RESUMO
Sequencing of the yeast genome has shown that about one-third of the yeast ORFs code for unknown proteins. Many other have similarity to known genes, but still the cellular functions of the gene products are unknown. The aim of the B1 Consortium of the EUROFAN project was to perform a qualitative phenotypic analysis on yeast strains deleted for functionally orphan genes. To this end we set up a simple approach to detect growth defects of a relatively large number of strains in the presence of osmolytes, ethanol, high temperature, inhibitory compounds or drugs affecting protein biosynthesis, phosphorylation level or nucleic acids biosynthesis. We have now developed this procedure to a semi-quantitative level, we have included new inhibitors, such as hygromycin B, benomyl, metals and additional drugs interfering with synthesis of nucleic acids, and we have performed phenotypic analysis on the deleted strains of 564 genes poorly characterized in respect to their cellular functions. About 30% of the deleted strains showed at least one phenotype: many of them were pleiotropic. For many gene deletions, the linkage between the deletion marker and the observed phenotype(s) was studied by tetrad analysis and their co-segregation was demonstrated. Co-segregation was found in about two-thirds of the analysed strains showing phenotype(s).
Assuntos
Genes Fúngicos/fisiologia , Genoma Fúngico , Saccharomyces cerevisiae/genética , Deleção de Genes , Ligação Genética/genética , Ligação Genética/fisiologia , Fases de Leitura Aberta/genética , Fases de Leitura Aberta/fisiologia , Fenótipo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/fisiologiaRESUMO
Previously we have shown that the Saccharomyces cerevisiae CCZ1 (YBR131w) gene encodes a protein involved in protein trafficking. Deletion of this gene leads to fragmentation of the vacuole typical of the class B vps mutants. Genetic and biochemical data indicated that Ccz1p is required for fusion of various transport intermediates with the vacuole. Here we present data indicating that CCZ1 is a close partner of the YPT7 gene, which encodes Rab GTPase and is required for fusion of transport vesicles to vacuole and homotypic vacuole fusion. We isolated extragenic suppressors of CCZ1 deletion. All these suppressors belong to one complementation group and correspond to mutated alleles of the YPT7 gene. The mutated residues are located in two Ypt7p domains responsible for guanine binding. These data suggest that Ccz1p and Ypt7p interact physically. Coimmunoprecipitation experiments provide direct evidence that this indeed is the case. A possible mechanism of Ccz1p action is discussed.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Proteínas de Transporte Vesicular , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Alanina/química , Alelos , Sequência de Aminoácidos , Ácido Aspártico/química , Transporte Biológico , Cafeína/farmacologia , Cálcio/metabolismo , Proteínas de Transporte/química , Estimulantes do Sistema Nervoso Central/farmacologia , DNA/metabolismo , Epitopos , Deleção de Genes , Biblioteca Gênica , Teste de Complementação Genética , Guanina/química , Hemaglutininas/química , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Fenótipo , Reação em Cadeia da Polimerase , Testes de Precipitina , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/química , Homologia de Sequência de Aminoácidos , Zinco/metabolismoRESUMO
We have introduced the concept of genomic 'style' of proteins. By style we understand those properties of a large set of proteins which are specific to the genome of one species (species primary-self) and different from the genome of another species (species contrasted-self). To characterise the style, we took advantage of the frequencies of amino acids and dipeptides present in non-identical segments of the complete set of orthologous ribosomal proteins encoded by 16 microbial species. We confirm the dependence of the overall amino acid composition on the genomic (G+C) content, and introduce a rectification procedure making it possible to extricate appropriate species-specific characteristics, which are no longer related to this content. The rectified frequencies are used to calculate inter-species distance matrices, and to build genomic evolutionary trees. Remarkably, the phylograms derived from the frequencies of non-identical residues in proteins closely resemble the classical phylograms based upon the conservation of identical residues in ribosomal RNAs. We believe that the concept of genomic style of proteins can be a useful tool for the study of evolution.
