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 LacZ␣ 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.

The c-type cytochromes are metalloproteins with a heme molecule covalently linked to the sulfhydryls of a CXXCH hemebinding 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 thioredoxinlike 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.

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 lacZ␣ 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 nonessential. Ccs1 is proposed to interact with other Ccs components based on its reduced accumulation in ccs2, ccs3, ccs4, and ccsA strains.

Nuclear genes play important regulatory roles in the biogenesis of the photosynthetic apparatus of eukaryotic cells by encoding factors that control steps ranging from chloroplast gene transcription to post-translational processes. However, the identities of these genes and the mechanisms by which they govern these processes are largely unknown. By using glass bead-mediated transformation to generate insertional mutations in the nuclear genome of Chlamydomonas reinhardtii, we have generated four mutants that are defective in the accumulation of the cytochrome b 6 f complex. One of them, strain abf3, also fails to accumulate holocytochrome c 6 . We have isolated a gene, Ccs1, from a C. reinhardtii genomic library that complements both the cytochrome b 6 f and cytochrome c 6 deficiencies in abf3. The predicted protein product displays significant identity with Ycf44 from the brown alga Odontella sinensis, the red alga Porphyra purpurea, and the cyanobacterium Synechocystis strain PCC 6803 (25-33% identity). In addition, we note limited sequence similarity with ResB of Bacillus subtilis and an open reading frame in a homologous operon in Mycobacterium leprae (11-12% identity). On the basis of the pleiotropic c-type cytochrome deficiency in the ccs1 mutant, the predicted plastid localization of the protein, and its relationship to candidate cytochrome biosynthesis proteins in Gram-positive bacteria, we conclude that Ccs1 encodes a protein that is required for chloroplast c-type holocytochrome formation.Studies in the unicellular green alga Chlamydomonas reinhardtii and vascular plants have demonstrated that the proteins required for photosynthesis are encoded by genes that reside in two distinct cellular compartments: the nucleus and the chloroplast (reviewed in Refs.

The light-induced assembly of light-harvesting complex (LHC) II PSI and PSII of higher plant thylakoid membranes include an evolutionarily conserved CC containing the photochemical reaction center and an LHC, the function of which is to absorb photons and to transfer excitation energy to the CC. The LHCs of both photosystems, LHC I and LHC 11, are composed of several distinct pigment-protein complexes. The proteins function to orient and space precisely their associated Chl and carotenoid molecules so that absorbed energy is not prematurely quenched but is efficiently transferred to the reaction center. Therefore, this function requires an exact arrangement of the individual pigment-proteins within each complex as well as between the LHC and the CC. Much is