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Originally published In Press as doi:10.1074/jbc.M404285200 on May 24, 2004

J. Biol. Chem., Vol. 279, Issue 31, 32474-32482, July 30, 2004
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A Homolog of Prokaryotic Thiol Disulfide Transporter CcdA Is Required for the Assembly of the Cytochrome b6f Complex in Arabidopsis Chloroplasts*

M. L. Dudley Page{ddagger}§, Patrice P. Hamel{ddagger}§, Stéphane T. Gabilly{ddagger}, Hicham Zegzouti||, John V. Perea**, José M. Alonso{ddagger}{ddagger}, Joseph R. Ecker{ddagger}{ddagger}, Steven M. Theg**, Sioux K. Christensen||, and Sabeeha Merchant{ddagger}§§

From the {ddagger}Department of Chemistry and Biochemistry and the ||Department of Molecular, Cell, and Developmental Biology, UCLA, Los Angeles, California 90095, the **Division of Biological Sciences, Section of Plant Biology, University of California, Davis, California 95616, and the {ddagger}{ddagger}Genomic Analysis Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037

Received for publication, April 19, 2004 , and in revised form, May 18, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The c-type cytochromes are defined by the occurrence of heme covalently linked to the polypeptide via thioether bonds between heme and the cysteine sulfhydryls in the CXXCH motif of apocytochrome. Maintenance of apocytochrome sulfhydryls in a reduced state is a prerequisite for covalent ligation of heme to the CXXCH motif. In bacteria, a thiol disulfide transporter and a thioredoxin are two components in a thio-reduction pathway involved in c-type cytochrome assembly. We have identified in photosynthetic eukaryotes nucleus-encoded homologs of a prokaryotic thiol disulfide transporter, CcdA, which all display an N-terminal extension with respect to their bacterial counterparts. The extension of Arabidopsis CCDA functions as a targeting sequence, suggesting a plastid site of action for CCDA in eukaryotes. Using PhoA and LacZ as topological reporters, we established that Arabidopsis CCDA is a polytopic protein with within-membrane strictly conserved cysteine residues. Insertional mutants in the Arabidopsis CCDA gene were identified, and loss-of-function alleles were shown to impair photosynthesis because of a defect in cytochrome b6f accumulation, which we attribute to a block in the maturation of holocytochrome f, whose heme binding domain resides in the thylakoid lumen. We postulate that plastid cytochrome c maturation requires CCDA, thioredoxin HCF164, and other molecules in a membrane-associated trans-thylakoid thiol-reducing pathway.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The c-type cytochromes are a virtually ubiquitous yet rather structurally diverse group of hemoproteins that reside on the so-called p-side1 of the energy-transducing membrane systems where they function typically as electron carriers (1, 2). The c-type cytochromes are defined by the occurrence of heme covalently attached to the polypeptide via two thioether linkages between the vinyl groups of heme and the cysteine sulfhydryls in the apocytochrome (1, 3). A CXXCH2 sequence in the apocytochrome provides the thiols for formation of the thioether bonds, and the imidazole of the histidine residue serves as one of the axial ligands of the heme. Remarkably, three distinct assembly pathways, referred to as systems I, II, and III, have emerged through genetic analysis of c-type cytochrome maturation in bacteria, chloroplasts, and mitochondria (for review, see Refs. 1 and 4–7), an unexpected finding for what appears on the surface to be a rather simple chemical reaction (i.e. the addition of apocytochrome cysteine thiols to the vinyls of heme). Each system can be distinguished on the basis of a sequence relationship of specific assembly factors. Yet, there is an underlying assumption that the three systems are united by common biochemical requirements for holocytochrome c biogenesis (for review, see Refs. 2, 4, and 8). For instance, the need for reducing conditions appears to be inherent to the chemistry of thioether bond formation (4). Both substrates, apocytochrome c sulfhydryls and heme, need to be maintained reduced prior to the ligation of heme as indicated from in vitro and in organello experiments (for review, see Refs. 4 and 9).

In bacteria, c-type cytochromes are assembled in the periplasm via system I or system II (10, 11). In the bacterial periplasm, a disulfide bond formation machinery (Dsb system in Gram-negative and Bdb system in Gram-positive) promotes the oxidative folding of proteins, which is counter to the requirement for apocytochrome cysteinyl thiols in the heme attachment reaction (12, 13). The current thinking is that apocytochromes c upon translocation to the p-side are first a substrate of the Dsb/Bdb system which introduces intramolecular disulfides between the two cysteines of the CXXCH sequence. Subsequently, the disulfide bond undergoes reduction to provide free sulfhydryls that are competent to react with heme.

The requirement for redox chemistry in the context of in vivo cytochrome c assembly was appreciated through the discovery in bacteria of thiol-metabolizing components in systems I and II that constitute a transmembrane thio-reduction pathway (12–14). The components of this pathway include a sulfhydryldisulfide membrane transporter that conveys reducing power from the cytoplasm to the periplasmic space and a dedicated membrane-anchored thioredoxin that reduces the heme binding site of apocytochrome c prior to the heme attachment (13–15).

Plastid c-type cytochromes are assembled through system II (5, 16–21), but the requirement for redox chemistry in cytochrome maturation has remained so far largely unexplored in this organelle. We suspected the operation of a redox subpathway in the plastid cytochrome assembly based on the occurrence of a CcdA homolog encoded in the plastid genome of a red alga, Porphyra purpurea, and in the nuclear genomes of green alga Chlamydomonas reinhardtii and vascular plant Arabidopsis thaliana. CcdA is related to the DsbD/DipZ family of membrane polytopic proteins (22) that functions in transmembrane transfer of thiol-reducing equivalents from the bacterial cytoplasm to the periplasm (12, 13, 23). Loss-of-function of CcdA in bacteria results in a pleiotropic deficiency in c-type cytochromes, presumably because of a block in the reduction of apocytochrome c thiols in the heme binding site (24–28). Interestingly, CcdA is physically linked to a gene encoding a homolog of Ccs1, a system II-specific cytochrome c biogenesis (19) in P. purpurea, Mycobacterium spp., and several cyanobacteria (6). This gene clustering speaks to a role for CcdA in the plastid cytochrome c maturation pathway, but no biochemical or genetic evidence is presently available to assign a function to the plant or cyanobacterial CcdAs.

