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

Beth Welty Dreyfuss, Patrice P. Hamel, Stacie S. Nakamoto, and Sabeeha MerchantDagger

From the Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095-1569

Received for publication, August 23, 2002, and in revised form, October 16, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (His274) within the last transmembrane domain, preceding the large lumenal domain, is required for c-type cytochrome assembly, whereas an invariant cysteine residue (Cys199) 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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Universal to all energy-transducing membrane systems is the presence of c-type cytochromes on the p-side of the membrane, which corresponds to the plastid lumen, the mitochondrial intermembrane space, and the bacterial periplasm. Their distinguishing feature is the covalent attachment of the heme prosthetic group through thioether linkage(s) between one or, in most cases, both of the cysteine residues lying in the CXXC(H/K) motif of the apocytochrome and the vinyl groups of heme. Genetic approaches to identify c-type cytochrome-specific assembly factors led to the conclusion that at least three distinct systems (I, II, and III) evolved for the conversion of these molecules to their holoforms (for review see Refs. 1-5). System I, also referred to as the Ccm1 pathway, is known from extensive studies in alpha - and gamma -proteobacterial models such as Rhodobacter capsulatus (6-9), Bradyrhizobium japonicum (10-12), Paracoccus denitrificans (13-16), and Escherichia coli (17, 18). 9 to 12 genes, whose products are dedicated to the assembly of all c-type cytochromes, define the Ccm pathway (for review see Refs. 1, 19, and 20). The Ccm2 proteins include subunits for a putative ABC-type transporter (6, 7, 10, 14, 21-23), components of a cytochrome biogenesis-specific thiol metabolism sub-pathway (8, 19, 24-29), a putative cyt c/heme lyase (30), a unique periplasmic heme chaperone (18, 31) and its accompanying heme delivery component (32-35). In contrast, system III, which was discovered through extensive genetic analysis in fungi and seems to be restricted to the mitochondria of vertebrates and invertebrates, is a minimal system with a single component, the so-called cytochrome c and c1 heme lyases (CCHL and CC1HL) (36-39). The system III CCHLs display no sequence similarity to system I and system II components; the similarity between individual CCHLs is itself limited to the occurrence in their N-terminal domains of between one to three CPV motifs that are believed to be involved in an interaction with heme (40, 41).

System II, the subject of this paper, operates in plastids, cyanobacteria, and some bacteria (42). Genetic studies in the green alga Chlamydomonas reinhardtii have assigned up to six loci, plastid ccsA and nuclear CCS1 to CCS5 (43-45), to the maturation of chloroplast c-type cytochromes, membrane-bound cyt f and soluble cyt c6. 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).

Genetic studies in Bacillus subtilis and Arabidopsis thaliana, and functional genomics in Bordetella pertussis, revealed two additional components required for c-type cytochrome biogenesis in system II (48-50). One is a membrane-anchored thioredoxin-like protein with its thiol-reducing active site on the p-side of the membrane, called ResA/HCF164/CcsX, respectively (49-51), and the other is CcdA, discovered originally in B. subtilis (48, 52). CcdA corresponds to the central portion of DipZ/DsbD, which functions in transmembrane thiol redox metabolism in E. coli and other bacteria (53). In B. pertussis, DipZ/DsbD is believed to function together with CcsX to provide the reductant to reduce apocytochromes, either directly or indirectly, on the p-side of the membrane before attachment of heme to the cysteinyl thiols (49).

For CcsA, CcdA, or CcsX, conserved sequence motifs suggest functional domains, but this has not been the case for Ccs1, which is a highly divergent protein. Its apparently essential function in popular bacterial model systems has hindered mutational analysis (54). Nevertheless, the fact that the cytochrome assembly pathways operate on multiple, divergent apoprotein substrates (two in C. reinhardtii and up to seven in B. pertussis (42)) may be suggestive of a direct interaction between Ccs1 and the apocytochrome. The limited sequence relationship among Ccs1 proteins would therefore be a consequence of co-evolution with the highly divergent apocytochromes. In this work, we undertake molecular and functional analyses of Ccs1. First, we confirm through molecular complementation that mutants previously assigned to the CCS1 locus are the result of lesions in the Ccs1 gene. Molecular characterization of each ccs1 allele reveals that a stromal loop appears to be functionally important, at least for the stability of Ccs1 in vivo. Second, we suggest that Ccs1 functions together not only with CcsA, as has been shown in an accompanying paper (55), but also with multiple other Ccs components to form a "CCS complex." Third, we undertake membrane topological analysis and site-directed mutagenesis to generate a functional model for Ccs1. We find that a single histidine residue, located within the final transmembrane domain, preceding the large soluble domain is necessary for c-type cytochrome assembly in chloroplasts.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Strains and Culture Conditions-- C. reinhardtii wild-type strain CC-125 (MT+) 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. Wild-type strains were grown at 22-25 °C in TAP medium (58) under cool fluorescent lights (15-125 µmol m-2 s-1) with agitation (225 rpm). Mutant strains were grown under the same conditions, except that the illumination was always reduced (15-25 µmol m-2 s-1). The C. reinhardtii mutant y-1 (yellow-in-the-dark; CC-735) strain was grown in TAP medium at 22 °C, wrapped in aluminum foil when necessary to prevent exposure to light. To de-green light-exposed green cells, one-half of the culture was diluted every day into fresh TAP medium. In 10 days green cells were de-greened to a Chl concentration of 0.029 µg/ml. To re-green the cells (to a Chl concentration of 3.6 µg/ml), the flasks were unwrapped and exposed to light (100-125 µmol m-2 s-1) at 22 °C.

Insertional Mutagenesis and Identification of ccs Mutants-- ccs1-6::ARG7 strain was generated by insertional mutagenesis as described previously (45). Briefly, arg7cw15A-recipient cells were transformed with EcoRI-linearized pARG7.8phi 3 by the glass bead transformation method (59, 60). Arginine prototrophic colonies were screened based on their variable fluorescence to identify candidate mutants blocked on the reducing side of Photosystem II (61). Candidate ccs mutants were screened for accumulation of holocyt f and holocyt c6 by heme staining and immunoblot analysis as described below. The ccs1-6::ARG7 strain was deposited into the Chlamydomonas Genetics Center (CC-3715/CC-3716).