Assuntos
Evolução Molecular , Genoma Bacteriano , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Sequência de Aminoácidos , Aminoácidos/análise , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Composição de Bases , Biologia Computacional/métodos , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Dados de Sequência Molecular , Filogenia , Proteoma/química , Proteoma/genética , Especificidade da EspécieRESUMO
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
CCZ1 was previously identified by the sensitivity of ccz1(delta) mutants to high concentrations of Caffeine and the divalent ions Ca(2+ )and Zn(2+). In this paper we show that deletion of CCZ1 leads to aberrant vacuole morphology, similar to the one reported for the family of vacuolar protein sorting (vps) mutants of class B. The ccz1(&Dgr;) cells display severe vacuolar protein sorting defects for both the soluble carboxipeptidase Y and the membrane-bound alkaline phosphatase, which are delivered to the vacuole by distinct routes. Ccz1p is a membranous protein and the vast majority of Ccz1p resides in late endosomes. These results, along with a functional linkage found between the CCZ1 and YPT7 genes, indicate that the site of Ccz1p function is at the last step of fusion of multiple transport intermediates with the vacuole.
Assuntos
Proteínas de Transporte de Cátions , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Trocadores de Sódio-Hidrogênio , Vacúolos/metabolismo , Proteínas de Transporte Vesicular , Proteínas rab de Ligação ao GTP/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/genética , ATPases Transportadoras de Cálcio/metabolismo , Proteínas de Transporte/análise , Proteínas de Transporte/metabolismo , Compartimento Celular/fisiologia , Endocitose/fisiologia , Proteínas Fúngicas/análise , Deleção de Genes , Dosagem de Genes , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Chaperonas Moleculares , Mutagênese/fisiologia , Fenótipo , ATPases Transportadoras de Cálcio da Membrana Plasmática , Plasmídeos , Receptores de Superfície Celular/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Vacúolos/química , Zinco/metabolismo , Proteínas rab de Ligação ao GTP/genéticaRESUMO
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
By now, the EUROFAN programme for the functional analysis of genes from the yeast genome has attained its cruising speed. Indeed, several hundreds of yeast mutants with no phenotype as tested by growth on standard media and no significant sequence similarity to proteins of known function are available through the efforts of various laboratories. Based on the methodology initiated during the pilot project on yeast chromosome III (Yeast 13, 1547-1562, 1997) we adapted it to High Throughput Screening (HTS), using robotics. The first 100 different gene deletions from EUROSCARF, constructed in an FY1679 strain background, were run against a collection of about 300 inhibitors. Many of these inhibitors have not been reported until now to interfere in vivo with growth of Saccharomyces cerevisiae. In the present paper we provide a list of novel growth conditions and a compilation of 49 yeast deletants (from chromosomes II, IV, VII, X, XIV, XV) corresponding to 58% of the analysed genes, with at least one clear and stringent phenotype. The majority of these deletants are sensitive to one or two compounds (monotropic phenotype) while a distinct subclass of deletants displays a hyper-pleiotropic phenotype with sensitivities to a dozen or more compounds. Therefore, chemotyping of unknown genes with a large spectrum of drugs opens new vistas for a more in-depth functional analysis and a more precise definition of molecular targets.
