RESUMO
The Leigh syndrome is a severe neurological disorder that has been associated with mutations affecting the mitochondrial energy transducing system. One of these mutations, T9176G, has been localized in the mitochondrial ATP6 gene encoding the Atp6p (or a) subunit of the ATP synthase. This mutation converts a highly conserved leucine residue into arginine within a presumed trans-membrane alpha-helical segment, at position 217 of Atp6p. The T9176G mutation was previously shown to severely reduce the rate of mitochondrial ATP production in cultured human cells containing high loads of this mutation. However, the underlying mechanism responsible for the impaired ATP production is still unknown. To better understand how T9176G affects the ATP synthase, we have created and analyzed the properties of a yeast strain bearing an equivalent of this mutation. We show that incorporation of Atp6p within the ATP synthase was almost completely prevented in the modified yeast. Based on previous partial biochemical characterization of human T9176G cells, it is likely that this mutation similarly affects the human ATP synthase instead of causing a block in the rotary mechanism of this enzyme as it had been suggested. Interestingly, the T9176G yeast exhibits important anomalies in mitochondrial morphology, an observation which indicates that the pathogenicity of T9176G may not be limited to a bioenergetic deficiency.
Assuntos
Doença de Leigh/enzimologia , Mitocôndrias/ultraestrutura , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Mutação de Sentido Incorreto , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Linhagem Celular , Humanos , Doença de Leigh/genética , Mitocôndrias/enzimologia , Mitocôndrias/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The longstanding question of the presence of mitochondria-bound polysomes has been recently revisited using new approaches. Genome-wide analyses provided evidence that many genes are actually translated on mitochondria-bound polysomes and GFP-labeling techniques have shown that, in vivo, the 3'UTR sequence of these genes contains signals which can target hybrid RNA molecules to the proximity of mitochondria. Evolutionary conservation of some of these signals will be presented. Interestingly, class I mRNA which are translated on free polysomes and class II mRNA which are translated on mitochondria-bound polysomes have, mostly, eukaryotic and prokaryotic origins respectively. Using ATP2, a typical prokaryotic-derived gene, as a model for class II mRNA, we showed that its 3'UTR sequence is essential both for a correct addressing of mRNA to mitochondria proximity and to a proper production of functional ATP synthases. These different observations suggest that prokaryotic-derived genes are, like the contemporary mitochondrial genes, translated near mitochondrial membranes. In both cases this locus specific translation process might be connected to a correct complex assembly program and the cases of ATP synthase and cytochrome c oxidase complexes will be considered in this respect.
Assuntos
Mitocôndrias/fisiologia , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Regiões 3' não Traduzidas/química , Regiões 3' não Traduzidas/genética , Sequência de Bases , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Filogenia , Polirribossomos/metabolismo , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Transdução de Sinais/fisiologiaRESUMO
Autophagy, a highly regulated programme found in almost all eukaryotes, is mainly viewed as a catabolic process that degrades nonessential cellular components into molecular building blocks, subsequently available for biosynthesis at a lesser expense than de novo synthesis. Autophagy is largely known to be regulated by nutritional conditions. Here we show that, in yeast cells grown under nonstarving conditions, autophagy can be induced by mitochondrial dysfunction. Electron micrographs and biochemical studies show that an autophagic activity can result from impairing the mitochondrial electrochemical transmembrane potential. Furthermore, mitochondrial damage-induced autophagy results in the preferential degradation of impaired mitochondria (mitophagy), before leading to cell death. Mitophagy appears to rely on classical macroautophagy machinery while being independent of cellular ATP collapse. These results suggest that in this case, autophagy can be envisioned either as a process of mitochondrial quality control, or as an ultimate cellular response triggered when cells are overwhelmed with damaged mitochondria.