Complementation of ccs1 Strains-- ccs1 strains grown in TAP medium (3-7 × 106 cells/ml) were collected by centrifugation, 1,500 × g for 5 min, and used directly for transformation (ccs1-6::ARG7) after resuspension in TAP medium (2 × 108 cells/ml) or (for strains ccs1-ac206 and ccs1-2 through 4) were resuspended in autolysin (prepared according to Ref. 62) at 2 × 108 cells/ml and incubated for 30-45 min to digest away the cell wall, after which autolysin was diluted by addition of 40 ml of TAP medium. The cells were recovered by centrifugation at 1,500 × g for 5 min. For glass bead transformation (60), 0.3 ml of cells were vortexed for 15 s in the presence of 0.3 mg of acid-washed glass bead and DNA. 1 µg of SalI-linearized wild-type pCcs1-2 DNA (simply referred to as pCcs1 for remainder of paper (47)) was used for complementation transformations. Co-transformation experiments included the addition of 1 µg of EcoRI-linearized pSP109 encoding the ble marker (63). 1 µg of EcoRI-linearized pTZ18U was used in control transformation reactions to assess the frequency of reversion. Vortexed cells were diluted in 10 ml of TAP and transferred to 50-ml flasks for recovery overnight in a shaking incubator. Cells were harvested by centrifugation and resuspended in 1 ml of minimal medium without acetate (58). 0.5 ml of cells were plated on minimal agar plates and incubated at 50-125 µmol m-2 s-1 until photosynthetic colonies appeared (about 1-2 weeks). The remaining 0.5 ml of cells were plated onto TAP + zeomycin (ZeocinTM, Invitrogen) (10 µg/ml) agar plates and incubated at 50-125 µmol m-2 s-1 until zeomycin-resistant transformants appeared (about 2-3 weeks). For the "empty vector" control transformations, the entire mix was plated onto minimal agar plates for selection for phototrophic growth.

The presence of introduced Ccs1 sequences was confirmed by amplification of the integrated pCcs1 DNA using a gene-specific primer (CCS1-7; see Table I of the Supplemental Material for all primers) and the Universal M13 -20 primer specific for the vector. 7 µl of genomic DNA, prepared as described previously (64), was amplified using Taq DNA polymerase in the presence of 5% Me2SO. Amplification conditions were 94 °C for 5 min prior to addition of polymerase, 25 cycles of 94 °C for 1 min, 52 °C for 45 s, 72 °C for 1 min, with a final 5-min extension at 72 °C.

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. 43. To increase efficiency of transfer of electrophoretically separated enriched thylakoid membrane proteins, 0.01% SDS was added to the transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol). Enriched thylakoid membrane proteins were transferred at 50 V, 4 °C for ~2 h to 0.2-µm polyvinylidene difluoride membranes (Immobilon PSQ, Millipore Corp., Bedford, MA). Polyclonal antisera raised against C. reinhardtii cytochrome c6 (1:1000), cytochrome f fusion protein (1:1000) (66), and Trx-Ccs1 fusion protein (1:100) (see Experimental Procedures in the Supplemental Material) were used for detection of cyt c6, cyt f, and Ccs1, respectively. Bound antibodies were detected chromagenically using alkaline phosphatase-conjugated secondary antibodies.

Southern Blot Analysis-- 3 µg of genomic DNA (see the Experimental Procedures for isolation in the Supplemental Material) was digested with restriction enzymes and analyzed by Southern blot hybridization. For strains ccs1-ac206 and ccs1-1, the probe was prepared using Genesis non-radioactive nucleic acid labeling kit (Roche Molecular Biochemicals), hybridized, and detected chromogenically following the manufacturer's procedure. For strains ccs1-3, ccs1-4, and the insertional mutant, ccs1-6::ARG7, the probe was prepared and detected as described previously (67, 68).

Sequencing of ccs1 Alleles and CC125-- Genomic DNA from ccs1-ac206, ccs1-2, ccs1-3, ccs1-4, and CC125 strains representing ~4 kb containing Ccs1 encoding DNA was sequenced. Sequences representing both DNA strands were obtained for the entire region from CC125. For the mutant alleles, the entire gene except intron 7 was sequenced. CCS1 was amplified from genomic DNA in five fragments (Fig. 1B) by using primers sets A = CCS1-1 + CCS1-10, B = CCS1-2 + CCS1-9, C = CCS1-3 + CCS1-12, D = CCS1-4 + CCS1-11, and intron 7 = CCS1-17 + CCS1-20 using either Taq DNA polymerase or ExpandTM DNA polymerase (Roche Molecular Biochemicals). Amplification reactions (25 µl) contained 0.2 mM dNTPs, 0.64 pmol of each primer, 1.5 mM MgCl2, 5% Me2SO in addition to the manufacturer's recommended components. Reactions were preheated at 94 °C for 2 min, prior to the addition of the polymerase, followed by 30 cycles as follows: 94 °C for 30 s; 56 °C for 45 s; 72 °C for 1 min; with a final 7-min extension at 72 °C. Amplification products were gel-purified by the freeze-squeeze method (69), sequenced directly by dye termination cycle sequencing using 3' dye-labeled dideoxynucleotide triphosphates according to the manufacturer's instruction, and run on an ABI PRISMTM DNA Sequencer (PerkinElmer Life Sciences). Sequences were compiled and compared using ABI PRISMTM AutoAssembler program (PerkinElmer Life Sciences). Mutations were confirmed by sequencing multiple independent amplification products.

RNA Preparation and Analysis-- The procedure for RNA isolation has been described previously (70). The abundance of Ccs1 mRNA was estimated by amplification of cDNA under conditions that were suitable for quantitative estimation of transcript abundance relative to Cpx1 transcript abundance. Total RNA was treated with RQ1-DNase (Promega, Madison, WI), phenol/chloroform-extracted, and ethanol-precipitated as preparation for template for reverse transcription. Five µg of treated RNA was used as template for Moloney murine leukemia virus-reverse transcriptase according to the manufacturer's suggested procedure (Invitrogen) using pdN6 random primers (Amersham Biosciences) (1.5 µl/20-µl reaction). Control reactions were set up with same input RNA but without the addition of reverse transcriptase (-RT). 1.5 µl of product was amplified directly in reactions (25 µl) containing 1.5 mM MgCl2, 0.2 mM dNTPs, 0.64 pmol of each primer, 5% Me2SO, and 1.25 units of Taq polymerase (Fisher) with other components as specified by the manufacturer of the enzyme. Ccs1 transcripts or Cpx1 transcripts were amplified with primer sets CCS1-5 and CCS1-6 (Fig. 1B) or CPX1-1 and CPX1-2, respectively (64). Amplifications conditions are as follows: 94 °C for 2 min; 30 cycles at 94 °C for 30 s, 50 °C for 30 s, 72 °C for 45 s and a final extension of 72 °C for 7 min (on a GeneAmp PCR system 2400; PerkinElmer Life Sciences). The yield of both products was dependent on the amount of input RNA (0.05-10 µg). The amount of pdN6 primer was determined to be saturating for synthesis of the cDNA, and the subsequent amplification reaction was in the exponential stage up to 35 cycles. The presence of the H274A mutation was confirmed by SacII digestion of the PCR product.

