Cysteine modification of a specific repressor protein controls the translational status of nucleus-encoded LHCII mRNAs in Chlamydomonas.
The cytosolic RNA-binding protein NAB1 represses translation of LHCII (light-harvesting complex of photosystem II) encoding mRNAs by sequestration into translationally silent mRNP complexes in the green alga Chlamydomonas reinhardtii. NAB1 contains 2 cysteine residues, Cys-181 and Cys-226, within its C-terminal RRM motif. Modification of these cysteines either by oxidation or by alkylation in vitro was accompanied by a decrease in RNA-binding affinity for the target mRNA sequence. To confirm the relevance of reversible NAB1 cysteine oxidation for the regulation of its activity in vivo, we replaced both cysteines with serines. All examined cysteine single and double mutants exhibited a reduced antenna at PSII caused by a perturbed NAB1 deactivation mechanism, with double mutations and Cys-226 single mutations causing a stronger and more distinctive phenotype compared with the Cys-181 mutation. Our data indicated that the responsible redox control mechanism is mediated by modification of single cysteines. Polysome analyses and RNA co-immunoprecipitation experiments demonstrated the interconnection of the NAB1 thiol state and its activity as a translation repressor in vivo. NAB1 is fully active in its dithiol state and is reversibly deactivated by modification of its cysteines. In summary, this work is an example that cytosolic translation of nucleus encoded photosynthetic genes is regulated via a reversible cysteine-based redox switch in a RNA-binding translation repressor protein.
Wobbe
Lutz
L
Department of Biology, University of Bielefeld, Algae BioTech Group, Universitätsstrasse 25, D-33615 Bielefeld, Germany.
Blifernez
Olga
O
Schwarz
Christian
C
Mussgnug
Jan H
JH
Nickelsen
Jörg
J
Kruse
Olaf
O
D020418:Algal Proteins / Q000737:chemistry / Q000378:metabolism*; D019943:Amino Acid Substitution / Q000528:radiation effects; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism* / Q000528:radiation effects; D016825:Chlamydomonas reinhardtii / Q000166:cytology / Q000254:growth & development / Q000378:metabolism* / Q000528:radiation effects; D003545:Cysteine / Q000378:metabolism*; D015536:Down-Regulation / Q000528:radiation effects; D008027:Light; D009154:Mutation / Q000235:genetics; D018384:Oxidative Stress / Q000528:radiation effects; D010641:Phenotype; D045332:Photosystem II Protein Complex / Q000235:genetics / Q000378:metabolism*; D052817:Phototrophic Processes / Q000528:radiation effects; D011485:Protein Binding / Q000528:radiation effects; D014176:Protein Biosynthesis / Q000528:radiation effects; D055550:Protein Stability / Q000528:radiation effects; D017434:Protein Structure, Tertiary; D048030:RNA, Algal / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D012694:Serine / Q000235:genetics; D013329:Structure-Activity Relationship
D020418:Algal Proteins; D045332:Photosystem II Protein Complex; D048030:RNA, Algal; D012333:RNA, Messenger; D012097:Repressor Proteins; D012694:Serine; D003545:Cysteine