Assuntos
Trifosfato de Adenosina/metabolismo , Mitocôndrias/fisiologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Saccharomyces cerevisiae/fisiologia , Autofagia/fisiologia , Morte Celular/fisiologia , Potencial da Membrana Mitocondrial/fisiologia , Proteínas Mitocondriais/metabolismo , Mutação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Four totally conserved glycines are involved in the packing of the two cytochrome b hemes, b(L) and b(H), of the bc(1) complex. The conserved glycine 131 is involved in the packing of heme b(L) and is separated by only 3 A from this heme in the bc(1) complex structure. The cytochrome b respiratory deficient mutant G131S is affected in the assembly of the bc(1) complex. An intragenic suppressor mutation was obtained at position 260, in the ef loop, where a glycine was replaced by an alanine. This respiratory competent revertant exhibited a low bc(1) complex activity and was affected in the electron transfer at the Q(P) site. The k(min) for the substrate DBH(2) was diminished by an order of magnitude and EPR spectra showed a partially empty Q(P) site. However, the binding of the Q(P) site inhibitors stigmatellin and myxothiazol remained unchanged in the suppressor strain. Optical spectroscopy revealed that heme b(L) is red shifted by 0.8 nm and that the E(m) of heme b(L) was slightly increased (+20 mV) in the revertant strain as compared to wild type strain values. Addition of a methyl group at position 260 is thus sufficient to allow the assembly of the bc(1) complex and the insertion of heme b(L) despite the presence of the serine at position 131. Surprisingly, reversion at position 260 was located 13 A away from the original mutation and revealed a long distance interaction in the yeast bc(1) complex.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/genética , Saccharomyces cerevisiae/genética , Sítios de Ligação , Catálise , Grupo dos Citocromos b/química , Grupo dos Citocromos b/genética , Citocromos c1/química , Espectroscopia de Ressonância de Spin Eletrônica , Modelos Moleculares , Mutação , Oxirredução , Oxirredutases/metabolismo , Potenciometria , Espectrofotometria , Supressão GenéticaRESUMO
We have identified a yeast nuclear gene (FMC1) that is required at elevated temperatures (37 degrees C) for the formation/stability of the F(1) sector of the mitochondrial ATP synthase. Western blot analysis showed that Fmc1p is a soluble protein located in the mitochondrial matrix. At elevated temperatures in yeast cells lacking Fmc1p, the alpha-F(1) and beta-F(1) proteins are synthesized, transported, and processed to their mature size. However, instead of being incorporated into a functional F(1) oligomer, they form large aggregates in the mitochondrial matrix. Identical perturbations were reported previously for yeast cells lacking either Atp12p or Atp11p, two specific assembly factors of the F(1) sector (Ackerman, S. H., and Tzagoloff, A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4986--4990), and we show that the absence of Fmc1p can be efficiently compensated for by increasing the expression of Atp12p. However, unlike Atp12p and Atp11p, Fmc1p is not required in normal growth conditions (28--30 degrees C). We propose that Fmc1p is required for the proper folding/stability or functioning of Atp12p in heat stress conditions.
Assuntos
Chaperoninas , Genes Fúngicos , Temperatura Alta , Mitocôndrias/enzimologia , ATPases Mitocondriais Próton-Translocadoras , Chaperonas Moleculares , ATPases Translocadoras de Prótons/química , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe , Proteínas Fúngicas/fisiologia , Proteínas Mitocondriais , Consumo de Oxigênio , Dobramento de Proteína , ATPases Translocadoras de Prótons/fisiologia , Saccharomyces cerevisiae/enzimologiaRESUMO
Saccharomyces cerevisiae homologues of phosphotyrosyl phosphatase activator (PTPA) are encoded byRRD1 and RRD2, genes whose combined deletion is synthetic lethal. Previously we have shown that the lethality of rrd1,2delta can be suppressed by increasing the osmolarity of the medium. Here we show that the lethality of rrd1,2delta is also suppressed under oxygen-limited conditions. The absence of respiration per se is not responsible for the suppression since elimination of the mitochondrial genome or a block in heme biosynthesis fail to rescue the rrd1,2delta double mutation.