Generation and Analysis of CcsB-phoA and CcsB-lacZ Topological Reporters-- Eight CcsB-PhoA translational fusions were generated by PCR amplification of various segments of the Synechocystis ccsB gene (slr2087) with Pfu polymerase. The pccsB:phoA plasmids expressing translational fusions of CcsB to PhoA with fusions at positions 23, 67, 134, 225, 288, 349, 410, and 458 of the CcsB polypeptide were constructed as described in the Experimental Procedures of the Supplemental Material. CcsB-encoding PCR products were cloned into pRGK200 (8), in-frame with the downstream phoA gene encoding alkaline phosphatase to yield the series of ccsB:phoA fusion plasmids. The reciprocal ccsB:lacZalpha fusions for all junctions were generated from the series of pccsB:phoA plasmids by replacing a 2.6-kb SalI-PstI fragment including the entire phoA gene with a 0.7-kb PCR-amplified lacZ segment corresponding to the alpha  fragment of beta -galactosidase in-frame with the upstream CcsB moiety. Alkaline phosphatase and beta -galactosidase activities of each CcsB-PhoA and CcsB-LacZalpha fusion were measured for two different clones in three independent assays as described in the accompanying paper (55).

Generation of Site-directed Mutant Strains-- Cys199, His274, and Asp348 were each mutagenized to alanine by overlap extension PCR (71) using Pfu polymerase (Stratagene, La Jolla, CA) and complementary mutagenic primer C199A-1 and C199A-2, H274A-1 and H274A-2, and D348A-1 and D348A-2 (see details in Experimental Procedures of the Supplemental Material). All mutagenized fragments were subcloned into pCcs1 and sequenced to verify introduction of the desired mutation and absence of non-target mutations. In addition to the desired mutation, mutagenic primers also contained silent mutations, which were used to distinguish mutagenized DNA from wild-type DNA. For complementation experiments, 1 µg of SalI-linearized mutant Ccs1 plasmid DNA was transformed into ccs1-6::ARG7 or ccs1-4 as described above. Co-transformants of pCcs1-H274A were generated by transformation of ccs1-4arg7 strain after autolysin treatment with 1 µg of SalI-linearized pCcs1-H274A DNA and 1 µg of pARG7 as described above. Arginine prototrophs were selected by plating on TAP agar plates (-arginine). The presence of the mutated Ccs1 gene was confirmed by amplification of the region containing the mutation followed by diagnostic restriction enzyme digestion of the amplification product.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Ccs1 Gene Corresponds to the CCS1 Locus-- The Ccs1 gene was cloned originally from strain abf3 (now renamed ccs1-5::NIT1). The strain was proposed to be defective in c-type cytochrome synthesis based on a hallmark pleiotropic deficiency in cyt f and cyt c6 (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. Therefore, we assigned the Ccs1 gene to the CCS1 locus. Southern analysis (Fig. 1A) showed the following: (i) the ccs1-3 strain shows an RFLP relative to the wild-type (i.e. DNA from ccs1-3 displays 6.0- and 3.1-kb Ccs1 hybridizing bands instead of a single 6.5-kb hybridizing band in wild-type cells), and (ii) strain ccs 1-6::ARG7 fails to show any hybridizable Ccs1 sequences when probed with the entire 6.5-kb SalI fragment containing the Ccs1 gene (Fig. 1A) or even a larger 8-kb NotI fragment (data not shown). ccs1-6::ARG7 appears to contain at least two copies of the integrated Arg7 containing plasmid (Fig. 1A, marked with asterisks). The integrated Arg7 copies behave as a single locus, because arginine prototrophy co-segregates with the mutant ccs phenotype (data not shown). We conclude that the phenotype of ccs1-6::ARG7 results from the insertion of multiple Arg7 sequences coupled with the deletion of the Ccs1 gene.


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Fig. 1.   Southern analysis of DNA from ccs1 strains and schematic map of CCS1 loci. A, approximately 3 µg of nucleic acids from wild-type, arg7cw15A, and ccs1 strains was subjected to SalI restriction enzyme digestion and analyzed for the presence of Ccs1 or Arg7 by Southern hybridization. For Ccs1, an ~2.3-kb XhoI fragment containing the entire Ccs1 cDNA was labeled with digoxigenin-11-UTP (Roche Molecular Biochemicals) (for the blots shown for strains ccs1-ac206 and ccs1-2) or with 32P-nucleotides by random priming. For strain ccs1-6::ARG7, a radiolabeled 6.5-kb EcoRI/SalI fragment containing the Ccs1 genomic DNA from pCcs1 or a 200-bp NcoI/SacI exon 8 Arg7-specific probe from pKS-18 (86) was used. B, the Ccs1 gene represented as a thick line on map of the 6.5-kb SalI genomic fragment. Exons are represented as numbered large black boxes. The position of mutations in various ccs1 alleles is indicated above, and the position of site-directed mutations is indicated below the gene structure schematic. Lines below the map represent genomic fragments that were amplified to determine the molecular lesions in various alleles. The fragment amplified for RT-PCR analysis is indicated.

