Organisation of the yeast ATP synthase F(0):a study based on cysteine mutants, thiol modification and cross-linking reagents.

A topological study of the yeast ATP synthase membranous domain was undertaken by means of chemical modifications and cross-linking experiments on the wild-type complex and on mutated enzymes obtained by site-directed mutagenesis of genes encoding ATP synthase subunits. The modification by non-permeant maleimide reagents of the Cys-54 of mutated subunit 4 (subunit b), of the Cys-23 in the N-terminus of subunit 6 (subunit a) and of the Cys-91 in the C-terminus of mutated subunit f demonstrated their location in the mitochondrial intermembrane space. Near-neighbour relationships between subunits of the complex were demonstrated by means of homobifunctional and heterobifunctional reagents. Our data suggest interactions between the first transmembranous alpha-helix of subunit 6, the two hydrophobic segments of subunit 4 and the unique membrane-spanning segments of subunits i and f. The amino acid residue 174 of subunit 4 is close to both oscp and the beta-subunit, and the residue 209 is close to oscp. The dimerisation of subunit 4 in the membrane revealed that this component is located in the periphery of the enzyme and interacts with other ATP synthase complexes.

Evidence of a subunit 4 (subunit b) dimer in favor of the proximity of ATP synthase complexes in yeast inner mitochondrial membrane.

Yeast mitochondria having either the D54C or E55C mutations in subunit 4 (subunit b), which is a component of the ATP synthase stator, displayed a spontaneous disulfide bridge between two subunits 4. This dimer was not soluble upon Triton X-100 extraction either at concentrations which extract the yeast ATP synthase or at higher concentrations. Increasing detergent concentrations led to a lack of the oligomycin-sensitive ATPase activity, thus showing an uncoupling between the two sectors of the mutated enzymes due to the dissociation of the subunit 4 dimer from the mutant enzyme. There is only one subunit 4 (subunit b) per eukaryotic ATP synthase. As a consequence, the results are interpreted as the proximity of ATP synthase complexes within the inner mitochondrial membrane.

Direct protein sequencing of wheat mitochondrial ATP synthase subunit 9 confirms RNA editing in plants.

RNA editing, a process that results in the production of RNA molecules having a nucleotide sequence different from that of the initial DNA template, has been demonstrated in several organisms using different biochemical pathways. Very recently RNA editing was described in plant mitochondria following the discovery that the sequence of certain wheat and Oenothera cDNAs is different from the nucleotide sequence of the corresponding genes. The main conversion observed was C to U, leading to amino acid changes in the deduced protein sequence when these modifications occurred in an open reading frame. In this communication we show the first attempt to isolate and sequence a protein encoded by a plant mitochondrial gene. Subunit 9 of the wheat mitochondrial ATP synthase complex was purified to apparent homogeneity and the sequence of the first 32 amino acid residues was determined. We have observed that at position 7 leucine was obtained by protein sequencing, instead of the serine predicted from the previously determined genomic sequence. Also we found phenylalanine at position 28 instead of a leucine residue. Both amino acid conversions, UCA (serine) to UUA (leucine) and CUC (leucine) to UUC (phenylalanine), imply a C to U change. Thus our results seem to confirm, at the protein level, the RNA editing process in plant mitochondria.

Interactions with tRNA(Lys) induce important structural changes in human immunodeficiency virus reverse transcriptase.

Retroviral RNA-dependent DNA polymerase (reverse transcriptase or RT) uses the 3'OH end of a cellular tRNA as primer to initiate DNA synthesis. Previous work with avian retrovirus has shown that reverse transcriptase is implicated in the selection of cellular virion-encapsidated tRNAs and has shown that the primer tRNA is positioned on the primer binding site near the 5' end of the viral RNA. These mechanisms support the idea that the retroviral polymerase should form complexes with primer tRNA and the specific encapsidated ones. The genomic sequence of human immunodeficiency virus (HIV) allows the prediction that tRNA(Lys3) is the natural primer. In this article we show, using the mobility shift assay, that recombinant HIV reverse transcriptase is able to form a complex with bovine tRNA(Lys.) By fluorescence studies and alpha-chymotrypsin analysis we have observed a modification of the enzyme conformation when reverse transcriptase is bound to the putative primer tRNA. This structural change is specific for tRNA(Lys) although the retroviral polymerase is able to interact with other tRNAs.

