RESUMO
hUBC9, an E2 ubiquitin conjugating enzyme, was identified by yeast two-hybrid screening and coprecipitation studies to interact with MEKK1 and the type I TNF-alpha receptor, respectively. Because both of these proteins regulate NFkappaB activity, the role of hUBC9 in modulating NFkappaB activity was investigated. Overexpression of hUBC9 in HeLa cells stimulated the activity of NFkappaB as determined by NFkappaB reporter and IL-6 secretion assays. hUBC9 also synergized with MEKK1 to activate NFkappaB reporter activity. Thus, hUBC9 modulates NFkappaB activity which, at least in part, can be attributed to its interaction with MEKK1 and the type I TNF-alpha receptor.
Assuntos
Antígenos CD/metabolismo , Ligases/metabolismo , MAP Quinase Quinase Quinase 1 , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Enzimas de Conjugação de Ubiquitina , Regulação da Expressão Gênica/genética , Genes Reporter/genética , Células HeLa , Humanos , Interleucina-6/metabolismo , Ligases/genética , Mutagênese , Receptores Tipo I de Fatores de Necrose Tumoral , Proteínas Recombinantes de Fusão/metabolismo , Transfecção/genéticaRESUMO
Screening for resistance to fenpropimorph was undertaken in order to isolate yeast mutants affected in the regulation of the ergosterol pathway. Among the mutants isolated, one bearing the recessive fen1-1 mutation was characterized by a 1.5-fold increase in the ergosterol level and a general resistance to sterol biosynthesis inhibitors. The fen1-1 mutation was linked to MAT locus on chromosome III. The measurement of enzyme activities involved in the ergosterol pathway revealed that isopentenyl diphosphate (IPP) isomerase activity was specifically increased 1.5-fold as compared to the wild type strain. However, overexpression of IPP isomerase in the wild type strain was not by itself sufficient to lead to sterol increase or resistance to sterol biosynthesis inhibitors, showing that IPP isomerase is not a limiting step in the pathway. The fen1-1 mutation permits viability in aerobiosis of yeast disrupted for sterol-14 reductase in absence of exogenous ergosterol supplementation, whereas the corresponding strain bearing the wild type FEN1 allele grows only in anaerobiosis. This result shows that ignosterol is able to efficiently replace ergosterol as bulk membrane component and that the fen1-1 mutation eliminates the specific ergosterol requirement in yeast.
Assuntos
Antifúngicos/toxicidade , Isomerases de Ligação Dupla Carbono-Carbono , Resistência Microbiana a Medicamentos , Genes Fúngicos , Saccharomyces cerevisiae/efeitos dos fármacos , Esteróis/biossíntese , Mapeamento Cromossômico , Cromossomos Fúngicos , Primers do DNA , Resistência Microbiana a Medicamentos/genética , Ergosterol/biossíntese , Expressão Gênica , Hemiterpenos , Isomerases/biossíntese , Isomerases/metabolismo , Metionina/metabolismo , Reação em Cadeia da Polimerase , Mapeamento por Restrição , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
The effects of fenpropimorph on sterol biosynthesis and growth of Saccharomyces cerevisiae were examined to pinpoint the mode of action of fungicides that inhibit ergosterol biosynthesis. Taking advantage of sterol auxotrophy and sterol permeability in mutant strains, we show that growth inhibition is strongly correlated with inhibition of sterol biosynthesis. We confirm that in vivo and at low concentrations, fenpropimorph inhibits delta 8----delta 7-sterol isomerase, and in addition, when it is used at higher concentrations, it inhibits delta 14-sterol reductase. We show also that the fungistatic effect of fenpropimorph is not due to the accumulation of abnormal sterols in treated cells but is linked to the specific inhibition of ergosterol biosynthesis, leading to the arrest of cell proliferation in the unbudded G1 phase of the cell cycle.
Assuntos
Fungicidas Industriais/farmacologia , Morfolinas/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Mutação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Esteróis/biossíntese , Esteróis/isolamento & purificaçãoRESUMO
We have transformed Saccharomyces cerevisiae with a genomic library contained in the replicative vector pFL44. The resulting transformants were screened for resistance to fenpropidin, a specific inhibitor of sterol 14-reductase. A plasmid was isolated that transformed yeast both to resistance to fenpropidin and to an increased specific activity of sterol 14-reductase. Sterol analysis of transformed cells grown in the presence of increasing concentrations of the inhibitor confirmed that resistance was a consequence of over-production of sterol 14-reductase. By chromosomal gene disruption, we have, for the first time, constructed yeast strains defective in sterol 14-reductase. As expected, since yeast in unable to take up sterols in aerobiosis, the disrupted strains do not grow in the presence of oxygen, even if exogenous sterols are supplied. However, disrupted cells grow in anaerobiosis with exogenous oleic acid and ergosterol supplements. They also grow in aerobiosis if they bear an additional mutation allowing sterol uptake. In this last growth condition the cells require a "sparking" ergosterol supplementation (25 nM) and accumulate ignosterol (ergosta-8,14-dienol) as the end-product of the sterol pathway. These results reveal that ignosterol is not obviously toxic to yeast membranes and strongly suggest that the molecular basis of the antifungal-activity morpholine and piperidine is directly related to the specific inhibition of ergosterol formation.
Assuntos
Ergosterol/metabolismo , Genes Fúngicos , Oxirredutases/genética , Piperidinas/farmacologia , Saccharomyces cerevisiae/enzimologia , Clonagem Molecular/métodos , Resistência Microbiana a Medicamentos , Ergosterol/análogos & derivados , Ergosterol/biossíntese , Mutagênese Sítio-Dirigida , Oxirredutases/antagonistas & inibidores , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transformação GenéticaRESUMO
Yeast mutants blocked in farnesyl diphosphate (FPP) synthetase have been isolated. Their specific phenotype is likely linked to a lowering in the FPP pool required for protein prenylation. The structural gene of FPP synthetase has been isolated. Complete inactivation of FPP synthetase by gene disruption is letal for the yeast cells.