In this work, we investigated the function of Arabidopsis CCDA in plastid cytochrome c assembly. In vitro import experiments and topological analysis using PhoA and LacZ established that the CCDA polypeptide is a polytopic membrane protein that is targeted to the plastid organelle. We have isolated T-DNA and Ds (dissociation) element insertions in the CCDA gene and shown that loss-of-function alleles resulted in photosynthetic incompetence at the level of cytochrome b6f assembly. We propose that impaired accumulation of cytochrome b6f is a consequence of a defect in holocytochrome f maturation, a c-type cytochrome resident in the plastid lumen. We hypothesize that CCDA is a component of a thylakoid transmembrane thioreduction pathway that operates in the delivery of reducing equivalents to apocytochrome f prior to its conversion to the holoform.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains and Molecular Biology Techniques—Bacterial strain Escherichia coli XL1-Blue and DH5-{alpha} were used as hosts for recombinant techniques. Strain LMG194 (phoA–) was used to measure PhoA activity of the sandwich constructs, and strain CC118 (phoA– lacZ–) was used to measure PhoA and LacZ activities. The sources of DNAs used in this work are provided in the on-line supplement.

Constructs for Chloroplast Import Experiments—The AtCCDA, NAtCCDA-DHFR,3 and N-AtPC-DHFR constructions were designed in the pGEM2 vector. AtCCDA corresponds to the entire ORF of the CCDA gene. N-AtCCDA-DHFR expresses the first 141 amino acids of Arabidopsis CCDA fused to the entire coding sequence of the mouse DHFR. N-AtPC-DHFR was engineered by fusing the sequence encoding the 77 amino acids of the plastocyanin chloroplast-targeting sequence plus the first 5 residues of the mature protein to the DHFR sequence. All gene fusions were under the control of the SP6 promoter that enables in vitro transcription. The details of the molecular cloning are described in the supplemental material.

Chloroplast Import Experiments—In vitro protein import reactions were performed with chloroplasts isolated from pea seedlings (29). Radiolabeled proteins were translated in wheat germ extract (Promega). Reactions were performed in the light for 20 min prior to thermolysin treatment (29).

Construction of CCDA-phoA, CCDA-lacZ, and CCDA-phoA Sandwich Fusions—Plasmid pRGK200 (30) was used to generate seven CCDA-phoA and seven CCDA-lacZ translational fusions (I–VII) by a PCR-based strategy. The phoA sandwich fusion approach consists of the design of fusion proteins where PhoA is inserted into an otherwise intact target protein (31). Two CCDA-phoA sandwich fusions carrying an insertion of phoA into CCDA at position 202 (SWA) or 242 (SWB) of the CCDA protein were constructed in the expression vector pBAD22, a plasmid that harbors an arabinose-inducible phoA gene (32). The details of the molecular cloning are provided in the supplemental material.

Measurement of PhoA and LacZ Activities—PhoA assays were performed as described previously in (33), except that isopropyl-1-thio-{beta}-D-galactopyranoside was not used for induction. LacZ activity was measured as detailed in Ref. 34. PhoA assays of the sandwich fusions were performed on overnight grown cultures supplemented with 100 µM L-arabinose for induction. PhoA activity expressed in Miller units was calculated using Equation 1,

(Eq. 1)
where t is time in minutes. LacZ activity is expressed in units and was calculated by Equation 2,

(Eq. 2)
where t is time in minutes.

Growth of A. thaliana—Col-0 seeds originated from Lehle Seeds, Round Rock, TX. Plants were propagated on solid minimal media comprising either the commercial soluble fertilizer Peters Professional General Purpose 20-10-20 (35) (Western Farm Services) or 0.5x Murashige & Skoog (MS) minimal medium, solidified with 0.8% (w/v) Select agar (Invitrogen), and containing 0.5 or 5% sucrose, or on a soil mix (Sunshine Professional Basic Mix 2; Sun Gro, Canada). Plants were grown at 24 ± 0.5 °C under white light (80–120 µE/m2/s) with a light/dark cycle of 16/8 h.

Extraction of Plant Genomic DNA and mRNAs—A. thaliana genomic DNA for Southern hybridization was prepared as described in Ref. 36, except that plant material was frozen in liquid nitrogen and ground to a powder prior to extraction. For PCR amplification, genomic DNAs were extracted as in Ref. 37. Total RNA from plants was isolated with an RNeasy Plant Mini Kit (Qiagen) starting from 100 mg of plant tissue according to the manufacturer's procedure. Following the elution step, purified RNAs were treated for 1 h with RNase-free DNase (Stratagene) to remove any contaminating genomic DNA and phenol-chloroform extracted. To confirm the absence of any contaminating DNA, 50 ng of RNA was subjected to a PCR amplification using the same primers that were used in the RT-PCR analysis.

PCR and RT-PCR Amplifications—PCR amplifications performed as part of the University of Wisconsin-Madison screening process were carried out using TAKARA Ex-Taq under the conditions recommended by the manufacturer. The reverse transcription reaction was performed using the RT system (Promega); see the supplemental material.

Screening and Molecular Characterization of ccda Mutant Lines— The PCR-based service provided by the Arabidopsis Knockout Facility at the University of Wisconsin-Madison Biotechnology Center was used to screen 60,480 T-DNA insertion lines, and one insertion in the CCDA gene was identified (ccda-1). The ET5250 line carrying a Ds element insertion (ccda-2), the SALK_025766 line (ccda-3) and SALK_014416 line (ccda-4), both harboring a T-DNA insertion in CCDA, were identified through querying data bases of sequenced insertion sites (Cold Spring Harbor laboratory and Salk Institute Genomic Analysis Laboratory, respectively). The details of the screening and molecular characterization of the mutant lines are available in the supplemental material.

Fluorescence Measurements—The fluorescence imaging system, Fluorcam 700 MF (Photon Systems Instruments Ltd.), was used to record chlorophyll fluorescence induction and decay kinetics of Arabidopsis plants that had been dark-adapted for at least 5 min. Plants were illuminated for 20 s under actinic light of 50 µE/m2/s. Emitted fluorescence was captured by the camera, and the collected data were converted into a graph using Excel software (Microsoft).