Assuntos
Saccharomyces cerevisiae/metabolismo , Cafeína/farmacologia , Diploide , Ativação Enzimática , Deleção de Genes , Heme/biossíntese , Mitocôndrias/metabolismo , Mutação , Oxigênio/metabolismo , Fenótipo , Plasmídeos/metabolismo , Sirolimo/farmacologia , TemperaturaRESUMO
In the context of the cooperative project for functional analysis of novel genes uncovered during the systematic sequencing of the Saccharomyces cerevisiae genome, we deleted two paralogous ORFs: YIL153w and YPL152w. Based on the resulting phenotypes, the corresponding genes were named RRD1 and RRD2, respectively. Rrd proteins show significant similarity to the human phosphotyrosyl phosphatase activator (PTPA). Both single mutants, rrd1delta and rrd2delta, were viable. Deletion of RRD1 caused pleiotropic phenotypes under a wide range of conditions, including sensitivity to Ca2+, vanadate, ketoconazole, cycloheximide and Calcofluor white, and resistance to caffeine and rapamycin. The only phenotypes found for rrd2delta - resistance to caffeine and rapamycin - were weaker than the corresponding phenotypes of rrd1delta. The double mutant rrd1,2delta was inviable on rich glucose medium, but could grow in the presence of an osmotic stabilizer. The rrd1,2delta mutant was partially rescued by inactivation of HOG1 or PBS2, suggesting an interaction between the RRD genes and the Hog1p signal transduction pathway. Introduction of slt2delta into the rrd1,2delta background improved the growth of rrd1,2delta on sorbitol-containing medium, indicating that the Rrd proteins also interact with the Slt2p/Mpk1p signaling pathway. Suppression of the lethal phenotype of the rrd1,2delta mutant by overexpression of PPH22 suggested that the products of the RRD genes function positively with catalytic subunits of PP2A. The synthetic lethality was also suppressed by the "viable" allele (SSD1-v1) of the SSD1 gene.
Assuntos
Genes Fúngicos , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Alelos , Meios de Cultura , Ativação Enzimática/genética , Deleção de Genes , Glucose , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Mutação , Concentração Osmolar , Consumo de Oxigênio , Peptidilprolil Isomerase , Fenótipo , Proteínas/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transdução de Sinais , SorbitolRESUMO
In the framework of the B1 Consortium of the EUROFAN-1 project, we set up a series of simple phenotypic tests that can be performed on a large number of strains at a time. This methodological approach was intended to help assign functions of putative genes coding for unknown proteins to several specific aspects of cell biology. The tests were chosen to study phenotypes which should be affected by numerous genes. In this report, we examined the sensitivity/resistance or the adaptation of the cell to physical or chemical stresses (thermotolerance, osmotolerance and ethanol sensitivity), the effects of the alteration of the level of protein phosphorylation (sensitivity or resistance to compounds affecting the activity of protein kinases or phosphatases) and the effects of compounds interfering with synthesis of nucleic acids or proteins. Deletions in 66 genes of unknown function have been tested in 21 different conditions. In many deletant strains, phenotypes were observed and, for the most promising candidates, tetrad analysis was performed in order to verify co-segregation of the deletion marker with the phenotype.
Assuntos
Adaptação Fisiológica , Proteínas Fúngicas/fisiologia , Genes Fúngicos/fisiologia , Saccharomyces cerevisiae/genética , Adaptação Fisiológica/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Etanol/farmacologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos/genética , Ligação Genética/genética , Marcadores Genéticos/genética , Temperatura Alta , Mutação/efeitos dos fármacos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Concentração Osmolar , Fenótipo , Fosforilação/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/fisiologia , Deleção de SequênciaRESUMO
A yeast mutant (cor2-45) in which approximately half of the C terminus of core protein 2 of the cytochrome bc1 complex is lacking due to a frameshift mutation that introduces a stop at codon 197 in the COR2 gene fails to assemble the cytochrome bc1 complex and does not grow on non-fermentable carbon sources that require respiration. The loss of respiration is more severe with this frameshift mutation than with the complete deletion of the COR2 gene, suggesting deleterious effects of the truncated core 2 protein. A search for extragenic suppressors of the nuclear cor2-45 mutation resulted (in addition to the expected nuclear suppressors) in the isolation of a suppressor mutation in the mitochondrial DNA that replaces serine 223 by proline in cytochrome b. Assembly of the cytochrome bc1 complex and the respiratory deficient phenotype of the cor2-45 mutant are restored by the proline for serine replacement in cytochrome b. Surprisingly, this amino acid replacement in cytochrome b corrects not only the phenotype resulting from the cor2-45 frameshift mutation, but it also obviates the need for core protein 2 in the cytochrome bc1 complex since it alleviates the respiratory deficiency resulting from the complete deletion of the COR2 gene. This is the first report of a homoplasmic missense point mutation of the mitochondrial DNA acting as a functional suppressor of a mutation located in a nuclear gene and the first demonstration that the supernumerary core protein 2 subunit is not essential for the electron transfer and energy transducing functions of the mitochondrial cytochrome bc1 complex.