To assign unequivocally the Ccs1 gene to the CCS1 locus, we tested all members of the CCS1 complementation group, including four UV-generated mutants (ccs1-ac206, ccs1-2 to ccs1-4) plus ccs1-6::ARG7 by molecular complementation for restoration of phototrophic growth. For all alleles, transformation of the mutants with a plasmid containing 6.5 kb of genomic Ccs1 DNA yielded phototrophic colonies on minimal medium (Table I). No phototrophic colonies were observed when the stains were transformed with empty vector DNA. When individual phototrophic colonies were tested for the presence of the integrated plasmid copy of Ccs1, all were found to be positive relative to the untransformed recipient (representative examples in Fig. 2A). Strain ccs1-2 could not be tested directly for rescue with pCcs1 because it carries a leaky allele and displays appreciable growth on minimal medium. Therefore, we introduced pCcs1 by co-transformation with the dominant ble marker, conferring resistance to zeomycin. Co-transformants of interest were identified among the zeomycin-resistant colonies by specific amplification of the introduced copy of Ccs1. These co-transformants, ccs1-2 (pCcs1), displayed wild-type phototrophic growth and fluorescence rise and decay kinetics (not shown). Several rescued colonies from each transformation were tested by immunoblot and heme stain analysis and found consistently to accumulate wild-type (or near wild-type) levels of cyt f (representative transformant shown in Fig. 2B). Occasionally, slight variability in holocyt f abundance was noted in a particular strain, but this was attributed to positional effects resulting from unique integration of pCcs1 in individual transformants. Because selection for phototrophic growth relies only on restoration of cyt f function, complemented transformants were also tested for cyt c6 accumulation. As expected, copper-deficient transformants accumulated holocyt c6 to approximately wild-type levels, confirming that the transformants were rescued for Ccs function.

                              
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Table I
Complementation of ccs1 alleles by transformation with wild-type Ccs1 gene
ccs1 strains were transformed with either pCcs1 or vector DNA (pTZ18U) and selected for restoration of photosynthetic growth on minimal medium.


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Fig. 2.   Complementation of ccs1 strains by transformation with wild-type genomic Ccs1 DNA. A, genomic DNA from ccs1 strains and representative complemented transformants was amplified. The introduced pCcs1 DNA was distinguished by using a Ccs1-specific primer (CCS1-7) and a plasmid specific primer (Universal). pCcs1 was amplified in parallel to generate a product standard. B, extracts were prepared from copper-deficient cultures of each strain. Proteins from the insoluble membrane fraction (equivalent to 5 µg of chlorophyll) were separated in a 12% polyacrylamide gel under denaturing conditions, and the immunoblots were probed with antiserum against Chlamydomonas cyt f. Total soluble proteins (equivalent to 5 µg of chlorophyll) were separated in 15% polyacrylamide gels under nondenaturing conditions, and immunoblots were probed with antisera against Chlamydomonas cyt c6. Heme-containing proteins immobilized on the membranes were detected by chemiluminescence. Because cyt c6 expression is strictly regulated by copper availability, the extent of holocytochrome c6 accumulation reflects minor differences in cellular copper nutritional status (87).

Ccs1 Accumulation during Cytochrome Biogenesis-- To monitor Ccs1 abundance, we raised antibodies against the putative C-terminal lumenal domain of Ccs1 (see below for topological model). The antiserum recognized a protein of ~60 kDa (Fig. 3). The signal is quite weak and is detected only when freshly prepared membranes were analyzed. When the membranes were purified on gradients or when they were stored (even frozen at -80 °C), the signal became weaker and was difficult to visualize over the noise. Therefore, for immunoblot analysis, we used a rapid method for preparing a thylakoid membrane-enriched fraction, which was solubilized directly and immediately used for electrophoretic separation.


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Fig. 3.   Abundance of Ccs1 under various growth conditions. Enriched thylakoid membrane fractions (corresponding to 50 µg of Chl for each lane) from wild-type cultures were tested for the accumulation of Ccs1 by immunoblot analysis. Wild-type cells were grown in the presence or absence of copper (A) and to different cell densities (B). Solubilized proteins were separated by electrophoresis on SDS-containing polyacrylamide (10%) gels, transferred to polyvinylidene difluoride membranes, incubated overnight with anti-Ccs1 antisera, and detected chromogenically using an alkaline phosphatase-conjugated secondary antibody. Arrows indicate position of Ccs1-specific species. The anti-Ccs1 antiserum specifically recognizes a protein of ~60 kDa that is present in wild-type samples and absent in ccs1 mutants (see Fig. 9).

Previously, we found that coprogen oxidase, a tetrapyrrole biosynthetic enzyme, was induced in copper deficiency, and we attributed this to an increased demand for heme synthesis when cyt c6 was induced (72). Therefore, we wondered whether Ccs1 accumulation might similarly be affected by copper nutritional status. However, we noted that cells adapted to either copper-replete or copper-deficient conditions accumulate the same amount of Ccs1 (Fig. 3A). On the other hand, the abundance of Ccs1 did increase on a per cell basis as the culture grew from log phase to stationary phase (Fig. 3B). At low cell density, we also observed a faster migrating band (marked with faint arrow) whose appearance correlated with cell density, i.e. more predominant in cultures in early exponential growth than stationary phase cultures (Fig. 3B). We know that the faster migrating band is Ccs1-specific because it is absent in immunoblots of ccs1 null mutants (see Fig. 9). At present, it is unclear if the faster migrating molecule is a physiologically relevant species or simply represents a degradation product generated during sample preparation.

The y-1 strain (deficient in the light-independent protochlorophyllide reductase, see Ref. 73) has been used as a model system to study thylakoid membrane biogenesis by light-initiated greening of de-greened cells (74-76). In contrast to chlorophyll proteins, cytochromes do accumulate in dark grown and non-green plastids (77), but their abundance increases along with other components of the thylakoid membrane as the de-greened cells re-assemble their photosynthetic apparatus (78, 79). We hypothesized that Ccs1 would be present in non-green plastids. As expected, both Ccs1 and cyt f are present in dark grown y-1 cells. The abundance of both proteins increased in parallel to each other and with the synthesis of chlorophyll (Fig. 4). Nevertheless, although the accumulation of cyt f requires Ccs1 function, the accumulation of Ccs1 is independent of cyt f. For instance, a petA deletion mutant (FIEB1, 80) accumulates Ccs1 to wild-type levels (data not shown).


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Fig. 4.   Ccs1 and cytochrome f accumulation during greening. A, Ccs1, cyt f, and CF1 (as loading control) were monitored during the greening of y-1 (yellow in the dark) mutant cells. Enriched thylakoid membranes fractions were prepared after the indicated number of hours of light exposure. Protein samples corresponding to 5.25 × 106 cells per lane were separated electrophoretically on an 8.75% SDS-containing gel for immunoblot analysis. B, the parallel accumulation of Chl was determined by organic extraction of Chl and spectrophotometric quantitation.

