Pierre-Vincent Graves

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.

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.

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.

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.

Isolation of Supernumerary Yeast ATP Synthase Subunits e and i CHARACTERIZATION OF SUBUNIT i AND DISRUPTION OF ITS STRUCTURAL GENE ATP18

Two subunits of the yeast ATP synthase have been isolated. Subunit e was found loosely associated to the complex. Triton X-100 at a 1% concentration removed this subunit from the ATP synthase. The N-terminal sequencing of subunit i has been performed. The data are in agreement with the sequence of the predicted product of a DNA fragment of Saccharomyces cerevisiae chromosome XIII. The ATP18 gene encodes subunit i, which is 59 amino acids long and corresponds to a calculated mass of 6687 Da. Its pI is 9.73. It is an amphiphilic protein having a hydrophobic N-terminal part and a hydrophilic C-terminal part. It is not apparently related to any subunit described in other ATP synthases. The null mutant showed low growth on nonfermentable medium. Mutant mitochondria display a low ADP/O ratio and a decrease with time in proton pumping after ATP addition. Subunit i is associated with the complex; it is not a structural component of the enzyme but rather is involved in the oxidative phosphorylations. Similar amounts of ATP synthase were measured for wild-type and null mutant mitochondria. Because 2-fold less specific ATPase activity was measured for the null mutant than for the wild-type mitochondria, we make the hypothesis that the observed decrease in the turnover of the mutant enzyme could be linked to a proton translocation defect through F0.

Organisation of the yeast ATP synthase F0: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.

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.

SUBUNIT F OF THE YEAST MITOCHONDRIAL ATP SYNTHASE : TOPOLOGICAL AND FUNCTIONAL STUDIES

Modified versions of subunit f were produced by mutagenesis of theATP17 gene of Saccharomyces cerevisiae. A version of subunit f devoid of thelast 28 amino acid residues including the unique transmembranous domaincomplemented the oxidative phosphorylation of the null mutant. However, atwo-fold decrease in the specific ATP synthase activity was measured andattributed to a decrease in the stability of the mutant ATP synthase complexas shown by the low oligomycin-sensitive ATPase activity at alkaline pH. Themodification or not by non-permeant maleimide reagents of cysteine residuesintroduced at the N and C termini of subunit f indicated aNin-Cout orientation. From the C terminus of subunit fit was possible to cross-link subunit 4 (also called subunit b), which isanother component of the F0 sector and which also displays a shorthydrophilic segment exposed to the intermembrane space.

Kinetics of formation of tryptophanyl-adenylate by tryptophanyl-tRNA synthetase from beef pancreas

The formation of an aminoacyl-adenylate has been shown to occur for most aminoacyl-tRNA synthetases as a first step in the tRNA aminoacylation reaction [ 1,2]. In the particular case of tryptophanyl-tRNA synthetase from beef pancreas a tryptophanyl-adenylate-enzyme complex has been evidenced by gel filtration [3] and spectroscopic changes [4]. Two moles of adenylate can be bound per mole of dimeric enzyme, though under some conditions it has been suggested that tryptophan can also be convalently linked to the protein in a 1: 1 ratio when the enzyme is obtained after fast purification procedure [5]. In the case of the non-covalent complex of the enzyme with L-tryptophan 2 mol tryptophan are bound in an apparent anti-cooperative way to the protein [6]. The formation of the adenylate-enzyme complex has not been studied under prestationary conditions which could show whether or not it is rate determining in the overall ATP-PP, isotope exchange and in the tRNA aminoacylation reactions. Since the formation of the adenylate-enzyme complex can be evidenced by spectroscopic changesof the enzyme [4], this reaction can be followed kinetically using the fluorescence changes of the system. This formation can also be studied by the depletion of [j’P]ATP according to [7] and by the measurement of the stoichiometry of appearance of [‘4C]tryptophanyl-adenylate [8] in order to ascertain that the recorded variations of the spectroscopic signal correspond really to the chemical step of carboxyl activation of tryptophan. This paper presents a preliminary report of the study of such a pre-stationary phase.

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.