Two disulfide-reducing pathways are required for the maturation of plastid c-type cytochromes in Chlamydomonas reinhardtii

Abstract

In plastids, conversion of light energy into ATP relies on cytochrome f, a key electron carrier with a heme covalently attached to a CXXCH motif. Covalent heme attachment requires reduction of the disulfide-bonded CXXCH by CCS5 and CCS4. CCS5 receives electrons from the oxidoreductase CCDA, while CCS4 is a protein of unknown function. In Chlamydomonas reinhardtii, loss of CCS4 or CCS5 yields a partial cytochrome f assembly defect. Here, we report that the ccs4ccs5 double mutant displays a synthetic photosynthetic defect characterized by a complete loss of holocytochrome f assembly. This defect is chemically corrected by reducing agents, confirming the placement of CCS4 and CCS5 in a reducing pathway. CCS4-like proteins occur in the green lineage, and we show that HCF153, a distant ortholog from Arabidopsis thaliana, can substitute for Chlamydomonas CCS4. Dominant suppressor mutations mapping to the CCS4 gene were identified in photosynthetic revertants of the ccs4ccs5 mutants. The suppressor mutations yield changes in the stroma-facing domain of CCS4 that restore holocytochrome f assembly above the residual levels detected in ccs5. Because the CCDA protein accumulation is decreased specifically in the ccs4 mutant, we hypothesize the suppressor mutations enhance the supply of reducing power through CCDA in the absence of CCS5. We discuss the operation of a CCS5-dependent and a CCS5-independent pathway controlling the redox status of the heme-binding cysteines of apocytochrome f.

Keywords: Chlamydomonas reinhardtii; Plant Genetics and Genomics; cytochromes c; energy-transducing membranes; photosynthesis; thiol–disulfide chemistry.

Graphical Abstract

Reduction of a disulfide formed between the heme-linking cysteines of apoforms of c-type cytochromes in the thylakoid lumen requires the provision of reducing power from stroma to apocytochrome c via thiol–disulfide exchange. Reducing power is supplied through two different pathways, pathways 1 and 2, with CCDA and CCS4 being common components to both pathways. In pathway 1, CCS5 directly reduces the disulfide-bonded heme-binding site. In pathway 2, one or several yet-to-be identified protein(s) (X) reduce(s) the disulfide. CCS4 functions in stabilizing CCDA. In the absence of CCS5, gain-of-function mutations altering the C terminus of CCS4 enhance the delivery of reducing power via CCDA to the unknown reductase(s) (X), underscoring the functional importance of the soluble domain in CCS4 (indicated by a yellow star). This domain might act by recruiting the stromal reductant to CCDA or control the reactivity of the redox-active cysteines in CCDA by providing an ideal chemical microenvironment for efficient thiol–disulfide exchange.

Fig. 1.

The ccs4Δccs5 mutant exhibits a photosynthetic growth defect due to a complete loss of cytochrome c assembly. a). Ten-fold dilution series of WT (CC-124), Δccs5 (CC-4129), ccs4 (ccs4(pCB412)), and ccs4Δccs5 [CC-4517, CC-4518, and ccs4 ccs5 (9) and (11)] strains were plated on acetate-containing (right panel) and minimal (left panel) medium. Cells grown phototrophically (CO₂) were incubated at 25°C for 7 days with 30–50 µmol/m²/s of light. Cells grown mixotrophically (acetate + CO₂) were incubated at 25°C for 7 days with 0.3 µmol/m²/s of light. b) The fluorescence induction and decay kinetics of ccs4Δccs5 is shown in comparison to that of Δccs5, ccs4, and WT. c) Heme staining and α-PsbO immunodetection were performed on total protein extracts. Strains are as in 1a except for the ccs4 (CC-4519) and ccs4Δccs5 (CC-4518). Cells were grown mixotrophically on acetate with 0.3 µmol/m²/s of light at 25°C for 5–7 days. Samples correspond to 30 µg of chlorophyll. Immunodetection of PsbO was used as a loading control. The vertical line indicates cropping from the same gel and same exposure.

Fig. 2.

The photosynthetic defect in ccs4Δccs5 is partially rescued by exogenous thiols. a) Ten-fold dilution series of WT (CC-4533), Δccs5 (CC-5922), ccs4 (CC-5925), ccs4Δccs5 (CC-5927), and ΔpetA (CC-5935) were plated on acetate and minimal medium with or without MESNA (2-mercaptoethane sulfonate sodium). Cells grown phototrophically (CO₂) were incubated at 25°C for 20 days with 30–50 µmol/m²/s of light. Cells grown mixotrophically (acetate + CO₂) were incubated at 25°C for 14 days with 0.3 µmol/m²/s of light. The horizontal lines indicate cropping from the same plate of serial dilution. b) Fluorescence transients were measured on cells grown on solid acetate-containing media for 5 days (with 0.3 µmol/m²/s of light) with or without MESNA. Strains are as in (a) except that the ccs4Δccs5 strain is CC-4518.

Fig. 3.

The abundance of CCDA is diminished in the ccs4 but not in the ccs5 mutant. a) Suppression of the photosynthetic growth defect in the ccs4 mutant by ectopic expression of CCDA was assessed by 10-fold dilution series. The WT (CC-124), ccs4 (CC-4520), CCS4-expressing [ccs4 (CCS4), CC-4522], and CCDA-expressing [ccs4 (CCDA), CC-5926] ccs4 strains were plated on acetate medium and grown mixotrophically (with 30–50 µmol/m²/s or 0.3 µmol/m²/s of light) at 25°C for 14 days. The horizontal lines indicate cropping from the same plate. b) and c) CCDA was immunodetected on total protein extracts of strains described in a) and the Δccs5 (CC-4129) and corresponding complemented [Δccs5(CCS5), CC-4527] strains. Cells were grown mixotrophically (on acetate with 0.3 µmol/m²/s of light) at 25°C for 5–7 days. The vertical line indicates cropping from the same blot and same exposure. Samples (100%) correspond to 10 µg of chlorophyll. Immunodetection with antisera raised against Chlamydomonas CF₁ (coupling factor 1 of chloroplast ATPase) and CCS5 was used as control (Gabilly et al. 2010).