Assuntos
Antifúngicos/farmacologia , Genoma Fúngico , Robótica , Saccharomyces/genética , Meios de Cultura , Resistência Microbiana a Medicamentos , Testes de Sensibilidade Microbiana , Mutação , Fenótipo , Saccharomyces/classificação , Saccharomyces/efeitos dos fármacos , Saccharomyces/fisiologiaRESUMO
A PCR-based method for targeted gene deletion by kanMX4 module was used to construct complete deletion mutants of six individual open reading frames from chromosome II: YBR128c, YBR131w, YBR133c, YBR137w, YBR138c and YBR142w. The ORFs were deleted in two diploid strains, FY1679 and W303. Sporulation and tetrad analysis revealed that only one ORF, YBR142w, encoding a putative DEAD-box RNA helicase, is an essential gene. A systematic phenotypic analysis of the deleted mutants was carried out. Homozygous diploids ybr128cDelta/ybr128cDelta and ybr131wDelta/ybr131wDelta did not sporulate. The ybr131cDelta mutant whether haploid or homozygous diploid, in addition displayed an increased sensitivity to Caffeine, Calcium and Zinc, and to emphasize this phenotype we named the gene CCZ1. ORF YBR133c was independently reported by others as Histone Synthetic Lethal (HSL7) (Ma et al., 1996). We found that the aberrant morphology characteristic for ybr133cDelta (hsl7Delta) cells was observed in W303 but not in FY1679 genetic background. Furthermore, we observed that deletion of YBR133c had a pleiotropic effect under a wide range of conditions, including increased sensitivity to calcium, caffeine, calcofluor white, vanadate and verapamil. The effects of the deletion were reinforced in W303 background. We found no phenotypic effects of the two remaining deletions, ybr137wDelta and ybr138cDelta.
Assuntos
Cromossomos Fúngicos/genética , Genes Fúngicos , Saccharomyces cerevisiae/genética , Antifúngicos/farmacologia , Cafeína/farmacologia , Cátions/farmacologia , Clonagem Molecular , Primers do DNA , Deleção de Genes , Genes Essenciais , Fenótipo , Reação em Cadeia da Polimerase , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/fisiologia , Esporos Fúngicos/fisiologia , Transformação Genética , Verapamil/farmacologiaRESUMO
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
MitBASE is an integrated and comprehensive database of mitochondrial DNA data which collects all available information from different organisms and from intraspecie variants and mutants. Research institutions from different countries are involved, each in charge of developing, collecting and annotating data for the organisms they are specialised in. The design of the actual structure of the database and its implementation in a user-friendly format are the care of the European Bioinformatics Institute. The database can be accessed on the Web at the following address: http://www.ebi.ac. uk/htbin/Mitbase/mitbase.pl. The impact of this project is intended for both basic and applied research. The study of mitochondrial genetic diseases and mitochondrial DNA intraspecie diversity are key topics in several biotechnological fields. The database has been funded within the EU Biotechnology programme.
Assuntos
DNA Mitocondrial/genética , Bases de Dados Factuais , Animais , Núcleo Celular/genética , Classificação , DNA Mitocondrial/classificação , Eucariotos/genética , Europa (Continente) , Fungos/genética , Código Genético , Doenças Genéticas Inatas/genética , Variação Genética , Humanos , Armazenamento e Recuperação da Informação , Internet , Invertebrados/genética , Mutação , Plantas/genética , Interface Usuário-Computador , Vertebrados/genéticaRESUMO
The Z-value is an attempt to estimate the statistical significance of a Smith-Waterman dynamic alignment score (SW-score) through the use of a Monte-Carlo process. It partly reduces the bias induced by the composition and length of the sequences. This paper is not a theoretical study on the distribution of SW-scores and Z-values. Rather, it presents a statistical analysis of Z-values on large datasets of protein sequences, leading to a law of probability that the experimental Z-values follow. First, we determine the relationships between the computed Z-value, an estimation of its variance and the number of randomizations in the Monte-Carlo process. Then, we illustrate that Z-values are less correlated to sequence lengths than SW-scores. Then we show that pairwise alignments, performed on 'quasi-real' sequences (i.e., randomly shuffled sequences of the same length and amino acid composition as the real ones) lead to Z-value distributions that statistically fit the extreme value distribution, more precisely the Gumbel distribution (global EVD, Extreme Value Distribution). However, for real protein sequences, we observe an over-representation of high Z-values. We determine first a cutoff value which separates these overestimated Z-values from those which follow the global EVD. We then show that the interesting part of the tail of distribution of Z-values can be approximated by another EVD (i.e., an EVD which differs from the global EVD) or by a Pareto law. This has been confirmed for all proteins analysed so far, whether extracted from individual genomes, or from the ensemble of five complete microbial genomes comprising altogether 16956 protein sequences.