Functional Analysis and Topology-- With the objective of deducing a functional model of Ccs1, we assembled a multiple alignment of all Ccs1-like sequences (see Supplemental Material Figs. 1 and 2). Based on these alignments, we predicted a topological arrangement of Ccs1 within the thylakoid membrane, and we also identified invariant residues (Fig. 5) (81). The topological predictions indicated that Ccs1 could contain three transmembrane segments in the N-terminal region of the protein followed by a large hydrophilic lumenal loop, followed by a fourth transmembrane span with a weak prediction rating at the C terminus (see hydropathy profiles in Ref. 47). To test the topology predictions, we used a cyanobacterial homologue of Ccs1, Synechocystis CcsB, in phoA and lacZalpha fusions. The relationship between Synechocystis CcsB and chloroplast Ccs1 is obvious, and the model of the cyanobacterial protein should be extendable to the chloroplast situation. Analysis of the fusion constructs confirmed three membrane spans domains clustered at the N terminus of the Ccs1 homologue (Table II). PhoA fusions on the p-side of the membrane, Syn2 and Syn4, show higher alkaline phosphatase activity compared with fusions Syn1 and Syn3 on the n-side. Conversely, Syn1 and Syn3 fusions are more active on the n-side as beta -galactosidase fusions (Table II). Fusion constructs Syn 4-8 (where Syn8 is at the very C terminus of the protein) all show high alkaline phosphatase activity and conversely low beta -galactosidase activity, which is consistent with the p-side location of the entire C-terminal domain. On this basis, we discount the weak prediction of a fourth transmembrane segment and favor the topology diagrammed in Fig. 5.


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Fig. 5.   Topological arrangement of Chlamydomonas Ccs1 in the thylakoid membrane. The predicted topology is based on phoA and lacZ fusion analysis of Synechocystis CcsB and the alignment of C. reinhardtii Ccs1 and Synechocystis CcsB sequences (see Supplemental Material Figs. 1 and 2). The equivalent positions of Synechocystis fusion constructs are indicated by numbered arrows (Syn1 through Syn8). This topology is consistent with the topological prediction based on the "positive-inside" rule (number of basic residues in loop 1: C. reinhardtii = +3, Synechocystis = +1. Number of basic residues in loop 2: C. reinhardtii = +12, Synechocystis = +14 (88)). The point mutations in ccs1 alleles are indicated by open arrows at the position of the mutations. Residues altered by site-directed mutagenesis are shown as follows: C199A and D348A mutations, which did not result in a discernible phenotype are indicated by circles. The H274A mutation, which did result in a ccs phenotype, is indicated by a square. The relative position of the insertion of pNIT1 in the insertional mutant ccs1-5::NIT1 and the breakpoint in the ccs1-3 allele is indicated by an * near the C terminus of protein. Regions within soluble lumenal domain displaying limited blocks of sequence conservation are indicated with bold lines. Region II containing an invariant signature motif VNxP, where x is a polar and generally positively charged residue (51 identifiable sequences, data base search May, 2002), is highlighted in black, whereas regions I and III showing less sequence conservation are highlighted in gray (see Supplemental Material Figs. 1 and 2 for alignment of Ccs1 sequences).

                              
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Table II
Topology analysis of Synechocystis sp. PCC 6803 CcsB by phoA and lacZ fusion analysis
Alkaline phosphatase and beta -galactosidase activities of CcsB fusion proteins expressed in E. coli were measured as described under "Experimental Procedures." At least two representatives of each CcsB fusion were tested for activity. The value is indicated as the mean ± S.D. of three independent measurements for the two representatives. n-side and p-side correspond to the negative and positive side of the membrane, respectively.

An Essential Histidine-- Multiple alignment of Ccs1-like sequences at the outset of these experiments revealed very few residues in Ccs1 that are absolutely conserved (see Ref. 47) and hence might be catalytically significant. Because biochemical analysis of ccs1 mutants suggested that Ccs1 participates in terminal steps of cytochrome synthesis involving attachment of heme to the apoprotein within the thylakoid lumen (43, 44), we considered that Ccs1 might be involved in substrate binding, either heme or apoprotein. Alignment of Ccs1 homologues highlighted three invariant residues, cysteine 199, histidine 274, and aspartic acid 348, with interesting functional groups and potential for interaction with heme. These three residues were chosen for site-directed mutagenesis and were changed to the neutral amino acid alanine. The corresponding alanine encoding mutated versions of Ccs1 were then tested for their ability to rescue strain ccs1-6::ARG7 for photosynthetic growth on minimal medium. Plasmids carrying the C199A and D348A mutations could complement ccs1-6::ARG7. Numerous photosynthetic colonies appeared after transformation (Table III) at frequencies comparable with wild-type (Table II). Representative C199A and D348A transformants were analyzed for the accumulation of holocyt f and holocyt c6 (Fig. 6). As expected from their ability to grow on minimal medium, C199A and D348A transformants were fully capable of synthesizing holocyt f, and under copper-deficient growth conditions, both C199A and D348A transformants were able to synthesize holocyt c6. Therefore, we conclude that cysteine 199 and aspartic acid 348 are not required for Ccs1 function under laboratory test conditions.

                              
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Table III
Complementation of ccs1 alleles by transformation with site-directed mutations in the Ccs1 gene
Site-directed mutations in Ccs1 were tested for their ability to complement ccs1 strains and restore photosynthetic growth when selected on minimal medium.


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Fig. 6.   Accumulation of c-type cytochromes in C199A and D348A mutants. Protein samples from cells transformed with plasmid carrying wild-type, C199A-encoding, or D348A-encoding copies of Ccs1 were prepared as described previously in Fig. 1 and analyzed by immunoblot or heme staining. For cyt f, membrane fractions were separated on a 10% SDS-containing polyacrylamide gel. An equivalent of 10 µg of Chl per sample was loaded. For cyt c6, soluble protein samples were separated on a 15% native gel. An equivalent of 10 µg of Chl per sample was loaded.