Protein Preparation and Analysis—To extract proteins, plant tissue was frozen in liquid nitrogen, ground to a powder, and ground further in ice-cold extraction buffer (50 mM Tris-HCl, pH 8.0, 5 mM EDTA). The homogenates were centrifuged at 13,000 x g for 15 min at 4 °C, and the pellets, enriched in membrane proteins, were washed twice and resuspended in extraction buffer (0.5–2.0 ml/g of fresh tissue). Protein concentration was determined using the bicinchoninic acid assay (38). In-gel heme stain of holocytochome f was performed using TMBZ (39). Immunoblotting of proteins separated by SDS-PAGE was performed as in Ref. 40, except that samples were heated at 37 °C for 30 min. Antibody dilutions were 1:1,000 for anti-CF1 (spinach), anti-Cox1, anti-cyt f, anti-subunit IV/PetD (C. reinhardtii), and anti-HCF164 (A. thaliana); 1:2,000 for anti-Sec12 (A. thaliana) and anti-cyt b6 (C. reinhardtii); and 1:3,000 for anti-Rieske FeS (maize). A 1:3,000 dilution of alkaline phosphatase-conjugated goat anti-rabbit IgG was used as the secondary antibody. Sample loading was adjusted so that each sample contained the same amount of the {alpha} subunit of CF1. Quantitation of the bands was performed using a MultiImage system running ChemImager 4400 software (Alpha Innotec).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
A Homolog of Prokaryotic Thiol Disulfide Transporter CcdA in Photosynthetic Eukaryotes—We had noted the occurrence of a CcdA-encoding gene in the plastid genome of a rhodophyte alga, P. purpurea and also in the genomes of several cyanobacteria (6). With red algal CcdA as our prototype, we searched the nucleotide sequence data bases and identified expressed sequence tags corresponding to CcdA-like proteins from a number of photosynthetic eukaryotes, including Chlamydomonas, Arabidopsis, and several other vascular plants.4 A full-length cDNA was identified for each and sequenced.5 A multiple alignment of the conceptual protein sequences (see Fig. 1 in the supplemental data) confirmed that the two cysteines that are essential for the redox activity of bacterial CcdA (26) are invariant in the plant homologs and revealed that all display an N-terminal extension relative to the plastid-encoded and prokaryotic CcdA proteins, suggesting plastid localization of the nucleus-encoded plant homologs. All eukaryotic CCDAs displayed the features of polytopic membrane proteins with six predicted transmembrane segments when analyzed in silico for the presence of hydrophobic domains (Fig. 1 in the supplemental data). To assess the function of plant CCDA, three specific questions were addressed: 1) Where is CCDA localized? 2) What is its topological arrangement? and 3) What is the phenotype of loss-of-function ccda plants?

Arabidopsis CCDA Is Targeted to the Plastid—The direct approach of immunolocalizing CCDA polypeptide after cell fractionation was not successful because the antisera we raised reacted very weakly if at all with native CCDA, perhaps because it is a low abundance hydrophobic protein (data not shown). Therefore, we chose to localize the in vitro synthesized radiolabeled protein after in vitro import into pea chloroplasts. We certainly expected CCDA to localize to chloroplasts based on the occurrence of the gene in the less derived red algal plastid genome of P. purpurea (6). Indeed, AtCCDA could be imported into isolated chloroplasts and was processed to the mature form, suggesting that the N-terminal extension functions as a chloroplast-targeting presequence in vitro (Fig. 1A). We further solidified this model by showing that the 141-amino acid N-terminal extension on CCDA could target a passenger protein like the widely used reporter, DHFR, to the chloroplast in vitro (Fig. 1B). As a positive control for import, we used the 77-amino acid targeting sequence of plastocyanin, a known thylakoid lumen resident protein. As expected, the targeting sequence of plastocyanin is able to drive import of DHFR into the chloroplast (Fig. 1C). We concluded that CCDA is targeted to the chloroplast via its N-terminal extension and that its plastid location is compatible with a function in chloroplast cytochrome c biogenesis.



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  		FIG. 1.
The N terminus of Arabidopsis CCDA contains a chloroplast transit peptide. A, pre-CCDA is imported into plastids and processed to a mature form. B and C, import of DHFR chimeras containing the N termini of the precursors of CCDA and plastocyanin (PC), respectively. Protein import reactions were performed as described under "Experimental Procedures"; the resulting fluorographs are shown. DHFR import driven by a known targeting sequence (AtPC) from preapoplastocyanin is shown for comparison (C). Lanes marked TP were loaded with translation product equivalent to 20% of that run in the import reaction; lanes marked + and – refer to the postreaction addition (or not) of thermolysin; p and m mark the positions of the precursor and mature forms of the translated proteins, and the positions of molecular mass markers are indicated as kDa on the left of each gel: AtCCDA (40 kDa) N-AtCCDA-DHFR (36.5 kDa), and N-AtPC-DHFR (29.5 kDa) run as expected from their calculated molecular masses.

 
Plastid CCDA Is a Polytopic Membrane Protein with Within-membrane Invariant Cysteines—To verify experimentally the predicted six-transmembrane segment model for chloroplast CCDA and, in particular, to locate the two universally conserved cysteines, we used PhoA and LacZ as topological reporters in bacteria. This approach relies on the topological analogy in a bioenergetic sense between the p-side and n-side compartments in bacteria (periplasm/cytoplasm) and those in plastids (lumen/stroma) and is justified by the previous establishment of its reliability for the analysis of CcsA, a thylakoid membrane polytopic protein (21). Translational fusions between CCDA and PhoA or LacZ were engineered at predicted periplasmic and cytoplasmic loops. The topology was deduced on the basis of measured LacZ and PhoA activities of the fusions proteins expressed in E. coli (Fig. 2 and Table I). High PhoA activities indicate a p-side location for the insertion site of the fusion because PhoA is active only in the periplasm. Reciprocally, fusions with high LacZ activity confirm association of the {alpha} and {omega} fragments of LacZ in the cytoplasm, and therefore an n-side location of the fusion site can be deduced. Location of the third extramembrane loop to the n-side or the p-side of the membrane could not be ascertained because both CCDA-PhoA and CCDA-LacZ fusions at position 236 of the polypeptide display high enzymatic activity (Table I, see fusion IV). We have ruled out that this is the result of a position effect at this particular site because fusions at positions 243, 249, and 254 in CCDA, all predicted to be located on the n-side, also led to high PhoA activity (data not shown). To overcome the ambiguity concerning the location of the third loop, we exploited the so-called sandwich approach, which offers greater reliability for examining membrane protein topology (31). In this method, PhoA is inserted in an otherwise intact CCDA protein (see arrows marked SW in Fig. 2). The measurement of PhoA activity of sandwich constructs SWA and SWB yielded a definitive placement of the third loop to the stromal n-side (Table I). Using an anti-PhoA antibody, we were able to detect the CCDA-PhoA sandwich protein in the membrane fraction of bacterial cells expressing the SWA construct (data not show). We could not detect the CCDA-PhoA sandwich protein expressed from the SWB construct, suggesting that the protein is not stable and undergoes degradation. This has been observed for fusion proteins where the cytoplasmic form of the alkaline phosphatase often fails to fold into a stable structure (26, 41). The experimentally deduced model is in agreement with the topological prediction and consists of a six-transmembrane protein with N and C termini on the lumen side.