Tryptophanyl-tRNA synthetase from beef pancreas. Spectroscopic analysis of the stoichiometry of formation of the enzyme-tryptophanyl-adenylate complex.

The dimeric enzyme tryptophanyl-tRNA synthetase from beef pancreas catalyses the stoichiometric formation of one mole of tryptophanyl-adenylate per subunit. This formation is associated with optical changes (absorbance, fluorescence, optical rotation) and is confirmed by analytical ultracentrifugation. An equal amplitude of the change is observed for each adenylation site at pH 8.0, 25 degrees C, regardless of the optical method used. The formation of two tryptophanyl adenylates per dimer corresponds to a molar absorbance change delta epsilon 291 = 12000 +/- 500 cm-1 M-1, to a fluorescence quenching of 24 per cent at 340 nm and to a variation in optical rotation of 6 per cent at 313 nm. The circular dichroic band of the adenosine moiety of ATP is strongly increased. The addition of sodium pyrophosphate to the tryptophanyl-adenylate-enzyme complex restores the absorbance and fluorescence amplitude observed prior to the addition of ATP to the enzyme. Magnesium ions are necessary to the reaction. A pertubation of the environment of both the protein and the substrates (tryptophan and ATP) have to be taken into account to explain the magnitude of the observed changes.

Ethidium bromide stimulation of DNA polymerase activity by stabilization of the primer-template duplex.

Plant DNA polymerases and E. coli DNA polymerase I, but not animal DNA polymerases or avian reverse transcriptase, are strongly stimulated by ethidium bromide (EtdBr) when TMP incorporation is followed using a short oligo dT primer at 37 degrees C. The effect is observed with a poly A template in the presence of Mg2+ or Mn2+ and of poly dA template only in the presence of magnesium ions. When a longer primer like poly dT is used, EdtBr inhibited wheat DNA polymerase C activity. This result prompted us to study the effect of the incubation temperature on the drug mediated stimulation. With oligo dT primer the stimulation by EdtBr is not observed at a temperature of incubation lower than 35 degrees C. It is shown that the Tm of poly A-dT12 is around 35 degrees C and that EdtBr will clearly increase this value. The stimulation is lost when the enzyme is preincubated with the primer alone whereas it is not affected when the enzyme is preincubated with the template.

Topography of the yeast ATP synthase F0 sector.

The interaction between the hydrophilic C-terminal part of subunit 4 (subunit b) and OSCP, which are two components of the connecting stalk of the yeast ATP synthase, was shown after reconstitution of the two over-expressed proteins and by the two-hybrid method. The organization of a part of the F0 sector was studied by the use of mutants containing cysteine residues in a loop connecting the two N-terminal postulated membrane-spanning segments. Labelling of the mutated subunits 4 by a maleimide fluorescent probe revealed that the sulfhydryl groups were modified upon incubation of intact mitochondria. In addition, non-permeant maleimide reagents labeled subunit 4D54C, thus showing a location of this residue in the intermembrane space. Cross-linking experiments revealed the proximity of subunits 4 and f. In addition, a disulfide bridge between subunit 4D54C and subunit 6 was evidenced, thus demonstrating near-neighbor relationships of the two subunits and a location of the N-terminal part of the mitochondrially-encoded subunit 6 in the intermembrane space.

Biochemical study of adduct synthesis between fibrin monomers and elastin.

Monomers of fibrin generated by thrombin from fibrinogen reacted with elastin to give a new addition product or adduct. Adduct formation resulted from a covalent bond between fibrin monomers and elastin. The kinetic studies of this reaction confirmed that the adduct was formed before fibrin precipitated to produce the clot. The reaction depended on elastin, fibrinogen and thrombin concentrations. When thrombin-induced and reptilase-induced fibrin were compared, it became obvious that fibrin monomers did intervene more commonly as Des AA-fibrin than as Des AA.BB-fibrin. The adduct synthesis was completely inhibited by 150 microM of the peptide Gly-Pro-Arg-Pro which was previously known to stabilize the fibrin monomers and consequently to inhibit the polymerization completely. It is shown that FXIII could intervene directly in the reaction where homological quality of elastin (human versus bovine) and purity of thrombin were other important factors.

ATP synthase of yeast mitochondria. Isolation of the subunit h and disruption of the ATP14 gene.