On the other hand, H274A failed to rescue either ccs1-6::ARG7 or ccs1-4 (which rescues at high frequency) (Table III), suggesting that histidine 274 is essential for Ccs1 function. To confirm the role of histidine 274, pH274A was introduced by co-transformation of ccs1-4arg7 with pArg7. Thirty-four H274A co-transformants were identified by specific amplification among 81 arginine prototrophs (two transformation experiments). Thirty of the 34 H274A co-transformants failed to show photosynthetic growth on minimal medium and were unable to accumulate cyt f (see Fig. 7, H274A lanes 1, 2, and 4 for representative examples). Four of the 34 H274A co-transformants displayed limited and spotty growth on minimal medium and accumulated ~5% of wild-type levels of cyt f, consistent with their limited photosynthetic capacity (see Fig. 7, H274A lane 3 for a representative). Four of the H274A co-transformants were analyzed in more detail and confirmed by RT-PCR amplification followed by diagnostic restriction digestion of the product to express the H274A mutated version of the Ccs1 mRNA (data not shown). All four confirmed H274A mutants accumulated very low levels of Ccs1 (~2-5% of wild type) (Fig. 7). Three of the four H274A mutants (1, 2, and 4) failed to accumulate either holocyt f or holocyt c6, although very low levels of an anti-cyt f immunoreactive species still accumulated in these transformants. We concluded that the immunoreactive species is the apoprotein form because it is ~0.7 kDa smaller than native holocyt f (corresponding to loss of the heme group) and also the band does not stain for heme. The H274A transformant 3 that displays very limited growth on minimal medium appears to accumulate both the apoprotein and holoprotein forms of cyt f based on the observation of a doublet in Fig. 7. Only the upper band shows heme staining, confirming its identity as holocyt f. Interestingly, transformant 3 does not show any accumulation of either apo or holo form of cyt c6. We conclude that the His274 residue is important for Ccs1 function.


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Fig. 7.   Mutation to invariant His274 results in c-type cytochrome-deficient phenotype. Enriched thylakoid membrane fractions were examined for the accumulation of Ccs1 and cyt f (as described previously). Soluble extracts from copper-deficient cultures were examined for the accumulation of cyt c6. The presence holocytochromes in the samples was examined by heme staining (as described in Fig. 1). Exposures of heme staining representing equivalent intensities for wild-type samples are shown. Dilution series of wild-type, ccs1-4arg7 (recipient strain for transformation), and four independent representative H274A site-directed mutant transformants are shown.

Molecular Analysis of ccs1 Alleles-- The existing collection of ccs1 alleles also provided an opportunity to distinguish functional domains in Ccs1. With this in mind, the mutations in each UV-generated ccs1 allele were identified by sequencing the Ccs1 genomic DNA from each strain (Fig. 1B) (GenBankTM accession numbers AY095299-AY095304). Ccs1 was sequenced also from the corresponding wild-type strain, CC125 (GenBankTM accession number AY095298) (44). The mutations in the ccs1 alleles are summarized in Table IV. In ccs1-ac206, the conserved guanine nucleotide at the 3' splice site junction of intron 4 and exon 5 is mutated to A (Table IV). Because intron 4 is 108 nucleotides in length, failure to splice intron 4 from the ccs1-ac206 mRNA would result in an mRNA encoding an additional 36 amino acids within the predicted stromal loop of Ccs1 (see Fig. 5). The longer species was not detected by RT-PCR; however, immunoblot analysis did reveal a slower migrating species, which could be a translation product from the unspliced longer transcript (discussed below). The ccs1-2 phenotype results from a missense mutation wherein non-conserved glycine 260 is changed to asparagine, ccs1-4 results from a nonsense mutation at codon 144, and the ccs1-3 phenotype appears to be the result of a rearrangement within intron 9, which would be expected to destroy the very C-terminal 77 amino acids of the protein. The re-arrangement was verified by amplification (Fig. 1B) and was consistent with Southern analysis (Fig. 1A). Specifically, fragments A-C could be amplified from ccs1-3 genomic DNA, and sequence analysis confirmed the wild-type sequence. Only the 5' portion of fragment D could be amplified using a primer annealing within exon 9. Primers annealing downstream of exon 9 used in conjunction with the upstream CCS1-12 primer consistently failed to produce amplification products from ccs1-3 DNA, whereas wild-type DNA yielded a product. On this basis, we placed the breakpoint for the genomic rearrangement in ccs1-3 within intron 9 ~6.0 kb from the 5' SalI site. We noted that exon 10 could be amplified from ccs1-3 to yield a product of the expected size, suggesting that the rearrangement event may be an inversion within the gene.

                              
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Table IV
Summary of ccs1 mutations

To assess the effect of each mutation on Ccs1 expression, we assayed for the presence of Ccs1 transcripts by a quantitative PCR-based method (Fig. 8). Reverse-transcribed cDNA was used as the template for amplification using primers that hybridized to exon 4 and exon 6. The amounts of Ccs1 cDNAs were normalized against the amounts of Cpx1 cDNAs, because Cpx1 is expressed constitutively under these conditions, and the level of expression is unaffected in mutants. A product with the size expected for the mature Ccs1 message was amplified from all strains (except ccs1-6::ARG7 in which Ccs1 has been completely deleted). Amplification products corresponding to templates derived from unspliced messages containing intron 4 and/or intron 5 were never observed from any RNA preparation. Strains ccs1-2 and ccs1-3 accumulate Ccs1 transcripts to wild-type levels (Fig. 8A) as do ccs2, ccs3, ccs4, and ccs5 alleles (Fig. 8B), but ccs1-ac206 (mutation at conserved splice site G), ccs1-4 (early nonsense), and ccs1-5::NIT1 (insert in exon 10) accumulate only about 25% of wild-type levels of Ccs1 transcripts. Surprisingly, the ccs1-ac206 mRNA that accumulates seems to be spliced correctly, despite the mutation in intron 4. A product representing mRNA containing the unspliced intron 4 was never observed in the ccs1-ac206 RNA population under the conditions used, even when intron 4-specific primers were used to target such a species (data not shown). The longer protein product (see below) implicates the existence of intron 4-containing mRNA in a translatable pool, but we conclude that the species must be short lived and hence not well represented in the mRNA pool. The decreased abundance of ccs1-4 mRNA is not surprising because non-sense-mediated mRNA decay (82) has been observed previously in Chlamydomonas (65). Previously, we could not detect Ccs1 mRNA in ccs1-5::NIT1 by RNA blot analysis (47). In this work, a small steady state amount is implicated by the RT-PCR results, which indicates that Ccs1 is still being transcribed in the insertional mutant.


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Fig. 8.   Expression of Ccs1 in ccs1 strains and other ccs mutants. C. reinhardtii total RNA was isolated from wild-type or ccs mutants, digested with RQ1 DNase, and used as template for reversed transcription with random pN6 primers. The cDNA corresponding to Ccs1 transcripts were detected by amplification with CCS1-5 and CCS1-6 primers to yield a 345-bp product. A cDNA corresponding to Cpx1 transcripts was amplified in parallel as an internal control using CPX1-1 and CPX1-2 primers resulting in a 634-bp product. Lanes marked -RT show the result of amplification reaction on the same RNA preparations but without reversed transcription. Plasmid DNA containing either Ccs1 cDNA or Cpx1 cDNA were used as templates for amplification with the same primers to generate a standard product (lane marked plasmid). A, Ccs1 transcript abundance in various ccs1 strains. B, Ccs1 transcript abundance in other ccs mutants.

