Ccs1, a Nuclear Gene Required for the Post-translational Assembly of Chloroplast c-Type Cytochromes

Abstract

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.

Functional Analysis of a Divergent System II Protein, Ccs1, Involved in c-Type Cytochrome Biogenesis

“…The strain was proposed to be defective in c-type cytochrome synthesis based on a hallmark pleiotropic deficiency in cyt f and cyt c 6 (47). Nevertheless, the relationship to previously characterized CCS loci (44) could not be ascertained by classical genetic methodologies because strain ccs1-5::NIT1 could not mate.…”

“…Strains and Culture Conditions-C. reinhardtii wild-type strain CC-125 (MTϩ) and mutant strains ccsA-B6 (CC-2695/CC-2934), ccs1-ac206 (CC-939/CC-1112), ccs1-2 (CC-3422/3423), ccs1-3 (CC-3424/CC-3425), ccs1-4 (CC-3426), abf3 (now ccs1-5::NIT1), ccs2-1 to ccs2-5 (CC-3428 to CC-3437), ccs3-F18 (CC-3092/CC-3093), ccs4-F2D8 (CC-3910/CC-3720), and ccs5-1::ARG7 (CC-3717/CC-3718), described previously (4,43,45,47,56,57), can be obtained from the Chlamydomonas Genetics Center (Duke University, Durham, NC). Arginine-auxotrophic strain arg7cw15A used for insertional mutagenesis was obtained from Prof. J.-D. Rochaix, University of Geneva, Switzerland.…”

“…Protein Preparation and Analysis-Cytochromes were detected after freeze-thaw fractionation and analysis of electrophoretically separated supernatant and pellet fractions by immunodecoration or by heme staining as described previously (43,47,65). Enriched thylakoid membrane fractions were prepared from sonicated cell lysates and analyzed immediately by denaturing PAGE according to Ref.…”

“…Two of these have been identified molecularly. CcsA, encoded by the ccsA locus (46), is a multiple membrane-spanning protein and contains the tryptophan-rich motif with the "WWD" signature first noted in CcmC and CcmF of system I (4,21,46), and Ccs1, which also displays characteristic features of a membrane protein (47). Ccs1 lacks any domains or structural features that might speak to a specific chemical function, and it appears to be unique to system II (20,47).…”

The GreenCut2 Resource, a Phylogenomically Derived Inventory of Proteins Specific to the Plant Lineage

“…For example, transcripts encoding proteins of the photosynthetic apparatus, such as CrPSAD (At1g03130/ At4g02770) and CrLHCA1 (At3g54890), are among the most abundant in Chlamydomonas (3200 and 4000 RPKM, respectively, in cells grown photoheterotrophically) (47). In contrast, regulatory proteins or assembly factors, such as AtMBB1 (At3g17040), an mRNA maturation factor for psbB (87), and CrCCS1 (At1g49380), which catalyzes the covalent attachment of heme to c-type cytochromes (88), are generally present in lower amounts, which is reflected by low mRNA abundances (ϳ16 and ϳ15 RPKM, respectively, for Chlamydomonas). The fact that many mRNAs and proteins have been identified through molecular screens that more readily recover abundant targets explains why many characterized proteins in the K and KI categories are encoded by high abundance transcripts.…”