Talk abstracts

Talk on Monday 03:00-03:15pm submitted by Stephane Gabilly

Emergence of a plastid-localized, disulfide-reducing pathway required for cytochrome c assembly.

Stephane Gabilly (MCDB, Departments of Molecular Genetics and Molecular and Cellular Biochemistry, The Ohio State University), Mohamed Karamoko, Vincent Corvest (Departments of Molecular Genetics and Molecular and Cellular Biochemistry, The Ohio State University), Sabeeha Merchant (Department of Chemistry and Biochemistry, University of California Los Angeles), Patrice Hamel (Departments of Molecular Genetics and Molecular and Cellular Biochemistry, The Ohio State University)

Abstract:
The plastid lumen has long been thought to be virtually empty of proteins. However, the discovery of luminal disulfide-bonded proteins implies that thiol-based redox chemistry does occur in the thylakoid lumen. While thiol-disulfide chemistry has been well studied in bacteria, the question of how this process is performed in the plastid has yet to be explored. From our study, using Chlamydomonas reinhardtii as model organism, we have identified three novel components that support the participation of a disulfide-reducing pathway on the luminal side of the thylakoid membrane, CCS4, CCS5 and CCDA.
The ccs4 and ccs5 mutants are photosynthetic deficient and exhibit a block in the assembly of cytochromes f and c6, two c-type cytochromes involved in electron transfer.
-CCS4 is a unique protein of 93 amino acids with a small, hydrophobic amino-terminus, a hydrophilic carboxyl-terminal domain with many charged residues and no noticeable motif suggestive of a biochemical activity. CCS4 is involved in the disulfide-reducing pathway based on 1) the thiol-dependent photosynthetic rescue of the ccs4 mutant and 2) the suppression of the ccs4 phenotype by overexpression of plastid CCDA, an ortholog of bacterial CcdA/DsbD, a thiol/disulfide transporter. We postulate that CCS4 interacts with CCDA to stabilize the protein and/or control its activity.
-CCS5 is a lumen-facing, thioredoxin-like protein that shows 35% sequence identity to Arabidopsis thaliana HCF164, a protein previously identified as being involved in cytochrome b6f biogenesis. CCS5 displays a disulfide-reducing activity, inferred from the findings that 1) the ccs5 mutant can be chemically rescued by exogenous thiols (DTT) and 2) recombinant CCS5 has the ability to reduce insulin and recombinant apocytochrome f in vitro.
By similarity to bacteria, we propose that the cysteines in the CXXCH motif of apocytochrome f and c6 are maintained reduced by the electrons conveyed from CCDA to CCS5 prior to the heme attachment in vivo.

References:
1. Gabilly ST, Dreyfuss BW, Karamoko M, Corvest V, Kropat J, Page MD, Merchant SS, Hamel PP. J Biol Chem. 2010 Sep 24;285(39):29738-49.
2. Gabilly ST, Kropat J, Karamoko M, Page MD, Nakamoto SS, Merchant SS, Hamel PP. Genetics. 2011 Mar;187(3):793-802.

Keywords: Thiol-based redox chemistry, Cytochrome c assembly, Chlamydomonas