The abundance of Ccs1 in the ccs mutants relative to wild-type cells was examined by immunoblot analysis of thylakoid membranes. The sensitivity was limited to detection of ~2-5% of wild-type levels of Ccs1 (Fig. 9A). Nevertheless, very low amounts of Ccs1 could clearly be seen in membranes from the missense ccs1-2 strain. We suggest that the mutation must de-stabilize the protein, indicating the structural importance of the stromal loop. Membranes from ccs1-ac206 contain a slower migrating form of Ccs1 in addition to very low levels of a wild-type sized Ccs1. The abundance of both forms is highly variable between sample preparations. The larger form is most likely the result of translational readthrough of the unspliced intron 4 in ccs1-ac206, which would add 36 amino acids to Ccs1, and the estimated size of the slower migrating form is consistent with a 4-kDa increase. Ccs1 was not detected in any other ccs1 strain, which is expected from the nature of their molecular lesions. Interestingly, when we examined mutants at other CCS loci, we noted that strains ccsA, ccs2, ccs3, and ccs4 each accumulated only 10-15% of wild-type levels of Ccs1 (Fig. 9B) even though Ccs1 mRNA accumulates to normal levels. The decreased accumulation of Ccs1 in these strains appears to be either translationally or post-translationally controlled (see "Discussion"). On the other hand, ccs5-1::ARG7, a leaky ccs strain that accumulates ~5-10% of wild-type levels of cyt f and cyt c6 (45), accumulates wild-type amounts of Ccs1. Perhaps this suggests significant functional differences in the site of action of Ccs5 versus CcsA, Ccs1 through Ccs4.


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Fig. 9.   Abundance of Ccs1 in various ccs1 strains and other ccs mutants. Enriched thylakoid membrane fractions (corresponding to 50 µg of chlorophyll) from wild-type or ccs strains were tested for the presence of Ccs1 by immunoblot analysis as described in Fig. 3. A, detection of Ccs1 in various ccs1 strains. B, detection of Ccs1 in other ccs mutants.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Topology and Functional Importance of Domains within Ccs1-- Topology studies of Synechocystis sp. PCC 6803 CcsB (cyanobacterial homologue of plastid Ccs1) suggest that Ccs1/CcsB is anchored in the thylakoid membrane by three transmembrane domains within the N-terminal half of the protein, which places the C-terminal half as an extramembrane lumenal domain (Fig. 5). This topology differs slightly from the model for B. pertussis, CcsB (49), in which an additional transmembrane domain at the very C terminus of the protein was proposed, based on positive (albeit low) alkaline phosphatase activity for the full-length C-terminal phoA fusion. In our work, we have used both phoA and lacZalpha fusion analysis to strengthen the model that plastid/cyanobacterial type Ccs1/CcsB has only three transmembrane domains, and the entire C-terminal domain resides on the lumen side of the thylakoid membrane.

The topological model was a prerequisite for deriving insight from molecular analysis of the ccs1 alleles and for building a functional model based on mutational analysis of conserved residues. Our first conclusion is that the stromal loop in Ccs1 is important (Fig. 5). The expected outcome of failure to splice intron 4 in strain ccs1-ac206 is the insertion of 36 codons in the region of the mRNA corresponding to the stromal loop, and indeed, a protein of larger size was observed in ccs1-ac206 (Fig. 9A). Strain ccs1-ac206 also appears to produce normal sized Ccs1 at very low levels, probably from a pool of normally spliced intron 4 (see "Results"). However, holocytochromes do not accumulate (Fig. 2), which is consistent with previous studies that showed by pulse-chase analysis that ccs1-ac206 is completely incapable of holocytochrome formation (43). We conclude that the insertion of additional amino acids in the stromal loop renders the larger form non-functional and that this larger form must exert a dominant-negative effect on the lesser abundant normal Ccs1 population, suggesting Ccs1 associations with other Ccs components in vivo. Blue native-PAGE indicates that Ccs1 is found in an ~200-kDa CcsA-dependent Ccs complex in the thylakoid membrane (see accompanying paper (55)). This size is more than adequate to accommodate two subunits of Ccs1 and/or the products of other CCS loci. The decreased abundance of Ccs1 in ccs2, ccs3, and ccs4 mutants (Fig. 9B) also argues in favor of Ccs1 interactions with additional Ccs components.

The importance of the stromal loop is underscored by molecular analysis of strain ccs1-2 in which mutation of a single residue in the stromal loop, due to a conversion of a non-conserved glycine to an asparagine, results in dramatically reduced Ccs1 accumulation (~2% of wild-type levels). The altered residue lies a mere two amino acids away from a highly conserved pair of residues, 262KG263 in the Chlamydomonas protein. The mutation must destabilize Ccs1 in the membrane, perhaps by affecting interactions with partner subunits. The G260N mutant form of Ccs1 is functional to the extent that it accumulates in the membrane (at least for assembly of holocyt f); between 1-5% of wild-type levels of holocyt f can be observed in thylakoid membranes from ccs1-2 (44), and the strain grows to a limited extent on copper-replete minimal media. However, in copper-deficient medium, ccs1-2 cannot synthesize holocyt c6, and this is clearly evident in pulse-chase experiments (44). The separate effect of the ccs1-2 mutation on cyt c6 versus cyt f accumulation is interesting because it shows that the role of Ccs1 in the assembly of cyt f can be separated from its role in cyt c6 assembly (see also discussion of site-directed mutants, below). Because the mutation occurs within a stromal loop of Ccs1 whereas biochemical evidence clearly places the apocytochrome substrates and site of cytochrome maturation within the lumen, we think it unlikely that the mutation contributes directly to altered interaction with apocytochromes. It is more likely that apocyt f is a kinetically favored substrate in the Ccs assembly pathway. A correlation between apoprotein abundance and the molecular lesions in Ccs1 was not observed in the Chlamydomonas mutants. Indeed, the four potentially null mutants (ccs1-3 through ccs1-6) all accumulate apocyt f and in some cases to appreciable levels (see apocyt f band observed in ccs1 strains in Fig. 2). The definitive evidence for the proposed chaperone function of Ccs1 therefore requires further study.