A new subunit of the yeast ATP synthase (termed subunit h) has been isolated. Amino acid composition and N-terminal sequencing were determined by chemical methods. These data were in agreement with the sequence of the hypothetical protein L8003.20 whose primary structure was deduced from DNA sequencing of the yeast chromosome XII. The amino acid sequence encoded by ATP14 gene is 32 amino acids longer than the mature protein, which contains 92 amino acids corresponding to a calculated mass of 10,408 Da. The protein is hydrophilic and acidic with a calculated pHi of 4.08. It is not apparently related to any subunit described in other ATP synthases. A null mutant was constructed. The mutation was recessive and the mutant strain was unable to grow on glycerol medium. A high percentage of rho- cells arose spontaneously. The mutant mitochondria had no detectable oligomycin-sensitive ATPase activity, but still contained ATPase activity with a catalytic sector dissociated from the membranous components. The mutant mitochondria did not contain subunit h, and the mitochondrially encoded hydrophobic subunit 6 was not present.

Substrate depletion analysis as an approach to the pre-steady-state anticooperative kinetics of aminoacyl adenylate formation by tryptophanyl-tRNA synthetase from beef pancreas.

The formation of tryptophanyl adenylate catalyzed by tryptophanyl-tRNA synthetase from beef pancreas has been studied by stopped-flow analysis under conditions where the concentration of one of the substrates was largely decreasing during the time course of the reaction. Under such conditions a nonlinear regression analysis of the formation of the adenylate (adenylate vs. time curve) at several initial tryptophan and enzyme concentrations gave an accurate determination of both binding constants of this substrate. The use of the jackknife procedure according to Cornish - Bowden & Wong [ Cornish - Bowden , A., & Wong , J.J. (1978) Biochem. J. 175, 969-976] gave the limit of confidence of these constants. This approach confirmed that tryptophanyl-tRNA synthetase presents a kinetic anticooperativity toward tryptophan in the activation reaction that closely parallels the anticooperativity found for tryptophan binding at equilibrium. Both sites are simultaneously forming the adenylate. The dissociation constants obtained under the present pre-steady-state conditions for tryptophan are KT1 = 1.6 +/- 0.5 microM and KT2 = 18.5 +/- 3.0 microM at pH 8.0, 25 degrees C. The rate constant kf of adenylate formation is identical for both active sites (kf = 42 +/- 5 s-1). The substrate depletion method presently used, linked to the jackknife procedure, proves to be particularly suitable for the determination of the kinetic constants and for the discrimination between different possible kinetic models of dimeric enzyme with high substrate affinity. In such a case this method is more reliable than the conventional method using substrate concentrations in high excess over that of the enzyme.

The inhibition of mitochondrial DNA polymerase gamma from animal cells by intercalating drugs.

DNA polymerase gamma from purified nuclei of EMT-6 cells (mice) seems to be identical to the mitochondrial DNA polymerase from the same source following several criteria. These two enzyme activities are strongly inhibited by ethidium bromide and acriflavin, while proflavin, acridine orange, daunomycin and chloroquine inhibition is less pronounced. In the case of DNA polymerases alpha and beta very little inhibition by ethidium bromide was observed. Intercalation of this dye in a poly dA-dT 12-18 template-primer was studied spectrophotometrically under conditions similar to those in the in vitro DNA polymerase assay. The polymerase assay. The inhibition by this drug of the mitochondrial DNA polymerase gamma activity was shown to be competitive at varying concentrations of TTP while the inhibition was of the non-competitive type at different concentrations of poly dA-dT 12-18. We conclude that the drug, most probably in the intercalated form, is able to interact with the active site (s) of mitochondrial DNA polymerase.

Magnesium enhances human pancreatic elastase digestion of 125I-labeled elastin.

The effect of some divalent cations, especially Mg++, on elastinolysis by porcine or human pancreatic elastase has been determined using 125Iodine-labeled elastin as substrate. Elastin degradation was significantly increased in the presence of 10(-3) M Mg++. If elastin was pre-incubated with 0.5 (w/v) Triton, there was a further increase in elastinolysis to 2.6 times the original rate.

Tryptophanamide formation by Escherichia coli tryptophanyl-tRNA synthetase.