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  		FIG. 2.
Topological model of Arabidopsis CCDA. The topological arrangement of Arabidopsis CCDA in the thylakoid membrane was deduced according to PhoA/LacZ fusion analysis in bacteria (Table I). The p-side corresponds to the lumen of the thylakoid, and the n-side corresponds to the stroma of the chloroplast. The transmembrane helices of CCDA as predicted from in silico analysis (67) are represented by gray rectangles within the lipid bilayer. Thick lines feature extramembranous regions of the CCDA polypeptide. The arrows indicate, within CCDA, the positions of in-frame alkaline phosphatase and {beta}-galactosidase fusions. Fusions at positions 107 (I), 181 (II), 202 (III and SWA), 236 (IV), 242 (SWB), 277 (V), 322 (VI), and 353 (VII) are indicated on the drawing by arrows. Strictly conserved cysteine residues that lie within the membrane are marked C.

 


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  		TABLE I
CCDA-PhoA and -LacZ activities

Alkaline phosphatase and {beta}-galactosidase activities of CCDA fusion proteins expressed in bacteria were measured as described under "Experimental Procedures." At least two representatives of each CCDA fusion that was generated by PCR were tested for activity. PhoA activities (Miller units) and LacZ activities (A.U.) values are the means of three independent measurements. n-side and p-side correspond to the negative and positive sides of the membrane, respectively. NA, not applicable.

 
Identification of Mutants with T-DNA and Ds Element Insertions in the CCDA Gene—To assess plant CCDA function, we sought next to take a reverse genetic approach. This was facilitated by the possibility to screen for T-DNA inserts in genes of interest in Arabidopsis (42, 43) and to obtain mutant lines from publicly available library stocks. Four candidate ccda mutant lines corresponding to one enhancer trap line with a Ds element (44) and three T-DNA lines (43) were identified experimentally by screening pooled genomic DNAs (42) at the PCR-based Arabidopsis Knockout Facility or by querying data bases of sequenced insertion sites. The exact positions of the insertion sites of the T-DNAs and the Ds elements were determined by sequencing PCR products encompassing the genomic border of the integration site (Table II). The Ds element insertion site in allele ccda-2 is in the 5'-untranslated region of the CCDA gene, 15 bp upstream from the putative start codon (Fig. 3 and Table II). The three T-DNA insertion sites mapped to the 5'-untranslated region, 23 bp upstream from the start codon (ccda-3 allele), to exon II (ccda-4 allele) and to intron III (ccda-1 allele) of the CCDA gene (Fig. 3 and Table II).


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  		TABLE II
T-DNA and Ds element integration sites in A. thaliana mutant lines

Underlining indicates the 8 bp of A. thaliana DNA duplication accompanying the insertion of Ds element in the CCDA gene. ATG- marks the CCDA start codon. {blacktriangledown} indicates the position of the integrated DNA, and {triangleup} (bp) shows the size of the deletion of A. thaliana genomic DNA upon integration of the interrupting element.

 



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  		FIG. 3.
Arabidopsis CCDA genomic locus. The genomic locus of Arabidopsis CCDA is drawn as a thick line and includes 12 exons (numbered black boxes) and 11 introns. Genomic DNA flanking the CCDA locus is drawn as a gray line. ATG and TAA indicate initiation and stop codon of CCDA ORF, respectively. The insertions of an interrupting element are indicated as gray (Ds) or white (T-DNA) arrows. The directionality of the interrupting element is indicated by the orientation of the arrow (left border -> right border for the T-DNA and 5' -> 3' for the Ds element). Primers used for molecular characterization of the ccda alleles are drawn as thin arrows.

 
Plants hemizygous for insertions at the CCDA locus were generated and self-fertilized to monitor Mendelian transmission of the interrupting elements. This was achieved by scoring phenotypically for kanamycin resistance conferred by the nptII marker present on the T-DNA and Ds elements and molecularly by DNA hybridization analyses (see "Experimental Procedures" and Fig. 2 in the supplemental data).

The progeny arising from self-fertilization of the heterozygous ccda-1 plant exhibited a kanr:kans ratio of 322:17, or ~15:1, suggesting the presence of two segregating T-DNA insertions/genome. This was confirmed by Southern hybridization (Fig. 2A in the supplemental data), and heterozygous plants carrying only the ccda-inserted T-DNA were identified (Fig. 2A in the supplemental data). These were grown to maturity and allowed to self-pollinate to generate the ccda-1 homozygous line.

The progeny of self-fertilized CCDA/ccda-4 plants exhibited a kanr:kans ratio of 72:26, or ~3:1, suggesting the presence of a single segregating T-DNA/genome. This was confirmed by Southern hybridization (Fig. 2B in the supplemental data). The progeny of self-fertilized CCDA/ccda-2 and CCDA/ccda-3 plants exhibited kanr:kans ratios of ~3:1 and 15:1, indicative of the segregation of one single Ds element and two T-DNA elements, respectively. Preliminary characterization revealed that plants homozygous for ccda-2 and ccda-3 alleles could be grown to maturity on soil (see below), and further clean-up of the ccda-3 allele carrying line was omitted.