Fig. 4.

Suppressors of ccs4Δccs5 are restored for phototrophic growth and cytochrome f assembly. a) Ten-fold dilution series of WT (CC-124), Δccs5 (CC-4129), ccs4 (CC-4519) and ccs4Δccs5 (CC-4518), SU9 (CC-5928), and SU11 (CC-5929) were plated on acetate medium and grown mixotrophically (with 250 or 0.3 µmol/m²/s of light) at 25°C for 14–18 days. The horizontal line indicates cropping from the same plate of serial dilutions. b) The fluorescence induction and decay kinetics observed in a dark-to-light transition of the SU9 and SU11 suppressors are shown in comparison to ccs4Δccs5, Δccs5, and WT. Strains are the same as in a). c) Heme staining and immunoblotting (α-cyt f and α-PsbO) were performed on total protein extracts prepared from cells (same strains as in (a)) grown mixotrophically (on acetate with 0.3 µmol/m²/s of light) at 25°C for 5–7 days. Samples (100%) correspond to 10 µg of chlorophyll. Immunodetection with antisera against PsbO was used as loading control.

Fig. 5.

Mutations in CCS4 suppress the cytochrome f assembly defect in the ccs5 mutant. a) Amino acid sequence of CCS4 deduced from the CCS4 gene in the original ccs4-F2D8 mutant; SU9 and SU11 are shown in black, green, and blue, respectively. The putative transmembrane domain, predicted using the TMHMM-2.0 prediction tool (Krogh et al. 2001), is underlined. Sequence analysis of the CCS4 gene shows that the original nonsense mutation in the ccs4 strain is changed to a missense mutation in SU9, resulting in the stop codon (at residue 50 of the protein) becoming a codon for tryptophan (WT codon is glutamine). In SU11, an in-frame deletion mutation encompassing the missense mutation resulted in a deletion of 3 residues A₄₉Q₅₀M₅₁ in the CCS4 gene product. b) WT (CC-124), ccs5 (Δccs5, CC-4129), ccs4Δccs5 (CC-4518), and the reconstructed ccs5 (CCS4-WT, CC-5932), SU9 (CCS4-SU9, CC-5933), and SU11 (CCS4-SU11, CC-5934) strains were used for 10-fold dilution series. The Δccs5, SU9, and SU11 were reconstructed by introducing the constructs pHyg3-CCS4(WT), pHyg3-CCS4(SU9) and pHyg3-CCS4(SU11) into a ccs4Δccs5 recipient strain. Two independent transformants for each construct are shown. Cells were plated on minimal medium and grown phototrophically (with 30–50 µmol/m²/s of light) or mixotrophically (with 0.3 µmol/m²/s of light) at 25°C for 14–18 days. The horizontal lines indicate cropping from the same plate of serial dilutions. c) In-gel heme staining was performed on total protein extracts corresponding to 30 µg of chlorophyll. Samples from the Δccs5 and original SU9 (CC-5933) and SU11 (CC-5934) were loaded on the gel for comparison to the reconstructed strains. The strains are as in b) and ΔpetA is CC-5935. The level of LHC complex is used as a loading control. One-microgram equine heart cytochrome c (∼10 kDa; Sigma) is used as a control for the heme-dependent peroxidase activity.

Fig. 6.

Mutations in CCS4 in SU9 and SU11 are dominant. a) Ten-fold dilution series of diploid (+/+, Δccs5/Δccs5, Δccs5/Δccs5  CCS4-SU9, and Δccs5/Δccs5 CCS4-SU11) strains were plated on minimal medium and grown phototrophically (with 30–50 µmol/m²/s of light) at 25°C for 14–18 days (left panel) and mixotrophically in 0.3 µmol/m²/s for 20–21 days (right panel). Two representatives of each diploid constructed with a Δccs5 strain are shown. b) The fluorescence induction and decay kinetics of one representative diploid strain. Strains are the same as in a).

Fig. 7.

CCS4-like proteins are present throughout the green lineage. Sequences of Arabidopsis thaliana (NP_194884.1), Picea sitchensis (Sitka spruce, ABK22505.1), C. reinhardtii (ADL27744.1), Chara braunii (Braun's stonewort, GBG73810.1), and Selaginella moellendorffii (spikemoss, XP_024543966.1) were aligned using Clustal Omega (Sievers and Higgins 2014) and manually edited in Word. The transmembrane domains are highlighted in gray; residues with positive and negative charges are highlighted in blue and red, respectively. The percentage of similarity followed by the percentage of charged residues is indicated.

Fig. 8.

Arabidopsis HCF153 complements the ccs4 mutant. a) Ten-fold dilution series of ccs4 (ccs4_empty, CC-4520), ccs4 transformed by the CCS4 gene (CC-4522), and two independent ccs4 transformants carrying the CCS4-HCF153 gene (CC-5592 and CC-5593) were plated on minimal medium and grown phototrophically (with 30–50 µmol/m²/s of light) at 25°C for 14 days. b) The fluorescence induction and decay kinetics of the one representative CCS4-HCF153 transformant (CC-5592) is shown in comparison to that of a WT (CC-124) and ccs4 strains (CC-4520). c) In-gel heme staining was performed on total protein extracts corresponding to 30 µg of chlorophyll. The strains are as in a) and b), and the ccs4 strain is CC-4519.