RESUMEN
To understand the biogenesis of the plastid cytochrome b(6)f complex and to identify the underlying auxiliary factors, we have characterized the nuclear mutant hcf164 of Arabidopsis and isolated the affected gene. The mutant shows a high chlorophyll fluorescence phenotype and is severely deficient in the accumulation of the cytochrome b(6)f complex subunits. In vivo protein labeling experiments indicated that the mutation acts post-translationally by interfering with the assembly of the complex. Because of its T-DNA tag, the corresponding gene was cloned and its identity confirmed by complementation of homozygous mutant plants. HCF164 encodes a thioredoxin-like protein that possesses disulfide reductase activity. The protein was found in the chloroplast, where it is anchored to the thylakoid membrane at its lumenal side. HCF164 is closely related to the thioredoxin-like protein TxlA of Synechocystis sp PCC6803, most probably reflecting its evolutionary origin. The protein also shows a limited similarity to the eubacterial CcsX and CcmG proteins, which are required for the maturation of periplasmic c-type cytochromes. The putative roles of HCF164 for the assembly of the cytochrome b(6)f complex are discussed.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/genética , Grupo Citocromo b/genética , Oxidorreductasas/genética , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Complejo de Citocromo b6f , Transporte de Electrón , Cinética , Datos de Secuencia Molecular , Oxidorreductasas/metabolismo , Proteínas de Plantas/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Tiorredoxinas/químicaRESUMEN
To understand the functional significance of RNA processing for the expression of plastome-encoded photosynthesis genes, we investigated the nuclear mutation hcf107 of Arabidopsis. The mutation is represented by two alleles, both of which lead to a defective photosystem II (PSII). In vivo protein labeling, in vitro phosphorylation, and immunoblot experiments revealed that the psbB gene product (CP47) and an 8-kD phosphoprotein, the psbH gene product (PsbH), are absent in mutant plants. PsbH and PsbB are essential requirements for PSII assembly in photosynthetic eukaryotes, and their absence in hcf107 is consistent with the PSII-less mutant phenotype. RNA gel blot hybridizations showed that the hcf107 mutation specifically impairs the accumulation of some but not all oligocistronic psbH transcripts that are released from the pentacistronic psbB-psbT-psbH-petB-petD precursor RNA by intergenic endonucleolytic cleavage. In contrast, neither the levels nor the sizes of psbB-containing RNAs are affected. S1 nuclease protection analyses revealed that psbH RNAs are lacking only where psbH is the leading cistron and that they are processed at position -45 in the 5' leader segment of psbH. These data and additional experiments with the cytochrome b(6)f complex mutant hcf152, which is defective in 3' psbH processing, suggest that only those psbH-containing transcripts that are processed at their -45 5' ends can be translated. Secondary structure analysis of the 5' psbH leader predicted the formation of stable stem loops in the nonprocessed transcripts, which are unfolded by processing at the -45 site. We propose that this unfolding of the psbH leader segment as a result of RNA processing is essential for the translation of the psbH reading frame. We suggest further that HCF107 has dual functions: it is involved in intercistronic processing of the psbH 5' untranslated region or the stabilization of 5' processed psbH RNAs, and concomitantly, it is required for the synthesis of CP47.
Asunto(s)
Arabidopsis/genética , Núcleo Celular/genética , ADN Intergénico/genética , Genes de Plantas/genética , Complejos de Proteína Captadores de Luz , Fosfoproteínas/genética , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Complejo de Proteína del Fotosistema II , Biosíntesis de Proteínas , Procesamiento Postranscripcional del ARN , ARN del Cloroplasto/metabolismo , Alelos , Secuencia de Aminoácidos , Arabidopsis/citología , Arabidopsis/metabolismo , Secuencia de Bases , Clorofila/genética , Genes/genética , Genes Recesivos , Datos de Secuencia Molecular , Peso Molecular , Mutación , Conformación de Ácido Nucleico , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/biosíntesis , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Filogenia , Estabilidad del ARN , ARN del Cloroplasto/química , ARN del Cloroplasto/genética , ARN de Planta/química , ARN de Planta/genética , ARN de Planta/metabolismo , Homología de Secuencia de Aminoácido , Tilacoides/metabolismo , Transcripción GenéticaRESUMEN
Lumenal proteins are transported across the thylakoid membrane by two very different pathways: Sec-dependent or twin-arginine translocase (Tat)-dependent, where the substrate protein can be transported in a folded state. We present the first evidence that a given protein can be targeted by different pathways in different organisms. Arabidopsis Hcf136 is targeted exclusively by the Tat pathway in pea chloroplasts and no Sec-dependent transport is evident even when the twin-arginine is replaced by twin-lysine. However, twin-arginine motifs are absent from the presequences of Hcf136 proteins encoded by plastid or cyanobacterial genomes, strongly implying translocation by another pathway (presumably Sec). We suggest that the Hcf136 protein was transferred to the Tat pathway when the gene became incorporated into the nuclear genome, possibly due to the tighter folding associated with the more involved, post-translational targeting pathway.
Asunto(s)
Proteínas de Arabidopsis , Proteínas Bacterianas , Núcleo Celular/genética , Cianobacterias/citología , Proteínas de Escherichia coli , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Pisum sativum/citología , Plastidios/genética , Tilacoides/genética , Adenosina Trifosfatasas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Arabidopsis/genética , Arginina/genética , Arginina/metabolismo , Evolución Biológica , Transporte Biológico , Proteínas Portadoras/metabolismo , Núcleo Celular/enzimología , Núcleo Celular/metabolismo , Cianobacterias/genética , Eucariontes/citología , Eucariontes/genética , Concentración de Iones de Hidrógeno , Membranas Intracelulares/enzimología , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Pisum sativum/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plastidios/enzimología , Plastidios/metabolismo , Señales de Clasificación de Proteína/química , Señales de Clasificación de Proteína/genética , Señales de Clasificación de Proteína/fisiología , Canales de Translocación SEC , Proteína SecA , Tilacoides/enzimología , Tilacoides/metabolismoRESUMEN
To understand the regulatory mechanisms underlying the biogenesis of photosystem II (PSII) we have characterized the nuclear mutant hcf136 of Arabidopsis thaliana and isolated the affected gene. The mutant is devoid of any photosystem II activity, and none of the nuclear- and plastome-encoded subunits of this photosystem accumulate to significant levels. Protein labelling studies in the presence of cycloheximide showed that the plastome-encoded PSII subunits are synthesized but are not stable. The HCF136 gene was isolated by virtue of its T-DNA tag, and its identity was confirmed by complementation of homozygous hcf136 seedlings. Immunoblot analysis of fractionated chloroplasts showed that the HCF136 protein is a lumenal protein, found only in stromal thylakoid lamellae. The HCF136 protein is produced already in dark-grown seedlings and its levels do not increase dramatically during light-induced greening. This accumulation profile confirms the mutational data by showing that the HCF136 protein must be present when PSII complexes are made. HCF136 homologues are found in the cyanobacterium Synechocystis species PCC6803 (slr2034) and the cyanelle genome of Cyanophora paradoxa (ORF333), but are lacking in the plastomes of chlorophytes and metaphytes as well as from those of rhodo- and chromophytes. We conclude that HCF136 encodes a stability and/or assembly factor of PSII which dates back to the cyanobacterial-like endosymbiont that led to the plastids of the present photosynthetic eukaryotes.