RESUMEN
The c-type cytochromes are metalloproteins with a heme molecule covalently linked to the sulfhydryls of a CXXCH heme-binding site. In plastids, at least six assembly factors are required for heme attachment to the apo-forms of cytochrome f and cytochrome c(6) in the thylakoid lumen. CCS5, controlling plastid cytochrome c assembly, was identified through insertional mutagenesis in the unicellular green alga Chlamydomonas reinhardtii. The complementing gene encodes a protein with similarity to Arabidopsis thaliana HCF164, which is a thylakoid membrane-anchored protein with a lumen-facing thioredoxin-like domain. HCF164 is required for cytochrome b(6)f biogenesis, but its activity and site of action in the assembly process has so far remained undeciphered. We show that CCS5 is a component of a trans-thylakoid redox pathway and operates by reducing the CXXCH heme-binding site of apocytochrome c prior to the heme ligation reaction. The proposal is based on the following findings: 1) the ccs5 mutant is rescued by exogenous thiols; 2) CCS5 interacts with apocytochrome f and c(6) in a yeast two-hybrid assay; and 3) recombinant CCS5 is able to reduce a disulfide in the CXXCH heme-binding site of apocytochrome f.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Citocromos c6/metabolismo , Citocromos c/metabolismo , Proteínas Protozoarias/metabolismo , Tiorredoxinas/metabolismo , Tilacoides/metabolismo , Animales , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sitios de Unión , Chlamydomonas reinhardtii/genética , Citocromos c/genética , Citocromos c6/genética , Citocromos f/genética , Citocromos f/metabolismo , Hemo/genética , Hemo/metabolismo , Mutación , Oxidación-Reducción , Proteínas Protozoarias/genética , Tiorredoxinas/genética , Tilacoides/genéticaRESUMEN
The assembly of chloroplast metalloproteins requires biochemical catalysis. Assembly factors involved in the biosynthesis of metalloproteins might be required to synthesize, chaperone, or transport the cofactor; modify or chaperone the apoprotein; or catalyze cofactor-protein association. Genetic and biochemical approaches have been applied to the study of the assembly of chloroplast iron-sulfur centers, cytochromes, plastocyanin, and the manganese center of photosystem II. These have led to the discovery of NifS-homologues and cysteine desulfhydrase for iron-sulfur center assembly, six loci (CCS1-CCS5, ccsA) for c-type cytochrome assembly, four loci for cytochrome b6 assembly (CCB1-CCB4), the CtpA protease, which is involved in pre-D1 processing, and the PCY2 locus, which is involved in holoplastocyanin accumulation. New assembly factors are likely to be discovered via the study of assembly-defective mutants of Arabidopsis, cyanobacteria, Chlamydomonas, maize, and via the functional analysis of candidate cofactor metabolizing components identified in the genome databases.
RESUMEN
The Ccs1 gene, encoding a highly divergent novel component of a system II type c-type cytochrome biogenesis pathway, is encoded by the previously defined CCS1 locus in Chlamydomonas reinhardtii. phoA and lacZalpha bacterial topological reporters were used to deduce a topological model of the Synechocystis sp. 6803 Ccs1 homologue, CcsB. CcsB, and therefore by analogy Ccs1, possesses a large soluble lumenal domain at its C terminus that is tethered in the thylakoid membrane by three closely spaced transmembrane domains in the N-terminal portion of the protein. Molecular analysis of ccs1 alleles reveals that the entire C-terminal soluble domain is essential for Ccs1 function and that a stromal loop appears to be important in vivo, at least for maintenance of Ccs1. Site-directed mutational analysis reveals that a single histidine (His(274)) within the last transmembrane domain, preceding the large lumenal domain, is required for c-type cytochrome assembly, whereas an invariant cysteine residue (Cys(199)) is shown to be non-essential. Ccs1 is proposed to interact with other Ccs components based on its reduced accumulation in ccs2, ccs3, ccs4, and ccsA strains.
Asunto(s)
Citocromos/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/fisiología , Alelos , Secuencia de Aminoácidos , Animales , Southern Blotting , Membrana Celular/metabolismo , Chlamydomonas reinhardtii/metabolismo , Proteínas de Cloroplastos , Genes Reporteros , Prueba de Complementación Genética , Histidina/química , Modelos Genéticos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Proteínas de Plantas , Estructura Terciaria de Proteína , Proteínas Protozoarias/genética , ARN/metabolismo , Tilacoides/metabolismo , Factores de TiempoRESUMEN
Three distinct systems (I, II, and III) for catalysis of heme attachment to c-type apocytochromes are known. The CcsA and Ccs1 proteins are required in system II for the assembly of bacterial and plastid cytochromes c. A tryptophan-rich signature motif (WWD), also occurring in CcmC and CcmF found in system I, and three histidinyl residues, all strictly conserved in CcsA suggest a function in heme handling. Topological analysis of plastid CcsA in bacteria using the PhoA and LacZalpha reporters placed the WWD motif, the conserved residues His(212) and His(347) on the lumen side of the membrane, whereas His(309) was assigned a location on the stromal side. Functional analysis of CcsA through site-directed mutagenesis enabled the designation of the initiation codon of the ccsA gene and established the functional importance of the WWD signature motif and the absolute requirement of all three histidines for the assembly of plastid c-type cytochromes. In a ccsA mutant, a 200-kDa Ccs1-containing complex is absent from solubilized thylakoid membranes, suggesting that CcsA operates together with Ccs1. We propose a model where the WWD motif and histidine residues function in relaying heme from stroma to lumen and we postulate the existence of a cytochrome c assembly machinery containing CcsA, Ccs1 and additional components.