The molecular arrangement of each insertion in the CCDA gene was analyzed by PCR using T-DNA/Ds-specific and CCDA-specific primers (see supplemental material). This analysis indicated that the T-DNA insertions in ccda-1, ccda-3, and ccda-4 are all tandem or complex insertions carrying two left borders at their extremities (45). In the case of the ccda-2 allele, the simplest interpretation is the integration of a single Ds element (Fig. 3 and Fig. 3 in the supplemental data).

Growth Characteristics and Fluorescence Transients of the ccda Mutant Lines—Because loss of CCDA function is anticipated to block assembly of plastid c-type cytochrome, holocytochrome f, and consequently impair photosynthesis, homozygous ccda-interrupted plants were examined at the level of growth and photosynthetic electron transfer by recording chlorophyll fluorescence induction and decay kinetics. For fluorescence measurements, plants were propagated as segregating populations on MS medium containing 0.5% sucrose, and each plant was subsequently genotyped by PCR to identify ccda homozygotes. Plants homozygous for either the ccda-2 or the ccda-3 allele were phenotypically indistinguishable from wild type lines (data not shown). The plants grew on soil, flowered, set seeds normally, and behaved as their corresponding wild type with respect to photosynthetic electron transfer (data not shown). Homozygotes for the ccda-1 allele grew very slowly on soil and remained pale green (Fig. 4 and not shown), but after 60 days the plants, although quite small, produced a few flowers and set seeds. Consistent with the growth defect, mutant plants displayed a leaky hcf (high chlorophyll fluorescence) phenotype (Fig. 4) typical of photosynthetic mutants that are not completely blocked in the function of the b6f complex (46). Of 77 plants analyzed in the segregating population, 63 exhibited wild type fluorescence and 14 a hcf phenotype, making a ~3:1 ratio ({chi}2 = 1.06). That the hcf phenotype cosegregated with the ccda-1 genotype suggested that the T-DNA integration in CCDA was responsible for the lesion we observed. Mutants homozygous for ccda-4 were seedling-lethal (not shown) and could only grow on a minimal synthetic medium supplemented with 0.5% sucrose, as expected for photosynthesisminus Arabidopsis plants (46). The ccda-4 homozygous plantlets obtained on 0.5% sucrose-containing medium were about half the size of the wild type and heterozygous segregants grown in the same conditions (not shown). They displayed a tight hcf phenotype consistent with a complete loss of the cytochrome b6f complex and hence photosynthetic growth (Fig. 4). Of 167 plants examined among the segregants, 131 exhibited wild type fluorescence and growth and 36 a hcf and reduced growth phenotype. This 3:1 distribution ({chi}2 = 0.554) and the fact that the hcf phenotype cosegregated with the ccda-4 genotype suggested that the observed phenotype is attributable to the T-DNA integration. Interestingly, growth in the presence of 5% sucrose resulted in ccda-4 homozygous plants that were similar in size to the corresponding wild-type and heterozygous segregants but which produced only a few small flowers that did not fully open (data not shown).



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  		FIG. 4.
ccda-1 and ccda-4 homozygous plants are photosynthetically incompetent. A, Col-0 ecotype wild type (WT), ccda-1 and ccda-4 homozygous plants are shown 20 days after germination on soil. B, the fluorescence induction and decay kinetics observed in a dark to light transition for ccda-1 and ccda-4 homozygous mutants are shown compared with those of Col-0 ecotype wild type. Ws ecotype wild type fluorescence transient is not shown but was found to be similar to Col-0 wild type. The continuously rising curve for the ccda mutant is the signature of a specific block in electron transfer at the level of cytochrome b6f complex because of its impaired assembly in the absence of membrane-bound holocytochrome f. When the energy absorbed by the chlorophyll cannot be utilized because of a block in photosynthetic electron transfer, an increase in the chlorophyll fluorescence is observed. Note the absence of the decay phase corresponding to reoxidation of the quinone pool (the primary electron acceptor of photosystem II) by the cytochrome b6f complex.

 
The direct correlation between the photosynthetic phenotype of the ccda mutant lines and the severity of the molecular lesions suggest that the level of expression of the CCDA gene product is directly responsible for the observed phenotype.

Analysis of CCDA Transcripts in ccda Mutant Lines—We monitored the level of expression of the CCDA gene in the different mutant lines by RT-PCR analysis. As shown in Fig. 5A, both ccda-2 and ccda-3 homozygous lines that display no visible phenotype still express the CCDA mRNA, suggesting that the insertion of T-DNA or Ds elements in the 5'-untranslated region did not abolish the transcriptional activity of the CCDA gene. By contrast, plants carrying the ccda-1 and ccda-4 mutations that result in impaired photosynthesis do not accumulate the CCDA mRNA, and consequently, those mutations correspond to loss-of-function type of alleles. However, the difference in the severity of the photosynthetic phenotypes caused by the ccda-1 or ccda-4 alleles suggests that those mutations do not both cause a complete lack of function of the CCDA protein. By RT-PCR, we were able to show that both mutant lines expressed a truncated form of the CCDA mRNA (Fig. 5B). One possible explanation for the phenotypic differences is that in ccda-4, the mRNA does not encode a functional protein, whereas in ccda-1 the truncated protein still exhibits some activity.



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  		FIG. 5.
Expression of the CCDA gene in the ccda mutant lines. RT-PCR experiments were performed as described under "Experimental Procedures." PCR amplification products were separated by agarose gel electrophoresis and visualized after staining with ethidium bromide. A, amplification of CCDA (from ATG to exon 7), petA (cytochrome f), and petB (cytochrome b6) transcripts in ccda mutant lines and corresponding wild type ecotypes using RT-F12, RT-R4, petA-L, petA-R, and petB-L, petB-R primers, respectively (see Table I supplemental data). B, left, amplification of T-DNA (Ds)/CCDA hybrid transcript in ccda-3 and ccda-4 lines using T-DNA-specific RT-LB1 primer and CCDA-specific RT-R4 primer and in ccda-2 line using Ds element-specific primer Ds-RT1 and CCDA primer RT-R4. Right, amplification of the 3'-end of the CCDA transcript (from exon 5 to exon 7) in the ccda-1 line and corresponding wild type ecotype with CCDA-specific primers RT-F5 and RT-R4 (see Table I supplemental data). The predicted sizes of the PCR products are: CCDA (from ATG to exon 7), 672 bp; petA, 158 bp; petB, 185 bp; Ds-CCDA hydrid in ccda-2, 697 bp; T-DNA-CCDA hybrid in ccda-3, 726 bp; T-DNA-CCDA hybrid in ccda-4, 599 bp, CCDA exons 5–7, 209 bp.