Assuntos
Dimetilaliltranstransferase/metabolismo , Saccharomyces cerevisiae/enzimologia , Divisão Celular , Dimetilaliltranstransferase/genética , Mutação/genética , Fenótipo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genéticaRESUMO
Exogenous sterols do not permeate wild-type Saccharomyces cerevisiae in aerobic conditions. However, mutant strain FKerg7, affected in lanosterol synthase, is a sterol auxotroph which is able to grow aerobically in the presence of ergosterol. Viability of this strain depends on the presence of an additional mutation, aux30, that leads to sterol permeability. Cells bearing the aux30 mutation fail to grow in standard yeast nitrogen base medium containing pyridoxine but grow normally if pyridoxine is replaced by either pyridoxal or pyridoxamine. These mutants are characterized by a lack in pyridoxine (pyridoxamine) phosphate oxidase [P(N/M)P oxidase] (EC 1.4.3.5) activity. The pleiotropic phenotype induced by the aux30 mutation includes a strong perturbation in amino acid biosynthesis. Strains bearing the aux30 mutation also display atypic fatty acid, sterol, and cytochrome patterns. Transformation of an aux30 strain with a replicative vector carrying the wild-type PDX3 gene encoding P(N/M)P oxidase restored wild-type fatty acid, sterol, and cytochrome patterns and suppressed exogenous sterol accumulation. It is proposed that sterol permeation of aux30 strains in mainly the consequence of their leaky Hem- character. The amino acid sequence of S. cerevisiae P(N/M)P oxidase inferred from the nucleotide sequence of PDX3 shows a high percentage of homology with the corresponding enzymes from Escherichia coli and Myxococcus xanthus. Several putative Gcn4p binding sequences are present in the PDX3 promoter region, leading to the assumption that transcription of this gene is under the general control of nitrogen metabolism.
Assuntos
Genes Fúngicos , Fosfato de Piridoxal/biossíntese , Piridoxaminafosfato Oxidase/genética , Saccharomyces cerevisiae/metabolismo , Esteróis/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA/química , Dados de Sequência MolecularRESUMO
In order to investigate ergosterol metabolism in S. cerevisiae we studied the CM8 mutant strain defective in the regulation of this pathway. A genomic multicopy library was screened to reverse the CM8 phenotype. This allowed us to characterize a new gene, FMS1, which relieves mutant phenotype by extragenic functional complementation. FMS1 may encode a 508 amino-acid protein. The predicted protein shares 35% identity with Cbp1p, a Candida albicans corticosteroid binding-protein. Fms1p also shows a weaker homology with monoamine oxidases. The construction of a FMS1 null-allele yeast strain demonstrated that this gene is not essential for yeast in normal usual laboratory culture conditions. The existence of a gene related to CBP1 of C. albicans in S. cerevisiae strongly suggests a possible function of steroid-binding proteins in yeast general physiology rather than in a process related to pathogenicity.
Assuntos
Candida albicans/genética , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Alelos , Sequência de Aminoácidos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Clonagem Molecular , Fungicidas Industriais/farmacologia , Genes Fúngicos , Teste de Complementação Genética , Vetores Genéticos , Dados de Sequência Molecular , Morfolinas/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Homologia de Sequência de AminoácidosRESUMO
We have compared Saccharomyces cerevisiae global gene expression in wild-type and mutants (Deltahap2 and Deltahap4) of the HAP transcriptional complex, which has been shown to be necessary for growth on respiratory substrates. Several hundred ORFs are under positive or negative control of this complex and we analyse here in detail the effect of HAP on mitochondria. We found that most of the genes upregulated in the wild-type strain were involved in organelle functions, but practically none of the downregulated ones. Nuclear genes encoding the different subunits of the respiratory chain complexes figure in the genes more expressed in the wild-type than in the mutants, as expected, but in this group we also found key components of the mitochondrial translation apparatus. This control of mitochondrial translation may be one of the means of coordinating mitochondrial and nuclear gene expression in elaborating the respiratory chain. In addition, HAP controls the nuclear genes involved in several other mitochondrial processes (import, mitochondrial division) that define the metabolic state of the cell, but not mitochondrial DNA replication and transcription. In most cases, a putative CCAAT-binding site is present upstream of the ORF, while in others no such sites are present, suggesting the control to be indirect. The large number of genes regulated by the HAP complex, as well as the fact that HAP also regulates some putative transcriptional activators of unknown function, place this complex at a hierarchically high position in the global transcriptional regulation of the cell.
RESUMO
We have isolated and characterized a pleiotropic recessive mutation. fen2-1, that causes resistance to fenpropimorph and a low level of ergosterol in Saccharomyces cerevisiae. Ergosterol synthesis in the mutant strain was 5.5-fold slower than in the wild type; however, in vitro assays of the enzymes involved in ergosterol biosynthesis could not account for this low rate in the mutant. The mutant phenotype was expressed only in media exerting both carbon and nitrogen catabolite repression. To our knowledge, this is the first locus in yeast that reveals a concerted regulation between different pathways (carbon and nitrogen catabolite repression and/or general control of amino acid biosynthesis and ergosterol biosynthesis). The yeast gene FEN2 has been isolated and contains an open reading frame (ORF) of 512 codons. This ORF was found to be identical to YCR28C of chromosome III. A possible function of the FEN2 gene product in yeast is discussed.