Assuntos
Grupo dos Citocromos b/genética , Complexo III da Cadeia de Transporte de Elétrons/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Mutação da Fase de Leitura , Dados de Sequência Molecular , Saccharomyces cerevisiae/metabolismoRESUMO
In 1993, a pilot project for the functional analysis of newly discovered open reading frames, presumably coding for proteins, from yeast chromosome III was launched by the European Community. In the frame of this programme, we have developed a large-scale screening for the identification of gene/protein functions via systematic phenotypic analysis. To this end, some 80 haploid mutant yeast strains were constructed, each carrying a targeted deletion of a single gene obtained by HIS3 or TRP1 transplacement in the W303 background and a panel of some 100 growth conditions was established, ranging from growth substrates, stress to, predominantly, specific inhibitors and drugs acting on various cellular processes. Furthermore, co-segregation of the targeted deletion and the observed phenotype(s) in meiotic products has been verified. The experimental procedure, using microtiter plates for phenotypic analysis of yeast mutants, can be applied on a large scale, either on solid or in liquid media. Since the minimal working unit of one 96-well microtiter plate allows the simultaneous analysis of at least 60 mutant strains, hundreds of strains can be handled in parallel. The high number of monotropic and pleiotropic phenotypes (62%) obtained, together with the acquired practical experience, have shown this approach to be simple, inexpensive and reproducible. It provides a useful tool for the yeast community for the systematic search of biochemical and physiological functions of unknown genes accounting for about a half of the 6000 genes of the complete yeast genome.
Assuntos
Cromossomos Fúngicos , Fases de Leitura Aberta/fisiologia , Saccharomyces cerevisiae/genética , Deleção de Genes , Genes Fúngicos/fisiologia , Fenótipo , Projetos Piloto , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
A cDNA carrying the Rip1 gene, which encodes the Rieske iron-sulfur protein of Schizosaccharomyces pombe, has been cloned by complementing the respiratory deficiency of a Saccharomyces cerevisiae strain in which the endogenous copy of the RIP1 gene has been deleted. The deduced amino acid sequences of the S. pombe and S. cerevisiae iron-sulfur proteins are 50% identical, with the highest region of identity being in the C termini of the proteins, where the 2Fe:2S cluster is bound. When expressed in the S. cerevisiae deletion strain, the S. pombe iron-sulfur protein restores 25-30% of the ubiquinol-cytochrome c reductase activity. The kinetics of cytochrome c reduction, the effects of inhibitors which act at defined sites in the cytochrome bc1 complex, and the optical properties of cytochrome b in membranes from the S. cerevisiae deletion strain complemented with S. pombe iron-sulfur protein indicate that the S. pombe protein interacts with cytochrome b to restore an apparently normal ubiquinol oxidase site, but that interaction between the iron-sulfur protein and cytochrome c1 is partially impaired. This is the first heterologous replacement of an electron transfer protein in a respiratory enzyme complex in S. cerevisiae.
Assuntos
Proteínas Ferro-Enxofre/genética , Saccharomyces cerevisiae/enzimologia , Schizosaccharomyces/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Genes Fúngicos , Teste de Complementação Genética , Dados de Sequência Molecular , Oxirredução , Estrutura Secundária de Proteína , Schizosaccharomyces/enzimologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Análise EspectralRESUMO
In the frame of the European Pilot Project for the functional analysis of newly discovered open reading frames (ORFs) from Saccharomyces cerevisiae chromosome III, we have deleted entirely the YCR46C ORF by a one-step polymerase chain reaction method and replaced it by the HIS3 marker in the strain W303. The deletion has been checked by meiotic segregation and Southern blot analyses. Characterization of the deleted strain indicates that YCR46C is essential for respiration and maintenance of the mitochondrial genome since its deletion leads to the appearance of 100% of cytoplasmic petites. Hybridization with molecular probes from mtDNA of individual clones of such petites showed that about 50% did hybridize (rho- clones) while others did not (possibly rho degrees clones). The wild-type gene has been cloned and shown to complement the deletion. The gene, which probably codes for a mitochondrial ribosomal protein, has been called petCR46.