 
As a control, we showed that the ccda mutations do not modify the expression of chloroplast petA and petB genes encoding apocytochrome f and apocytochrome b6, respectively (Fig. 5A).

Mutations in CCDA Result in a Defect in Cytochrome b6f Complex Accumulation—We assessed the abundance of holocytochrome f in the mutant lines by heme staining and immunoblotting and also that of Rieske FeS, cytochrome b6, and subunit IV, which are subunits of the cytochrome b6f complex. As expected from the lack of phenotype in the ccda-2 and ccda-3 lines, holocytochrome f accumulation is not affected by these mutations, and the molecule assembles to essentially wild type level (Fig. 6). By contrast, the ccda-1 mutation causes a severe reduction in the level of holocytochrome f. Only 20–25% of wild type levels of holocytochrome f is detected in the ccda-1 line. Concomitant with the diminution in holocytochrome f abundance, we observed a decrease in the accumulation of the subunits of the cytochrome b6f complex (Fig. 6). The ccda-4 mutation that leads to a more drastic phenotype than the ccda-1 mutation has a more pronounced effect on holocytochrome f accumulation. Only 10% of wild type level of cytochrome f is assembled in this mutant line, and the abundance of cytochrome b6f subunits is also likewise severely reduced (Fig. 6). We concluded that loss-of-function ccda alleles lead to a defect in plastid biogenesis that is the result of impaired accumulation of the cytochrome b6f complex in the thylakoid membranes.



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  		FIG. 6.
Accumulation of cytochrome b6f subunits in ccda mutants. Protein fractions were prepared from entire wild type (Ws-0 and Col-0) and homozygous ccda-1, ccda-2, ccda-3 plants grown on MS medium supplemented with 0.5% sucrose, or from wild type (Col-0) and ccda-4 grown on MS medium supplemented with 5% sucrose. Samples were loaded on the basis of an equal amount of subunit {alpha} of CF1 and separated by SDS-PAGE (12% acrylamide). For an estimation of the subunit abundance, dilutions of the wild type sample were loaded on the gel. In-gel heme staining of cytochrome f was performed using TMBZ (39). Proteins were transferred to nitrocellulose membranes after electrophoresis, and immunodecoration was carried out using antisera against spinach cytochrome f and CF1, Chlamydomonas cytochrome b6 and subunit IV, and maize Rieske FeS. Equal loading of samples was further confirmed by verifying that the abundance of Cox1 (mitochondria) and Sec12 (endoplasmic reticulum) was unchanged in all samples (not shown). The extent of reduction in the protein abundance in ccda mutants is different for each subunit (compare, e.g. cytochrome b6 and Rieske FeS) because the steady-state level of each cytochrome b6f subunit (whether assembled in a complex or unassembled/disassembled in the membrane) reflects its individual rate of synthesis and degradation.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In an attempt to determine whether a thio-reduction pathway operates in the chloroplast, we have investigated the function of an Arabidopsis homolog of the prokaryotic sulfide-disulfide transporter CcdA that is known to function in the analogous pathway in bacteria. In this paper, we provide evidence that Arabidopsis CCDA is 1) a chloroplast protein, 2) a polytopic membrane protein with within-membrane cysteine residues and that 3) mutations in the CCDA gene lead to a defect in plastid biogenesis associated with a loss in cytochrome b6f assembly.

Arabidopsis CCDA Is a Chloroplast Polytopic Membrane Protein—All vascular plant CCDA homologs that we identified exhibit an additional stretch of 100–115 amino acids in the N-terminal part of the polypeptide that is missing in the rhodophyte homolog encoded in the plastid genome (see Fig. 1 in the supplemental data). Chlamydomonas CCDA displays the shortest N-terminal extension (61 amino acids), a feature that is common to algal transit peptides that are 32 amino acids shorter, on average, than those from vascular plants (47). The N-terminal extensions present typical features of bipartite transit sequences that direct proteins to the thylakoid membrane via the so-called "spontaneous import pathway" (48, 49). The first domain directs the protein across the envelop into the stroma, whereas the second domain carries targeting information to the thylakoid. Both domains in the N-terminal extensions exhibit the expected characteristics: the former is hydrophilic and rich in hydroxylated residues, and the latter contains a positively charged N terminus followed by a hydrophobic segment, which is a crucial determinant for thylakoid translocation and ends with a processing site (AXA{downarrow}) for the thylakoid processing peptidase (see Fig. 1 in the supplemental data). Our in vitro experiments have established that this N-terminal extension indeed serves as a chloroplast-targeting sequence and is cleaved off upon import as expected for nuclearly encoded proteins that function in the plastid organelle. The sublocalization of CCDA could not be examined by conventional cell fractionation techniques because we were unable to generate an immunoreactive probe for the polypeptide. However, based on the presence of the bipartite chloroplast-targeting sequence and the polytopic model of CCDA that we have generated from PhoA-LacZ fusion analysis, we can deduce indirectly that CCDA is localized to the thylakoid membrane. Our topogical model is compatible with the experimentally deduced topology of bacterial Rhodobacter capsulatus CcdA (26), and it is noteworthy that in both plastid and bacterial CcdAs, the critical cysteine residues are assigned a within-membrane location in the first and the fourth transmembrane segments. The exact position of these cysteines, whether embedded or in proximity to the stromal or luminal sides, cannot be resolved by our topological experiments. The function of the within-membrane cysteines in CCDA is not really known, but functional dissection of the related bacterial DsbD/DipZ transporter has demonstrated that the redox active cysteines face the cytoplasmic side of the membrane (50). These cysteines have the potential to form a disulfide bond, an indication of their physical vicinity, and are key residues in the relay of reducing equivalents originating from the cytoplasm (50). It is likely that conserved cysteines in CCDA are also facing the stromal side of the thylakoid membrane and operate in a similar manner.

