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bioRxiv · 10.1101/2022.10.14.512171

TWO DISULFIDE-REDUCING PATHWAYS ARE REQUIRED FOR THE MATURATION OF PLASTID C-TYPE CYTOCHROMES IN CHLAMYDOMONAS REINHARDTII

Das, A.·Subrahmanian, N.·Gabilly, S. T.·Andrianova, E. P.·Jouline, I.·Motohashi, K.·Hamel, P. P.
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Abstract

AO_SCPLOWBSTRACTC_SCPLOWIn 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 motif by CCS5 and CCS4, a protein of unknown function. CCS5 receives electrons from the oxido-reductase CCDA at the thylakoid membrane. In Chlamydomonas reinhardtii, loss of CCS4 or CCS5 function yields a partial cytochrome f assembly defect. Here we report that the {Delta}ccs4ccs5 double mutant displays a synthetic photosynthetic defect due to a complete loss of holocytochrome f assembly, a phenotype that can be chemically corrected by reducing agents. In {Delta}ccs4, the CCDA protein accumulation is decreased, indicating that one function of CCS4 is to stabilize CCDA. Dominant suppressor mutations mapping to the CCS4 gene were identified in photosynthetic revertants of the {Delta}ccs4ccs5 mutants. The suppressor mutations correspond to changes in the stroma-facing domain of CCS4 and restore holocytochrome f assembly above the residual levels detected in {Delta}ccs5. Because disulfide reduction via CCS5 no longer takes place in {Delta}ccs5, we hypothesize the suppressor mutations enhance the supply of reducing power independently of CCS5, uncovering the participation of CCS4 in a distinct redox pathway. CCS4-like proteins occur in the green lineage and are related to mitochondrial COX16, a protein involved in a disulfide reducing pathway. We discuss the operation of two pathways controlling the redox status of the heme-binding cysteines of apocytochrome f and the possible function of CCS4 as a shared component between the two pathways. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/512171v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@7f496forg.highwire.dtl.DTLVardef@ae5229org.highwire.dtl.DTLVardef@942b20org.highwire.dtl.DTLVardef@19b7576_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract.C_FLOATNO The {Delta}ccs4ccs5 mutant exhibits a photosynthetic growth defect due to a complete loss of cytochrome c assembly. Reduction of apocytochrome f in the thylakoid lumen requires the provision of reducing power through two different pathways, pathway 1 and 2. CCDA and CCS5, components of pathway 1, deliver electrons from stroma to apocytochrome f via thiol - disulfide exchange. CCS4 is involved in pathway 1 by stabilizing CCDA, but also functions through a CCS5 - independent pathway (pathway 2). In the absence of CCS5, gain - of - function mutations in the C terminus of CCS4 (indicated by a yellow star) enhance the delivery of reducing power either via CCDA or independently of CCDA to yet-to-be-discovered reductases C_FIG

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Das, A., Subrahmanian, N., Gabilly, S. T., Andrianova, E. P., Jouline, I., Motohashi, K., Hamel, P. P.. 2022-10-17. TWO DISULFIDE-REDUCING PATHWAYS ARE REQUIRED FOR THE MATURATION OF PLASTID C-TYPE CYTOCHROMES IN CHLAMYDOMONAS REINHARDTII. https://doi.org/10.1101/2022.10.14.512171

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