Assuntos
DNA Mitocondrial/genética , Fases de Leitura/genética , Saccharomyces cerevisiae/genética , Sequência de Bases , Southern Blotting , Genoma , Meiose , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Saccharomyces cerevisiae/citologia , Deleção de SequênciaRESUMO
Trp-142 is a highly conserved residue of the cytochrome b subunit in the bc1 complexes. To study the importance of this residue in the quinol oxidation catalyzed by the bc1 complex, we characterized four yeast mutants with arginine, lysine, threonine, and serine at position 142. The mutant W142R was isolated previously as a respiration-deficient mutant unable to grow on non-fermentable carbon sources (Lemesle-Meunier, D., Brivet-Chevillotte, P., di Rago, J.-P, Slonimski, P.P., Bruel, C., Tron, T., and Forget, N. (1993) J. Biol. Chem. 268, 15626-15632). The mutants W142K, W142T, and W142S were obtained here as respiration-sufficient revertants from mutant W142R. Mutant W142R exhibited a decreased complex II turnover both in the presence and absence of antimycin A; this suggests that the structural effect of W142R in the bc1 complex probably interferes with the correct assembly of the succinate-ubiquinone reductase complex. The mutations resulted in a parallel decrease in turnover number and apparent Km, with the result that there was no significant change in the second-order rate constant for ubiquinol oxidation. Mutants W142K and W142T exhibited some resistance toward myxothiazol, whereas mutant W142R showed increased sensitivity. The cytochrome cc1 reduction kinetics were found to be severely affected in mutants W142R, W142K, and W142T. The respiratory activities and the amounts of reduced cytochrome b measured during steady state suggest that the W142S mutation also modified the quinol-cytochrome c1 electron transfer pathway. The cytochrome b reduction kinetics through center P were affected when Trp-142 was replaced with arginine or lysine, but not when it was replaced with threonine or serine. Of the four amino acids tested at position 142, only arginine resulted in a decrease in cytochrome b reduction through center N. These findings are discussed in terms of the structure and function of the quinol oxidation site and seem to indicate that Trp-142 is not critical to the kinetic interaction of ubiquinol with the reductase, but plays an important role in the electron transfer reactions that intervene between ubiquinol oxidation and cytochrome c1 reduction.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Saccharomyces cerevisiae/enzimologia , Ubiquinona/análogos & derivados , Sequência de Aminoácidos , Sítios de Ligação , Evolução Biológica , Transporte de Elétrons , Complexo III da Cadeia de Transporte de Elétrons/química , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Mutação Puntual , Estrutura Secundária de Proteína , Análise Espectral , Relação Estrutura-Atividade , Triptofano , Ubiquinona/metabolismoRESUMO
The mutations C133-->Y133, L282-->F282 and G340-->E340 in yeast mitochondrial cytochrome b each lead to a dysfunction of the cytochrome bc1 complex and, consequently, to the absence of growth on non-fermentable substrates. We isolated and characterized, from these mutants, fourteen different intragenic pseudo-revertants of various respiratory sufficient phenotypes. Both first-site and second-site suppressor mutations were found. A novel type of suppressor mutation consisted of the three-base-pair deletion of the parental mutated codon (E340 delta). The results provide, for the first time, evidence for the transmembrane disposition of helices F and G of the current eight-helix cytochrome b model. These two helices are presumably in contact with helix C in the folded protein. A simple modelisation study suggests that the packing of helices C, F and G in cytochrome b may be similar to that of helices I, II and VII in bacteriorhodopsin, respectively. We observed from the study of second-site revertants that compensation across the membrane never occurs. For each revertant, the suppressor mutation and the corresponding target mutation are on the same side of the membrane. This membrane sidedness strengthens the topological constraints imposed by the Q-cycle, namely the necessity of spatial separation of two catalytic reaction sites for ubiquinone.