Assuntos
Genoma Bacteriano , Genoma Fúngico , Alinhamento de Sequência , Metodologias Computacionais , Escherichia coli/genética , Matemática , Método de Monte Carlo , Saccharomyces cerevisiae/genéticaRESUMO
In the yeast Saccharomyces cerevisiae, the product of the nuclear gene CBP2 is required exclusively for the splicing of the terminal intron of the mitochondrial cytochrome b gene. The homologous gene from the related yeast, Saccharomyces douglasii, has been shown to be essential for respiratory growth in the presence of a wild-type S. douglasii mitochondrial genome and dispensable in the presence of an intronless mitochondrial genome. The two CBP2 genes are functionally interchangeable although the target intron of the S. cerevisiae CBP2 gene is absent from the S. douglasii mitochondrial genome. To determine the function of the CBP2 gene in S. douglasii mitochondrial pre-RNA processing we have constructed and analyzed interspecific hybrid strains between the nuclear genome of S. cerevisiae carrying an inactive CBP2 gene and S. douglasii mitochondrial genomes with different intron contents. We have demonstrated that inactivation of the S. cerevisiae CBP2 gene affects the maturation of the S. douglasii LSU pre-RNA, leading to a respiratory-deficient phenotype in the hybrid strains. We have shown that the CBP2 gene is essential for excision of the S. douglasii LSU intron in vivo and that the gene is dispensable when this intron is deleted or replaced by the S. cerevisiae LSU intron.
Assuntos
Proteínas Fúngicas/fisiologia , RNA Ribossômico/genética , Ribonucleoproteínas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces/fisiologia , Divisão Celular , Íntrons , RNA , Precursores de RNA , RNA MitocondrialRESUMO
We describe a new nuclear gene, CBT1 (Cytochrome B Termination), specifically involved in the generation of mature mRNA of cytochrome b in yeast mitochondria. Disruption of CBT1 (corresponding to ORF YKL 208W) results in a respiratory deficiency (no growth on acetate and ethanol, a reduced growth on glycerol, and a moderate growth on lactate). Cytochrome b is practically undetectable spectrally, while cytochromes a and a3 (cytochrome oxidase) appear unaffected by the disruption. Analysis of mitochondrial transcripts shows a reduced abundance of cytb mRNA, which in addition is approximately 200 nucleotides longer than that of the wild-type. Sequencing of the 3' region of the mutant cytb mRNA with an oligonucleotide primer positioned 148 nt downstream from the dodecamer sequence ("end-of-messenger" signal), demonstrates that the mutant transcript is extended beyond this position and is not processed at the conserved dodecamer cleavage site. The CBT1 gene product may be one of the components required for the exact 3' cleavage of the cytb messenger and may also be related to RNA splicing, since the intron-containing cytb gene is not as well expressed as the intron-less gene and the respiratory deficiency is more severe. We propose, that the CBT1 protein is necessary for the correct trimming of the end of cytb pre-mRNA and may be a part of the multi-component complex involved in this process.