Mutations in CCDA Indicate a Function in the Biogenesis of the Cytochrome b6f Complex—The severity of the photosynthetic phenotype in the ccda lines correlates nicely with the impact of the interrupting DNA elements on the expression of the CCDA gene. The two insertions in ccda-2 and ccda-3 lines which lie in the 5'-untranslated region of the CCDA gene did not result in any observable phenotype under our conditions, either at the level of growth on soil or when examined for the accumulation of the cytochrome b6f complex. Neither insertion prevented the transcription of the CCDA gene, a rather surprising finding considering that the interrupting elements are only 15 and 23 bp away from the initiation codon of the CCDA gene. One likely explanation is that both the T-DNA and the Ds element harbor some promoter-like activity that enables transcription of CCDA. The Ds element is known to carry a promoter-like sequence at its 3'-end that can be activated upon integration (51). Indeed, we could detect a hybrid RNA in the ccda-2 line using a Ds element-specific primer and a CCDA-specific primer (Fig. 5B), confirming that the element does not block expression of CCDA. Despite several attempts, a transcript originating from the T-DNA in the ccda-3 line could not be detected by RT-PCR (Fig. 5B). However, based on the observation that the T-DNA in the ccda-4 line allows transcription of a truncated CCDA mRNA (Fig. 5B), we assume that the CCDA mRNA produced in the ccda-3 line also originates from the T-DNA. Lack of phenotype because of insertion of interrupting element in proximity to the initiation codon has also been reported previously in Arabidopsis (52).

The ccda-1 and ccda-4 lines do not express the full-length CCDA mRNA and result in photosynthetic incompetence that is correlated to decreased level of assembled cytochrome b6f complex. The ccda-1 and ccda-4 alleles cause distinct phenotypes: the former leads to a partial loss of function with 25% of assembled holocytochrome f that enables photosynthetic growth, and the latter results in complete loss of function with only 10% of accumulated holocytochrome f, which prevents photosynthetic growth. It is interesting to note that 25% of the level of holocytochrome f is sufficient to sustain germination and photosynthetic growth, whereas only 10% of the level of cytochrome f is insufficient for photosynthetic growth.

Unexpectedly, truncation of the first three exons in the ccda-1 allele does not produce a complete knock-out of the CCDA gene. Somehow, despite the lack of the first 90 amino acids that carry the targeting information, the truncated protein is still able to function in the assembly of the cytochrome b6f complex. It is possible that targeting information lies also in the hydrophobic core of the polypeptide or that the N-terminal sequence created by the fusion between the T-DNA and the CCDA gene is compatible with import into the thylakoid membrane.

CCDA May Correspond to an Eighth Component in the System II Pathway in Plastid—Our data support the implication of CCDA in a process required for the biogenesis of cytochrome b6f complex but do not elucidate whether heme attachment to apocytochrome f is compromised by ccda mutations. The defect in cytochrome b6f biogenesis in the absence of CCDA is most likely the result of compromised assembly of the complex, consistent with the observation that holocytochrome f is required for the assembly of the b6f complex in Chlamydomonas (53). A definite placement of CCDA in the plastid c-type cytochrome assembly pathway also relies on the examination of the assembly of cytochrome cX, the other c-type cytochrome occurring in the thylakoid lumen in Arabidopsis (54). This was not feasible because of the extraordinarily low abundance of this molecule in the plastid, whose holoform can only be detected in plants engineered to overexpress the protein (55). The green alga C. reinhardtii remains the ideal system to study plastid c-type cytochrome assembly because of the availability of ccs mutants (cytochrome c synthesis) that are blocked at the level of heme attachment to the apoforms of cytochrome f and cytochrome c6, the two c-type cytochromes resident in the lumen (56–59). Seven loci, plastid ccsA and nuclear CCS1 to CCS6, are required for the accumulation of both plastid holocytochrome f and c6 (17, 20).6 The presence of a CCDA homolog in Chlamydomonas prompted us to examine whether unidentified CCS2 to CCS6 loci could correspond to the CCDA gene. Two cosmids (pARG7-CCDA) carrying the ARG7 selection marker and containing genomic DNA7 corresponding to Chlamydomonas CCDA plus 5'- and 3'-flanking DNA were identified. The pARG7-CCDA cosmids were tested directly for their ability to rescue representative mutants corresponding to each of the CCS2 through CCS6 loci or indirectly for restoration of photosynthetic growth in arginine prototrophs resulting from transformation of ccs arg7 mutants. For most strains, we also analyzed the CCDA locus physically by blot hybridization and sequence analysis of the entire ORF. Based on the lack of complementation and absence of physical change in the CCDA gene in the ccs mutants, we concluded that if CcdA is involved in cytochrome c biogenesis, then it must correspond to a component that is not yet genetically defined.

There is the formal possibility that plastid CCDA is necessary for other processes controlling the biogenesis of cytochrome b6f complex, such as, for instance, maturation of the recently discovered covalent heme on holocytochrome b6 (60, 61). However, a function of CCDA in holocytochrome b6 assembly seems unlikely based on the fact that the covalent heme in holocytochrome b6 is on the stromal side (60, 61) and that covalent binding of heme to bacterial holocytochrome b is not dependent upon CcdA (25). That CCDA is recruited to complete plastid cytochrome c maturation seems in our sense the most compelling hypothesis, and we propose that it corresponds to an eighth assembly factor in the system II plastid pathway.

Emergence of a Thioreduction Pathway in Plastid c-Type Cytochrome Biogenesis—The existence of a thio-reduction pathway in the chloroplast was also suggested recently with the discovery of HCF164, a thylakoid membrane-anchored thioredoxin-like protein with disulfide reductase activity which is required for cytochrome b6f biogenesis in Arabidopsis (62). HCF164 displays similarity to CcsX/ResA, a thioredoxin-like protein involved in system II bacterial cytochrome c assembly pathway (63, 64). Remarkably, CcsX, ResA, and HCF164 proteins share the same membrane topology with the thioredoxin-like domain facing the p-side of the membrane, suggesting that their relevant targets of action are also located in this compartment (62–64). The definitive placement of HCF164 in plastid cytochrome c maturation is still uncertain because the defect in cytochrome b6f assembly could not be localized specifically to a block in the maturation of a particular subunit (i.e. conversion of apocytochrome f to its holoform) (62). A nucleus-encoded HCF164-like protein is also present in Chlamydomonas, but functional assignment in chloroplast cytochrome c biogenesis could not be provided in the algal system because none of the ccs2 to ccs6 mutations appears to lie in the HCF164-encoding gene.8

