An intermolecular disulfide‐based light switch for chloroplast psbD gene expression in Chlamydomonas reinhardtii

SUMMARYExpression of the chloroplast psbD gene encoding the D2 protein of the photosystem II reaction center isregulated by light. In the green alga Chlamydomonas reinhardtii, D2 synthesis requires a high-molecular-weight complex containing the RNA stabilization factor Nac2 and the translational activator RBP40. Based onsize exclusion chromatography analyses, we provide evidence that light control of D2 synthesis depends ondynamic formation of the Nac2/RBP40 complex. Furthermore, 2D redox SDS–PAGE assays suggest anintermolecular disulfide bridge between Nac2 and Cys11 of RBP40 as the putative molecular basis forattachment of RBP40 to the complex in light-grown cells. This covalent link is reduced in the dark, most likelyvia NADPH-dependent thioredoxin reductase C, supporting the idea of a direct relationship betweenchloroplast gene expression and chloroplast carbon metabolism during dark adaption of algal cells.Keywords: Chlamydomonas, chloroplast gene expression, Nac2, redox control, ribonucleoprotein particle.INTRODUCTIONDue to the endosymbiotic originsof the chloroplast, its geneexpression machinery is basically of prokaryotic origin.However, during the evolutionary development of chlorop-lasts, this machinery was extensively modified by recruit-ment of nucleus-encoded regulatory factors, which nowconstituteanintracellularnetworkdedicatedtocoordinationof gene expressionin thenucleus andthe organelle(Barkan,2011). While recent years have seen the identification andcharacterization of a number of these trans-acting factors,much less is known about their precise molecular modes ofaction with regard to light-dependent regulation.In this context, the idea of redox control of chloroplastgene expression has attracted much attention, as it providesan appealing basis for a direct link between photosyntheticactivity and expression of photosynthesis-related chloro-plast genes (Dietz and Pfannschmidt, 2011). Indeed, manyelements of chloroplast gene expression, including RNAtranscription, stabilization, processing and splicing, andtranslation have been shown to be affected directly orindirectly by the redox state of the organelle (Barnes andMayfield, 2003). However, translation appears to representthe rate-limiting step for synthesis of chloroplast-encodedproteins (Eberhard et al., 2002; Zerges and Hauser, 2009).In the green alga Chlamydomonas reinhardtii, synthesisof the large subunit of ribulose-1,5-bisphosphate carboxyl-ase/oxygenase(Rubisco)encodedbytherbcLgenehasbeenshown to be regulated via the redox state of the chloroplastglutathione pool, which in turn is modulated by light-induced oxidative stress (Irihimovitch and Shapira, 2000).Interestingly, the RbcL protein possesses an intrinsic non-specific RNA binding activity located within its N-terminalregion (Yosef et al., 2004). It has therefore been postulatedthatbindingofRbcLtoitsownmRNAblocksitstranslationifeither its redox-controlled interaction with the chloroplastchaperone system or the Rubisco subunit assembly isdisturbed (Cohen et al., 2005).The most elaborate (but also the most controversial)model for redox-controlled translational regulation in chlo-roplasts has been described in C. reinhardtii for the psbAgenethatencodestheD1proteinofthephotosystem II(PSII)reaction center (Barnes and Mayfield, 2003; Zerges andHauser, 2009). This model postulates that redox-controlledbinding of a protein complex to the 5¢ UTR of the psbAmRNA leads to recruitment of ribosomes. The heart of thiscomplex is the RNA-binding protein RB47, whose activity ismodulated by RB60, a disulfide isomerase homolog (Kim