Assuntos
Grupo dos Citocromos b/química , Estrutura Secundária de Proteína , Supressão Genética , Leveduras/química , Sequência de Aminoácidos , Simulação por Computador , Grupo dos Citocromos b/genética , Genes Fúngicos/genética , Mitocôndrias/química , Modelos Moleculares , Dados de Sequência Molecular , Mutação Puntual/fisiologia , Dobramento de Proteína , Leveduras/genética , Leveduras/crescimento & desenvolvimentoRESUMO
Four modified cytochrome b's carrying mononucleotide substitutions affecting center N residues were analysed. The mutant carrying a G33D change does not incorporate heme into the apocytochrome b and fails to grow on non-fermentable carbon sources. Out of 85 genetically independent revertants derived from this mutant, 82 were true back-mutants restoring the wild type sequence (D33G). The remaining three replaced the aspartic acid by an alanine (D33A) indicating that small size residues are best tolerated at this position which is consistent with the perfect conservation of the G33 during evolution. This glycine may be of crucial importance for helix packing around the hemes. The replacement of methionine at position 221 by lysine (M221K) produced a non-functional cytochrome b [(1993) J. Biol. Chem. 268, 15626-15632]. Non-native revertants replacing the lysine 221 by glutamic acid (K221E) or glutamine (K221Q) expressed a selective resistance to antimycin and antimycin derivatives having a modified dilactone ring moiety. Cytochrome b residues in 33 and in 221 seemed to contribute to the quinone reduction (QN) site of the cytochrome bc1 complex. Possible intramolecular interactions between the N-terminal region and the loop connecting helices IV and V of cytochrome b are proposed.
Assuntos
Grupo dos Citocromos b/genética , Mutação , Saccharomyces cerevisiae/genética , Antimicina A/análogos & derivados , Antimicina A/farmacologia , Sequência de Bases , Mapeamento Cromossômico , Códon , Grupo dos Citocromos b/química , DNA Fúngico/química , DNA Mitocondrial/química , Resistência Microbiana a Medicamentos , Dados de Sequência Molecular , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
Yeast mutants modifying the C-terminal region of mitochondrial cytochrome b were isolated and characterized. A nonsense mutation of the leucine codon 335 (TTA-->TAA), 50 residues before the normal C-terminus, blocks incorporation of heme into the apocytochrome b and prevents growth on non-fermentable substrates. The same defects were observed in a frameshift mutant (after codon 348, TAT-->TATT) in which the last 37 C-terminal residues are predicted to be replaced by a novel sequence of 33 amino acids. Function was regained in the nonsense mutant only by true back mutations restoring a protein of the wild-type sequence. The respiratory capacity was restored to wild-type levels in the frameshift mutant by a variety of single base subtractions located within a window of 24 bases before or after the original +T addition, these pseudo-reversions resulted in single or multiple (up to five) consecutive amino acid replacements between positions 346 and 354 and restored the wild-type sequence from position 355 to 385. These data, combined with hydropathy calculations and sequence comparisons, suggest that the C-terminal domain of cytochrome b forms a transmembrane segment essential for the correct assembly of the cytochrome bc1 complex.
Assuntos
Grupo dos Citocromos b/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/enzimologia , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Códon , Eletroforese em Gel de Poliacrilamida , Leucina/genética , Dados de Sequência Molecular , Mutação , Fenótipo , Saccharomyces cerevisiae/genéticaRESUMO
We characterized six novel missense mutations in mitochondrial cytochrome b (C133Y, W142R, S206L, M221K, L282F, and G340E) which impair the respiratory growth of yeast and which have differential effects on the functioning and assembly of the bc1 complex. The mutations have been mapped genetically in exons of the mitochondrial gene coding for apocytochrome b and their nucleotide sequence established. The mutants help to better define the topographical and primary sequence location of the ubiquinol oxidase (center P) and ubiquinone reductase (center N) sites on cytochrome b. Two mutants (C133Y and S206L) resulted in an active assembled complex, with selective disturbances of heme 565 and heme 562, respectively, which is consistent with the assignment of the axial ligands of these hemes; the C133Y mutation induced myxothiazol resistance, whereas the S206L did not modify the antimycin binding site, although perturbing the center N. These two amino acid replacements, along with those described elsewhere (Tron, T., and Lemesle-Meunier, D. (1990) Curr. Genet. 18, 413-419), constitute a novel class of mutants exhibiting appreciable electron transfer activity, despite their impaired ability to grow on respiratory substrates, raising the possibility that these mutants carry alleles which result in "decoupling" of proton translocation from electron transfer. Mutants W142R and M221K had an inactive but well assembled bc1 complex, whereas the G34OE and L282F mutations impaired the assembly of the bc1 complex.
