RESUMEN
The disruption of the two thioredoxin genes in Saccharomyces cerevisiae leads to a complex phenotype, including the inability to use methionine sulfoxide as sulfur source, modified cell cycle parameters, reduced H(2)O(2) tolerance, and inability to use sulfate as sulfur source. Expression of one of the multiple Arabidopsis thaliana thioredoxins h in this mutant complements only some aspects of the phenotype, depending on the expressed thioredoxin: AtTRX2 or AtTRX3 induce methionine sulfoxide assimilation and restore a normal cell cycle. In addition AtTRX2 also confers growth on sulfate but no H(2)O(2) tolerance. In contrast, AtTRX3 does not confer growth on sulfate but induces H(2)O(2) tolerance. We have constructed hybrid proteins between these two thioredoxins and show that all information necessary for sulfate assimilation is present in the C-terminal part of AtTRX2, whereas some information needed for H(2)O(2) tolerance is located in the N-terminal part of AtTRX3. In addition, mutation of the atypical redox active site WCPPC to the classical site WCGPC restores some growth on sulfate. All these data suggest that the multiple Arabidopsis thioredoxins h originate from a totipotent ancestor with all the determinants necessary for interaction with the different thioredoxin target proteins. After duplications each member evolved by losing or masking some of the determinants.
Asunto(s)
Arabidopsis/enzimología , Saccharomyces cerevisiae/enzimología , Tiorredoxinas/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Arabidopsis/genética , Sitios de Unión , Western Blotting , Ciclo Celular , Evolución Molecular , Prueba de Complementación Genética , Peróxido de Hidrógeno/farmacología , Metionina/análogos & derivados , Metionina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Oxidación-Reducción , Fenotipo , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Especificidad por Sustrato , Sulfatos/metabolismo , Tiorredoxinas/química , Tiorredoxinas/genéticaRESUMEN
Thioredoxins and glutaredoxins are ubiquitous proteins that reduce disulphide bridges of oxidized target proteins in vitro. In contrast to the situations in other organisms, phylogenic analysis has indicated that plant thioredoxins and glutaredoxins are present as multigenic families, and that thioredoxins have several subclasses. Thioredoxins and glutaredoxins are probably involved in similar physiological events - the major challenge is to identify their specific targets and establish the function of these proteins in vivo.
RESUMEN
Disruption of the two thioredoxin genes in yeast dramatically affects cell viability and growth. Expression of Arabidopsis thioredoxin AtTRX3 in the Saccharomyces thioredoxin Delta strain EMY63 restores a wild-type cell cycle, the ability to grow on methionine sulfoxide, and H2O2 tolerance. In order to isolate thioredoxin targets related to these phenotypes, we prepared a C35S (Escherichia coli numbering) thioredoxin mutant to stabilize the intermediate disulfide bridged complex and we added a polyhistidine N-terminal extension in order to purify the complex rapidly. Expression of this mutant thioredoxin in the wild-type yeast induces a reduced tolerance to H2O2, but only limited change in the cell cycle and no change in methionine sulfoxide utilization. Expression in the Delta thioredoxin strain EMY63 allowed us to isolate a complex of the thioredoxin with YLR109, an abundant yeast protein related to PMP20, a peroxisomal protein of Candida. No function has so far been attributed to this protein or to the other numerous homologues described in plants, animals, fungi, and prokaryotes. On the basis of the complementation and of low similarity with peroxiredoxins, we produced YLR109 and one of its Arabidopsis homologues in E. coli to test their peroxiredoxins activity. We demonstrate that both recombinant proteins present a thioredoxin-dependent peroxidase activity in vitro. The possible functions of this new peroxiredoxin family are discussed.
Asunto(s)
Proteínas de Arabidopsis/genética , Peroxidasas/genética , Tiorredoxinas/genética , Secuencia de Aminoácidos , Arabidopsis/enzimología , Proteínas de Arabidopsis/química , ADN/química , Ditiotreitol/farmacología , Escherichia coli/enzimología , Citometría de Flujo , Proteínas Fúngicas/química , Regulación de la Expresión Génica , Peróxido de Hidrógeno/metabolismo , Cinética , Datos de Secuencia Molecular , Mutagénesis , Peroxidasas/metabolismo , Peroxirredoxinas , Filogenia , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/enzimología , Alineación de Secuencia , Tiorredoxina h , Tiorredoxinas/químicaRESUMEN
Whereas vertebrates possess only two thioredoxin genes, higher plants present a much greater diversity of thioredoxins. For example, Arabidopsis thaliana has five cytoplasmic thioredoxins (type h) and at least as many chloroplastic thioredoxins. The abundance of plant thioredoxins leads to the question whether the various plant thioredoxins play a similar role or have specific functions. Because most of these proteins display very similar activities on artificial or biological substrates in vitro, we developed an in vivo approach to answer this question. The disruption of both of the two Saccharomyces cerevisiae thioredoxin genes leads to pleiotropic effects including methionine auxotrophy, H2O2 hypersensitivity, altered cell cycle characteristics, and a limited ability to use methionine sulfoxide as source of methionine. We expressed eight plant thioredoxins (six cytoplasmic and two chloroplastic) in yeast trx1, trx2 double mutant cells and analyzed the different phenotypes. Arabidopsis type h thioredoxin 2 efficiently restored sulfate assimilation whereas Arabidopsis type h thioredoxin 3 conferred H2O2 tolerance. All thioredoxins tested could complement for reduction of methionine sulfoxide, whereas only type h thioredoxins were able to complement the cell cycle defect. These findings clearly indicate that specific interactions between plant thioredoxins and their targets occur in vivo.