Assuntos
Núcleo Celular/metabolismo , Grupo dos Citocromos b/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Mitocôndrias/química , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Bases , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Análise Espectral , Transformação GenéticaRESUMO
The nuclear ABC1 gene was isolated as a multicopy suppressor of a cytochrome b mRNA translation defect. Its inactivation leads to a respiratory deficiency suggesting a block in the bc1 segment of the respiratory chain [Bousquet, I., Dujardin, G. & Slonimski, P. P. (1991) EMBO J. 10, 2023-2031]. In the present study, we established that deleting the ABC1 chromosomal gene from Saccharomyces cerevisiae does not prevent the assembly of the bc1 complex (complex III) but markedly impairs the kinetics of its high-potential electron transfer pathway occurring on the positive, outer, side of the membrane, which results in reduced activity of the bc1 complex. In addition, the activity of complex II and its cytochrome b560 decrease drastically and complex IV activity is halved. It is also observed that the binding of the quinol to the bc1 complex ubiquinol oxidation site is affected and that adding exogenous quinones partially compensates for the respiratory deficiency in vitro, although the quinone content of mutant and wild-type mitochondria are similar. Lastly, complexes II, III and IV are found to be thermosensitive and the bc1 complex exhibits greater sensitivity than the wild-type strain to center N and P inhibitors, suggesting that the three multisubunit complexes have undergone structural modifications. The data suggest that the ABC1 gene product acts as a chaperone-like protein essential for the proper conformation and efficient functioning of the bc1 complex and the effects of the Abc1 protein on the complexes II and IV might result from interactions with the modified bc1 complex.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Escherichia coli , Proteínas Fúngicas/genética , Complexos Multienzimáticos/metabolismo , Oxirredutases/metabolismo , Conformação Proteica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Succinato Desidrogenase/metabolismo , Grupo dos Citocromos b/metabolismo , Transporte de Elétrons , Complexo I de Transporte de Elétrons , Complexo II de Transporte de Elétrons , Complexo III da Cadeia de Transporte de Elétrons/química , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática , Proteínas Fúngicas/fisiologia , Genes Fúngicos , Genes Supressores , Cinética , Mitocôndrias/metabolismo , Chaperonas Moleculares/metabolismo , Complexos Multienzimáticos/genética , Mutação , NADH Desidrogenase/metabolismo , NADH NADPH Oxirredutases/metabolismo , Saccharomyces cerevisiae/genética , Deleção de Sequência , Succinato Citocromo c Oxirredutase/metabolismo , Temperatura , Ubiquinona/análogos & derivados , Ubiquinona/metabolismoRESUMO
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
The NAM2 gene of Saccharomyces cerevisiae encodes the mitochondrial leucyl tRNA synthetase (mLRS), which is necessary for the excision of the fourth intron of the mitochondrial cytb gene (bI4) and the fourth intron of the mitochondrial coxI gene (aI4), as well as for mitochondrial protein synthesis. Some dominant mutant alleles of the gene are able to suppress mutations that inactivate the bI4 maturase, which is essential for the excision of the introns aI4 and bI4. Here we report mutagenesis studies which focus on the splicing and suppressor functions of the protein. Small deletions in the C-terminal region of the protein preferentially reduce the splicing, but not the synthetase activity; and all the C-terminal deletions tested abolish the suppressor activity. Mutations which increase the volume of the residue at position 240 in the wild-type mLRS without introducing a charge, lead to a suppressor activity. The mutant 238C, which is located in the suppressor region, has a reduced synthetase activity and no detectable splicing activity. These data show that the splicing and suppressor functions are linked and that the suppressor activity of the mutant alleles results from a modification of the wild-type splicing activity.
Assuntos
Genes Fúngicos , Leucina-tRNA Ligase/genética , Splicing de RNA , Saccharomyces cerevisiae/genética , Supressão Genética , Endorribonucleases/genética , Mitocôndrias/enzimologia , Nucleotidiltransferases/genética , RNA Fúngico/genética , Saccharomyces cerevisiae/enzimologiaRESUMO
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
We demonstrate here that the open reading frame (ORF) YCL059c, discovered during the systematic sequencing of chromosome III [Oliver et al., Nature 357 (1992) 38-46], codes for a protein essential for yeast: neither spore germination nor cell division occur in strains deleted for this gene. We have cloned the wild-type (wt) gene and shown that it complements the deletion. A relatively abundant RNA transcript corresponds to the gene. The protein has no similarity to proteins of known function. Interestingly, however, it is homologous to several expressed sequence tags (EST) of unknown function from Caenorhabditis elegans, Oryza sativa and Homo sapiens. Thus, a novel family of proteins of presumably nuclear localization, with a characteristic highly basic motif, KRR-R, transcends various phyla, and plays an important role in cellular processes. We propose to call this essential gene KRR1.