When tryptophanyl-tRNA synthetase from Escherichia coli is allowed to react with L-tryptophan and ATP-Mg in the presence of inorganic pyrophosphatase, the fluorescence change of the reaction mixture reveals three or four sequential processes, depending on the buffer used. Quenched-flow and stopped-flow experiments show that the first two processes, which occur in the 0.001-1.0-s time scale, can be correlated to the formation of two moles of tryptophanyl-adenylate per mole of dimeric enzyme. These two processes are reversible by adding PPi, as seen in the fluorimeter. The third process leads to a reaction product that can no longer reform ATP after addition of PPi and that represents tryptophanyl-ATP ester, as demonstrated by thin-layer chromatography. This compound has been previously shown to be formed by tryptophanyl-tRNA synthetase from E. coli [K. H. Muench (1969) Biochemistry 8, 4872-4879]. Its formation is accompanied by a fluorescence decrease which reaches a minimum in about 30 min. The nature of the fourth process depends on the reaction conditions employed. In sodium bicarbonate or potassium phosphate buffer, the fourth process corresponds to the non-enzymatic hydrolysis of tryptophanyl-ATP ester. This spontaneous hydrolysis competes with formation of the ester and limits its concentration. Eventually, the progressive exhaustion of ATP brings the fluorescence intensity of the reaction mixture back to its initial value. In contrast, in ammonium bicarbonate buffer the previous third process is no longer visible, as evidenced by the absence of a fluorescence decrease beyond the fast initial quenching linked to the formation of tryptophanyl-adenylate. Instead, a fluorescence increase is observed. However, unlike the fourth process seen in sodium bicarbonate buffer, the fluorescence increase in ammonium bicarbonate is much larger than the initial fluorescence decrease linked to adenylate formation, the final fluorescence greatly surpassing the starting fluorescence signal. The reaction product of this process is tryptophanamide, as evidenced by high-performance liquid chromatography. Tryptophanamide formation is faster than that of tryptophanyl-ATP ester and is enzyme-catalyzed with a Km of 1 mM for ammonia and a rate constant of 5.7 min-1 at pH 8.3, 25 degrees C. The affinity of tryptophanamide for the protein is too weak to allow the formation of a significant concentration of enzyme-product complex. Tryptophanamide is therefore released in the reaction medium and its concentration reaches that of the limiting substrate.

Tryptophanyl adenylate formation by tryptophanyl-tRNA synthetase from Escherichia coli.

By gel filtration and titration on DEAE-cellulose filters we show that Escherichia coli tryptophanyl-tRNA synthetase forms tryptophanyl adenylate as an initial reaction product when the enzyme is mixed with ATP-Mg and tryptophan. This reaction precedes the synthesis of the tryptophanyl-ATP ester known to be formed by this enzyme. The stoichiometry of tryptophanyl adenylate synthesis is 2 mol per mole of dimeric enzyme. When this reaction is studied either by the stopped-flow method, by the fluorescence changes of the enzyme, or by radioactive ATP depletion, three successive chemical processes are identified. The first two processes correspond to the synthesis of the two adenylates, at very different rates. The rate constants of tryptophanyl adenylate synthesis are respectively 146 +/- 17 s-1 and 3.3 +/- 0.9 s-1. The third process is the synthesis of tryptophanyl-ATP, the rate constant of which is 0.025 s-1. The Michaelis constants for ATP and for tryptophan in the activation reaction are respectively 179 +/- 35 microM and 23.9 +/- 7.9 microM, for the fast site, and 116 +/- 45 microM and 3.7 +/- 2.2 microM, for the slow site. No synergy between ATP and tryptophan can be evidenced. The data are interpreted as showing positive cooperativity between the subunits associated with conformational changes evidenced by fluorometric methods. The pyrophosphorolysis of tryptophanyl adenylate presents a Michaelian behavior for both sites, and the rate constant of the reverse reaction is 360 +/- 10 s-1 with a binding constant of 196 +/- 12 microM for inorganic pyrophosphate (PPi).(ABSTRACT TRUNCATED AT 250 WORDS)

RNA editing of wheat mitochondrial ATP synthase subunit 9: direct protein and cDNA sequencing.

RNA editing of subunit 9 of the wheat mitochondrial ATP synthase has been studied by cDNA and protein sequence analysis. Most of the cDNA clones sequenced (95%) showed that editing by C-to-U transitions occurred at eight positions in the coding region. Consequently, 5 amino acids were changed in the protein when compared with the sequence predicted from the gene. Two edited codons gave no changes (silent editing). One of the C-to-U transitions generated a stop codon by modifying the arginine codon CGA to UGA. Thus, the protein produced is 6 amino acids shorter than that deduced from the genomic sequence. Minor forms of cDNA with partial or overedited sequences were also found. Protein sequence and amino acid composition analyses confirmed the results obtained by cDNA sequencing and showed that the major form of edited atp9 mRNA is translated.