Assuntos
Grupo dos Citocromos b/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Mutação , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Grupo dos Citocromos b/química , Grupo dos Citocromos b/metabolismo , Análise Mutacional de DNA , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Dados de Sequência Molecular , Consumo de Oxigênio , Dobramento de Proteína , Saccharomyces cerevisiae/genética , Análise EspectralRESUMO
Revertants of nonfunctional cytochrome b mutants were isolated and characterized to determine how specific deleterious mutations in cytochrome b can be suppressed by secondary mutations not restoring a wild type protein. It was recently shown that the cytochrome b function can be recovered following various pseudo-wild type reversions at the level of the original site mutation or adjacent positions (di Rago, J.-P., Netter, P., and Slonimski, P. P. (1990) J. Biol. Chem. 265, 3332-3339). In the present study, we describe how the cytochrome b function can be recovered by secondary mutations in positions which are removed from the original mutation by up to more than 100 amino acids. Such revertant mutants are useful for the study of the three-dimensional structure of cytochrome b. The results of the analysis of four deficient mutations which affect a short region of the protein (positions 131-138 of the polypeptide chain) lead us to propose a possible mode of interactive combination between the first five putative transmembrane segments of cytochrome b within the membrane.
Assuntos
Grupo dos Citocromos b/genética , Íntrons , Mitocôndrias/metabolismo , Mutação , Saccharomyces cerevisiae/genética , Supressão Genética , Sequência de Aminoácidos , Sequência de Bases , Códon/genética , Grupo dos Citocromos b/metabolismo , Éxons , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Saccharomyces cerevisiae/metabolismoRESUMO
Funiculosin is a well-known inhibitor of the mitochondrial respiratory chain, probably acting at the ubiquinone reducing site or center i of QH2-cytochrome c reductase. We report here the isolation, mapping and RNA sequence analysis of yeast apo-cytochrome b mutants resistant to this inhibitor. Funiculosin-resistance was found to be conferred, in 4 independent isolates, upon replacement of a leucine residue by phenylalanine in position 198 of the cytochrome b polypeptide chain.
Assuntos
Antifúngicos/farmacologia , Grupo dos Citocromos b/genética , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Mitocôndrias/metabolismo , Mutação , RNA Fúngico/genética , Saccharomyces cerevisiae/genética , Alelos , Sequência de Aminoácidos , Animais , Antraquinonas/farmacologia , Resistência Microbiana a Medicamentos/genética , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Cinética , Mitocôndrias/ultraestrutura , Dados de Sequência Molecular , NAD/metabolismo , Oxirredução , Conformação Proteica , RNA Fúngico/isolamento & purificação , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
We have analyzed the structure/function relationships of the yeast mitochondrial cytochrome b with a new methodology based upon the isolation of pseudo-wild type revertants from well-characterized cytochrome b respiratory deficient mutants. Our goal was to determine how cytochrome b function could be restored in such mutants, at least to some degree, by suppressor mutations within the protein. True wild type revertants were differentiated from pseudo-wild type revertants by the use of a simple and rapid screening technique based upon oligonucleotide hybridization. This can easily be used to analyze a large number of revertants. The suppressor mutations responsible for the restoration of respiratory competence were identified by sequencing the revertant's cytochrome b mRNA in crude mitochondrial RNA preparations. Using this new method we have analyzed 210 independent revertants. We report here nine novel cytochrome b structures conferring a variety of respiratory sufficient phenotypes, obtained from five respiratory deficient mutations affecting a short region of the protein (positions 131-138 of the polypeptide chain), presumably belonging to the ubiquinol oxidizing center of the bc1 complex.