it is postulated that F1- and F0F1-ATPases, respectively, can occur ubiquitously in all urkingdoms of organisms as functional units of energy-transducing membranes.