The subunit f of mitochondrial yeast ATP synthase--characterization of the protein and disruption of the structural gene ATP17.

The subunit f of the yeast F1F0ATP synthase has been isolated from the purified enzyme. Amino acid composition, protein and peptide sequencing were performed. The data are in agreement with the sequence of the predicted product of the gene D9481.21 identified on the Saccharomyces cerevisiae chromosome IV. A 303-bp open reading frame encoding a 101-amino acid polypeptide is described. The deduced amino acid sequence from the ATP17 gene is 6 amino acids longer than the mature protein, which displays a molecular mass of 10567 Da. The protein is basic with a short hydrophobic segment located in the C-terminal part of the subunit. Subunit f remained associated with other F0 subunits upon sodium bromide treatment of the whole enzyme. A null mutant was constructed. The disrupted strain was unable to grow on glycerol medium and the mutation was recessive; rho- cells arose spontaneously. The null mutant mitochondria were devoid of oligomycin-sensitive ATPase, but still contained an active F1, while the subunits f, 6 and 8 were absent.

The in vitro inhibition of DNA polymerase alpha and avian reverse transcriptase by novobiocin.

Novobiocin inhibits animal DNA polymerase alpha and avian reverse transcriptase activities when these enzymes are assayed in vitro with activated DNA as template. Under the same conditions DNA polymerase beta and gamma are much less inhibited. DNA polymerase alpha and reverse transcriptase are inhibited by different mechanisms: in the case of the retroviral enzyme the effect of novobiocin is not overcome by dilution of the drug, while in the case of polymerase alpha the inhibition disappeared after novobiocin dilution. The inhibition of polymerase alpha by novobiocin is non-competitive with respect to the TTP precursor or activated DNA. The irreversible inactivation of reverse transcriptase by novobiocin leads to the loss of the enzyme affinity for primer tRNATrp. Moreover, novobiocin inhibits the partial unwinding of the 3' end of tRNATrp by reverse transcriptase.

Anticooperative binding of L-tryptophan to tryptophanyl-tRNA synthetase from beef pancreas. Study at equilibrium by dialysis and changes in spectroscopic properties.

Equilibrium dialysis and gel filtration studies show that tryptophanyl-tRNA synthetase from beef pancreas binds two molecules of L-tryptophan per dimer in an anticooperative way. The binding of tryptophan ellicits a series of spectroscopic changes in the protein as seen by absorbance, fluorescence and circular dichroism. The molar absorption change of the protein-tryptophan system upon formation of the complex is delta epsilon292 = 10 400 +/- 1000 M(-1) cm(-1) per dimer. Taking an initial symmetrical dimeric protein the two dissociation constants for tryptophan at pH 8, 25 degrees C are respectively K1 = 2.0 +/- 0.5 muM and K2 = 10 +/- 4 muM. They are respectively K1 = 1 +/- 0.25 muM and K2 = 20 +/- 8 muM if one considers a sequenced binding of the two tryptophan molecules. The dichroic band at 290 nm of the free protein disappears when tryptophan is bound. All observed changes are characteristic of tryptophan perturbation and none of tyrosine perturbation. They all exceed the effect that can be expected from the change in environment of the bound tryptophan molecules and modifications of the tertiary structure of the protein have to be taken into account to explain the observed spectroscopic data.

Subunit f of the yeast mitochondrial ATP synthase: topological and functional studies.

Modified versions of subunit f were produced by mutagenesis of the ATP17 gene of Saccharomyces cerevisiae. A version of subunit f devoid of the last 28 amino acid residues including the unique transmembranous domain complemented the oxidative phosphorylation of the null mutant. However, a two-fold decrease in the specific ATP synthase activity was measured and attributed to a decrease in the stability of the mutant ATP synthase complex as shown by the low oligomycin-sensitive ATPase activity at alkaline pH. The modification or not by nonpermeant maleimide reagents of cysteine residues introduced at the N and C termini of subunit f indicated a Nin-Cout orientation. From the C terminus of subunit f it was possible to cross-link subunit 4 (also called subunit b), which is another component of the F0 sector and which also displays a short hydrophilic segment exposed to the intermembrane space.