CCS5, a Thioredoxin-like Protein Involved in the Assembly of Plastid c-Type Cytochromes

Abstract

The c-type cytochromes are metalloproteins with a heme molecule covalently linked to the sulfhydryls of a CXXCH hemebinding site. In plastids, at least six assembly factors are required for heme attachment to the apo-forms of cytochrome f and cytochrome c 6 in the thylakoid lumen. CCS5, controlling plastid cytochrome c assembly, was identified through insertional mutagenesis in the unicellular green alga Chlamydomonas reinhardtii. The complementing gene encodes a protein with similarity to Arabidopsis thaliana HCF164, which is a thylakoid membrane-anchored protein with a lumen-facing thioredoxinlike domain. HCF164 is required for cytochrome b 6 f biogenesis, but its activity and site of action in the assembly process has so far remained undeciphered. We show that CCS5 is a component of a trans-thylakoid redox pathway and operates by reducing the CXXCH heme-binding site of apocytochrome c prior to the heme ligation reaction. The proposal is based on the following findings: 1) the ccs5 mutant is rescued by exogenous thiols; 2) CCS5 interacts with apocytochrome f and c 6 in a yeast two-hybrid assay; and 3) recombinant CCS5 is able to reduce a disulfide in the CXXCH heme-binding site of apocytochrome f.

Regulatory factors for the assembly of thylakoid membrane protein complexes

“…In Arabidopsis, the loss of CcdA and HCF164 (high chlorophyll fluorescence 164, an orthologue of CcSX) impairs photosynthesis and results in a cyt b 6 f assembly defect, suggesting that these proteins are required for cyt b 6 f biogenesis in plant chloroplasts [54,55]. The role of HCF164 was further investigated through a study of CCS5, its orthologue in Chlamydomonas [56]. CCS5 interacted with apocytochrome f and c6 in a yeast two-hybrid analysis and reduced a disulphide in the CXXCH haem-binding site of apocytochrome f. Moreover, the ccs5 mutant was rescued by exogenous thiols [56].…”

“…The role of HCF164 was further investigated through a study of CCS5, its orthologue in Chlamydomonas [56]. CCS5 interacted with apocytochrome f and c6 in a yeast two-hybrid analysis and reduced a disulphide in the CXXCH haem-binding site of apocytochrome f. Moreover, the ccs5 mutant was rescued by exogenous thiols [56]. These results indicate that CCS5/HCF164 is a component of a trans-thylakoid redox pathway and operates by reducing the CXXCH haem-binding site of apocytochrome c before the haem ligation reaction [56].…”

Nitric Oxide–Triggered Remodeling of Chloroplast Bioenergetics and Thylakoid Proteins upon Nitrogen Starvation inChlamydomonas reinhardtii

“…Antibodies against D1, PsaA, and Rubisco LS were purchased from Agrisera. Other antibodies were described previously: cytochrome b 6 f subunits (Kuras and Wollman, 1994), ATP synthase subunit b (Drapier et al, 1992), CCBs , CCS1 (Dreyfuss et al, 2003), CCS5 (Gabilly et al, 2010), PTOX2 (HouilleVernes et al, 2011), and NDA2 (Desplats et al, 2009). TCA1-Flag and MCA1-HA were detected by ECL using monoclonal antibodies against the tags anti-Flag M2 (Sigma-Aldrich), anti HA.11 (Covance), and horseradish peroxidase-conjugated antibody against mouse IgG (Promega).…”

Lumen Thiol Oxidoreductase1, a Disulfide Bond-Forming Catalyst, Is Required for the Assembly of Photosystem II inArabidopsis

“…These proteins define a trans-thylakoid pathway for the delivery of reductants from stroma to lumen (Lennartz et al, 2001;Page et al, 2004;Hisabori, 2006, 2010;Gabilly et al, 2010Gabilly et al, , 2011. Operation of the trans-thylakoid pathway is needed to reduce disulfides in target proteins, a process essential for photosynthesis (Lennartz et al, 2001;Page et al, 2004;Gabilly et al, 2010Gabilly et al, , 2011.…”

“…Operation of the trans-thylakoid pathway is needed to reduce disulfides in target proteins, a process essential for photosynthesis (Lennartz et al, 2001;Page et al, 2004;Gabilly et al, 2010Gabilly et al, , 2011.…”