Erldensson and co-workers (28, 64) have proposed that ResA and CcdA are part of a redox relay required for the reduction of the cysteinyls in the heme binding site of apocytochrome c. Interestingly, we have noted that in cyanobacteria Gloeobacter violaceus and Geobacter sulfurreducens, CCDA and HCF164 homologs are encoded by adjacent genes (65, 66), giving support to the hypothesis that they might act together in a same redox pathway in the plastid. Immunoblotting experiments showed that HCF164 accumulation is not affected by loss-of-function alleles in CCDA (Fig. 7). This result, however, does not necessarily rule out a model in which CCDA and HCF164 are interacting partners involved in the same pathway, and we hypothesize that HCF164 is the ninth component involved in plastid cytochrome c formation. We propose that the transfer of electrons from the stroma to the lumen for maintenance of reduced CXXCH motif in apocytochrome c proceeds via sulfide/disulfide transporter CCDA and HCF164 thioredoxin (Fig. 8). We postulate that the provision of reducing equivalents in the plastid system is dependent upon other redox components, like for instance, a membrane-anchored thioredoxin-like protein on the n-side (Fig. 8). These redox components might correspond to the gene products of CCS4 and CCS5 loci in Chlamydomonas (17, 20). Indeed, a preliminary functional assessment of these genes in redox chemistry is based on the observation that ccs4 and ccs5 mutants, similar to ccda and resA/ccsx mutants in bacteria (26, 28, 63, 64), can be rescued for cytochrome c deficiency by exogenous reduced thiol compounds.9 It is conceivable that, because of the compartmentalization of the thylakoid lumen, the redox pathway evolved in plastids relies on several players (at least CCDA, HCF164, Ccs4, and Ccs5) and is therefore more complex than its bacterial counterpart.



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  		FIG. 7.
Accumulation of HCF164 thioredoxin in ccda-1 and ccda-4 mutants. Protein fractions were prepared from entire wild type (Ws) and homozygous ccda-1 plants grown on MS medium supplemented with 0.5% sucrose and from wild type (Col-0) and homozygous ccda-4 plants grown on MS medium supplemented with 5% sucrose. Samples were normalized for loading on the basis of an equal amount of subunit {alpha} of CF1 and separated by SDS-PAGE (12% acrylamide) to detect HCF164 thioredoxin (62). After electrophoresis, proteins were transferred to nitrocellulose membranes before immunodecoration with Arabidopsis anti-HCF164. Equal loading of samples was further confirmed by verifying that the abundance of Cox1 (mitochondria) and Sec12 (endoplasmic reticulum) was unchanged in all samples (not shown).

 



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  		FIG. 8.
Model for a thioreduction pathway in plastid c-type cytochrome maturation. Our current view is that delivery of thiol-reducing equivalents (e–) from the n-side to the p-side of the thylakoid membrane is necessary to maintain apocytochrome sulfhydryls in a reduced state prior to heme ligation. This thioreduction pathway includes CCDA and requires additional reaction partners on both sides of the membrane, like Nsr for the n-side reductant, which could be a membrane-anchored stroma facing thioredoxin, and HCF164, which is a candidate for the p-side reductant. The source of reducing power on the stromal side is not known and could be ferredoxin or NAD(P)H as in the bacteria (12, 13).

 


   FOOTNOTES
 
* This research was supported in part by United States Department of Agriculture Grant 98-35306-6975 (to S. M.), National Institutes of Health Grants GM48350 (to S. M.) and GM64584 (to S. K. C.), and by the National Science Foundation (to S. M. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains an additional Table I and Figs. 1–3 and supplemental experimental procedures. Back

§ These authors contributed equally to this work. Back

Supported in part by American Heart Association Fellowship 0120100Y and the Muscular Dystrophy Association Fellowship 3618. Back

§§ 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{at}chem.ucla.edu.

1 The p-side (for positive) corresponds to the plastid lumen, mitochondrial intermembrane space, or bacterial periplasm. The n-side (for negative) is the plastid stroma, mitochondrial matrix, or the bacterial cytoplasm. Back

2 The single letter amino acid code is used where X is any amino acid except cysteine. Back

3 The abbreviations used are: DHFR, dihydrofolate reductase; kanr, kanamycin-resistant; kans, kanamycin-sensitive; MS medium, Murashige & Skoog medium; ORF, open reading frame; RT, reverse transcription. Back

4 Descurainia sophia, Glycine max, Hordeum vulgare, Lotus japonicus, and Medicago truncatula (TBLASTN scores ranged from 10–17 to 10–55). Back

5 GenBankTM accession numbers: C. reinhardtii, AAL84598 [GenBank] A. thaliana, AAF35369 [GenBank] D. sophia, AAQ95739 [GenBank] G. max, AAP81162 [GenBank] H. vulgare, AAO16019 [GenBank] L. japonicus, AAO16018 [GenBank] and M. truncatula, AAO16020 [GenBank] Back

6 P. Hamel and S. Merchant, unpublished data. Back

7 We determined the genomic organization of the CCDA locus by sequencing. Back

8 S. T. Gabilly, P. P. Hamel, and S. Merchant, unpublished data. Back

9 P. P. Hamel and S. Merchant, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank L. Dolfini for technical assistance; Dr. U. Boronowsky for the generation of CCDA antisera, the sequencing 118B12T7 and F2D5T7 clones, and initial PhoA fusion constructs; and Dr. S. Nakamoto for sequencing LC074f09_r. We are grateful to Drs. A. Barkan (maize {alpha}-Rieske FeS), D. Krogmann (spinach {alpha}-cyt f), K. Meierhoff (Arabidopsis {alpha}-HCF164), J. L. Popot (Chlamydomonas {alpha}-Cox1), N. Raikhel (Arabidopsis {alpha}-Sec12), and F.-A. Wollman (Chlamydomonas {alpha}-cyt b6 and {alpha}-subunit IV/PetD) for the kind donation of antibodies. We also thank Dr. J. Gober for the gift of plasmid placZ290, Drs. J. Beckwith for the gift of plasmid pBAD22 and M. Ehrmann for the gift of strain LMG194.



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