Molecular analyses of ccs1-3, ccs1-5::NIT1, and a site-directed C-terminal deletion construct, Delta 542-613, highlight the functional importance of the very C-terminal region of the large lumenal domain. Strains ccs1-3 and ccs1-5 result from lesions within intron 9. Both strains do accumulate Ccs1 message and retain the potential to encode at least 536 of the 613 amino acids of Ccs1. However, both strains fail to synthesize either holocytochrome c6 or f as confirmed by radiolabeling experiments (47), and the C-terminal deletion construct (Delta 542-613) fails to rescue ccs1-4 (data not shown). In all three cases, the truncation in Ccs1 occurs after the third region of sequence conservation (Fig. 5, and see Supplemental Material Figs. 1 and 2), yet the protein is non-functional. In the B. pertussis study (49) as well, only the full-length fusion construct was able to complement the ccsB mutant. These results emphasize the functional importance of the entire C-terminal domain and argue for the same topological placement of the C terminus on the p-side of the membrane where cytochrome maturation occurs.

A Non-essential Cysteine and Identification of a Functionally Important Histidine-- Site-directed mutagenesis identified a histidinyl residue, His274, as important for Ccs1 function in the maturation of c-type cytochromes. Two other residues, cysteine 199 and aspartic acid 348 that were absolutely conserved at the start of these studies, were shown by mutagenesis to be non-essential for Ccs1 function. Analysis of 51 Ccs1-like proteins in the data bases (as of May, 2002) revealed that the cysteine residue is totally invariant, but the aspartic acid residue is not conserved. It remains possible that the invariant cysteine residue is important for Ccs1 function but may not be absolutely essential; therefore, the mutation does not present a visible phenotype under the conditions examined. The conserved cysteine is an attractive candidate for participation in the thioreduction pathway involved in cytochrome biogenesis (1, 19). Interestingly, an additional N-terminal domain containing four clustered cysteines, two within a thioredoxin motif, has been identified in the Ccs1 homologue of B. subtilis, ResB (83). This cysteine-rich region is present in all Bacillus ResB proteins currently identified and in Geobacillus stearothermophilus but is lacking in all other identified Ccs1 homologues (data base search May, 2002). The function of this domain remains speculative. Identification and multiple alignments of additional Ccs1 homologues have identified few other conserved residues with attractive functional potential. However, an invariant signature sequence, VNXP, located within the large stromal loop (see Fig. 5) that had not been aligned previously (see Ref. 47) has been highlighted. Whether this sequence element could be involved in an interaction with heme is of future research interest.

The H274A mutated version of Ccs1 is clearly non-functional. The mutated constructs failed to complement ccs1 mutants regardless of the particular allele used for the transformation experiments, and even when a period of recovery in acetate-supplemented medium was allowed after transformation. Detailed analysis of four strains carrying H274A constructs showed that three of the four strains were completely devoid of holocytochromes in the plastid. The mutations must affect a catalytic function of Ccs1 because the mutated protein does accumulate. A fourth transformant was capable of limited holocyt f formation but failed to form holocyt c6, a phenotype that is similar to that of strain ccs1-2. We cannot explain why one transformant makes a small amount of holocyt f. The four H274A transformants appear to contain comparable amounts of Ccs1, but it is possible that the subtle variations in Ccs1 abundance are not revealed by immunoblot analysis, and it may be that small differences in expression of mutant Ccs1 genes (resulting from independent integration events in each transformant and accompanying variations in transgene expression (84)) contributes to variations in the severity of the phenotype. We note that all four H274A transformants accumulate very low levels of apocyt f but no apocyt c6. Perhaps apocyt c6 is more susceptible to proteolysis relative to apocyt f which may be sheltered within the membrane or within the cyt b6f complex. The presence of a small steady state pool of apocyt f but not apocyt c6 may contribute to the preferential synthesis of holocyt f in the histidine transformant discussed above and in the ccs1-2 allele.

In Wolinella succinogenes, a protein designated NrfI is responsible for heme attachment to the unique CXXCK motif of the pentaheme cytochrome c catalytic subunit, NrfA, of nitrite reductase (85). Even though pairwise BLAST analysis (GenBankTM) does not reveal significant sequence similarity between any NrfI and any Ccs1, topology predictions based on multiple alignments of NrfI homologues predicts a similar transmembrane structure for the N-terminal two-thirds of NrfI as for Ccs1. Therefore, NrfI and its homologues may be structurally analogous to a Ccs1/CcsA fusion with three tightly spaced transmembrane domains at the N terminus followed by a large extramembrane domain on the p-side, followed by a C-terminal CcsA-related portion containing multiple membrane-spanning segments and a conserved WWD domain. Alignment of NrfI homologues identifies a conserved histidine in the third transmembrane domain, preceding the large soluble domain and positioned toward the n-side of the membrane span. We wonder if this histidine is analogous to the essential histidine identified in Ccs1 in this work and indicates a common ancestral origin between NrfI and the CcsA/Ccs1 components in system II.

    ACKNOWLEDGEMENTS

We acknowledge the past and present members of the Merchant laboratory for support and intellectual input during the course of this study; J. Leichman and S. Gabilly for technical assistance; Dr. J. Quinn for help during the analysis of Ccs1 expression; and Dr. J. M. Moseley for generation of the ccs1-4arg7 strain and for other help during the course of the project. We thank Prof. J.-D. Rochaix, University of Geneva, for hosting B. W. D in his laboratory during which period the ccs1-6::ARG7 strain was identified.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM48350, NRSA Grant GM17483 from the National Institutes of Health (to B. W. D.), American Heart Association Post-doctoral Fellowship 0120100Y (to P. H.), and a United States Public Health Service NRSA Award GM07185 from the National Institutes of Health (to S. S. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Experimental Procedures, Figs. 1 and 2, and Table I.

Dagger To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, UCLA, Box 951569, Los Angeles, CA 90095-1569. Tel.: 310-825-8300; Fax: 310-206-1035; E-mail: merchant@chem.ucla.edu.

Published, JBC Papers in Press, November 9, 2002, DOI 10.1074/jbc.M208652200

2 For consistency, the ccm nomenclature for the system I genes will be employed throughout.

    ABBREVIATIONS

The abbreviations used are: Ccm, cytochrome c maturation; ccs, cytochrome c synthesis; Chl, chlorophyll; cyt, cytochrome; Me2SO, dimethyl sulfoxide; RT, reverse transcription.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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