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. 2009 Aug 11;106(32):13290-5.
doi: 10.1073/pnas.0900670106. Epub 2009 Aug 3.

Cysteine modification of a specific repressor protein controls the translational status of nucleus-encoded LHCII mRNAs in Chlamydomonas

Lutz Wobbe  1 , Olga Blifernez, Christian Schwarz, Jan H Mussgnug, Jörg Nickelsen, Olaf Kruse
Affiliations

Cysteine modification of a specific repressor protein controls the translational status of nucleus-encoded LHCII mRNAs in Chlamydomonas

Lutz Wobbe et al. Proc Natl Acad Sci U S A. .

Abstract

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
In silico and in vitro analyses to examine a potential cysteine regulation of NAB1. (A) Homology model of the C-terminal RRM domain of NAB1. Cys-181 and Cys-226 within the RRM domain are indicated. (B) In vitro RNA binding studies. Autoradiogram of UV-cross-linked and SDS/PAGE separated NAB1-LHCBM6-CSDCS complexes. Recombinant NAB1 was either left untreated (Top) or oxidized with 5 mM glutathione disulfide (GSSG, Middle) before addition of a radioactively labeled LHCBM6-CSDCS probe alone (−) or of a mixture containing labeled LHCBM6-CSDCS and a 5-fold molar excess of unlabeled psbD competitor RNA (+). N-ethylmaleimide (NEM, third panel from Top) or 4-vinylpyridine (4-vinylpyridine, Bottom) was applied for cysteine alkylation. (C) Treatment of recombinant NAB1 with different concentrations of glutathione disulphide (GSSG) and immunodetection of protein-glutathione adducts (Upper). Coomassie blue (CBB) stain of recombinant NAB1 after treatment with the indicated GSSG concentrations (Lower).
Fig. 2.
Fig. 2.
Phenotypical analyses of NAB1-cysteine mutants. (A) Total chlorophyll content of NAB1-cysteine mutants relative to the control strain C expressing Wt-NAB1 (left y axis; black bars) and Chl. a/b ratios of cysteine mutants and control strain (right y axis; white bars). The data represent mean values of three independent chlorophyll measurements (using triplicates) performed with different strains for each cysteine mutation (10 strains expressing NAB1Cys181Ser; three strains expressing NAB1Cys226Ser; two strains expressing NAB1Cys-181/226Ser). Error bars indicate standard deviations (n = 30 for NAB1Cys181Ser; n = 9 for NAB1Cys226Ser; n = 6 for NAB1Cys-181/226Ser). (B) Anti-NAB1 immunoblot analyses to assess the NAB1 expression level in the Wt control strain and the cysteine mutant strains. (Upper) Representative immunoblot. (Lower) Coomassie blue stain (loading control). (C) Anti-HA-tag immunoblots to determine the expression of HA-epitope tagged LHCBM6 protein (Upper). (Lower) Coomassie blue-stained SDS protein gel (loading control).
Fig. 3.
Fig. 3.
Growth and photosynthetic low light acclimation of control strain (C) and NAB1 cysteine mutants. (A) Growth rates within the exponential phase observed under phototrophic low light conditions (HSM medium; 40 μmol m−2 s−1). The growth rate was determined by measurements of the increase of the optical density per hour. Error bars indicate the standard deviation of three independent growth experiments. (B) Representative immunoblot using a LHCII-specific antiserum and Coomassie blue-stained SDS/PAGE gel. Protein samples were taken from cells grown under photoautotrophic dim light conditions (HSM medium; 40 μmol m−2 s−1). (C) Results from three independent LHCII immunoblot analyses after phototrophic growth (HSM medium; 40 μmol m−2 s−1) using samples of the control strain and the cysteine mutants. Signal intensities were quantified by densitometry and used to calculate the mean values represented by black bars. The amount of LHCII protein in the control strain was set to 100%. Standard errors are indicated by error bars.
Fig. 4.
Fig. 4.
Polysome analysis of control strain and NSM2 cells grown phototrophically under dim light conditions. Cytosolic extracts were centrifuged through a 15–45% continuous sucrose gradient to separate subpolysomal mRNPs, monosomes and polysomes. RNA was extracted from 18 gradient fractions and analyzed by formaldehyde-agarose gel electrophoresis and ethidium bromide staining. The amount of LHCBM6 and β-ACTIN mRNA in each fraction was assessed by Northern slot-blot analysis. Slot-blot signals of LHCBM6 were quantified by densitometrical scanning and normalized to the corresponding ß-ACTIN signal. The strongest LHCBM6 blot signal obtained for each strain was set to 100%. Standard errors are based on three independent polysome fractionations.
Fig. 5.
Fig. 5.
Effects of cysteine oxidation on the RNA-binding capacity of Wt-NAB1 (control strain), NAB1Cys181Ser, and NAB1Cys226Ser analyzed in vivo. (A) Examination of the Wt-NAB1 thiol state after 60 and 120 min after diamide addition (2 mM) to a liquid cell culture. The degree of cysteine modification was assessed by mPEG-MAL-labeling and subsequent anti-NAB1 immunoblot detection. (B) Coimmunoprecipitation of LHCBM6-mRNA using a NAB1-specific antiserum before and after diamide-induced oxidation of Wt -NAB1 (control strain), NSM1 (Cys181Ser) and NSM2 (Cys226Ser). The amount of coprecipitated LHCBM6-mRNA was quantified by RT-Q-PCR and the t0-value was set to 100% for each strain. Error bars indicate the standard error of four independent RT-Q-PCR measurements.
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References

    1. Walters RG. Towards an understanding of photosynthetic acclimation. J Exp Bot. 2005;56:435–447. - PubMed
    1. Pursiheimo S, Mulo P, Rintamaki E, Aro EM. Coregulation of light-harvesting complex II phosphorylation and lhcb mRNA accumulation in winter rye. Plant J. 2001;26:317–327. - PubMed
    1. Chen Y-B, Durnford DG, Koblizek M, Falkowski PG. Plastid regulation of Lhcb1 transcription in the Chlorophyte Alga Dunaliella tertiolecta. Plant Physiol. 2004;136:3737–3750. - PMC - PubMed
    1. Mussgnug JH, et al. Nab1, an RNA-binding protein involved in the light-regulated differential expression of the light-harvesting antenna of Chlamydomonas reinhardtii. Plant Cell. 2005;17:3409–3421. - PMC - PubMed
    1. McKim SM, Durnford DG. Translational regulation of light-harvesting complex expression during photoacclimation to high-light in. Chlamydomonas reinhardtii. Plant Physiol Biochem. 2006;44:857–865. - PubMed

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