The Chloroplast Protein Disulfide Isomerase RB60 Reacts with a Regulatory Disulfide of the RNA-binding Protein RB47

Alergand, Tal; Peled-Zehavi, Hadas; Katz, Yael; Danon, Avihai

doi:10.1093/pcp/pcj023

Cited by 40 publications

(31 citation statements)

References 32 publications

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“…A redox-active site monothiol mutant, in which the resolving Cys is replaced with a Ser or an Ala, is typically used to inhibit the second step and to increase the stability of the reaction intermediate ( Figure 1B). This approach facilitated the isolation in vivo (Walker et al, 1996;Frand and Kaiser, 1999;Senkevich et al, 2002) or in vitro (Motohashi et al, 2001;Balmer et al, 2003) of Trx targets and the identification in vitro of the Cys residues participating in the intermolecular disulfide (Alergand et al, 2006). Importantly, protein disulfide isomerase (PDI) and its oxidizing protein, ENDOPLASMIC RE-TICULUM OXIDOREDUCTIN1 (ERO1), were trapped by this approach in an intermolecular disulfide complex in yeast and human cells (Pollard et al, 1998;Frand and Kaiser, 1999;Mezghrani et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

A Chloroplast Light-Regulated Oxidative Sensor for Moderate Light Intensity in Arabidopsis

et al. 2012

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The transition from dark to light involves marked changes in the redox reactions of photosynthetic electron transport and in chloroplast stromal enzyme activity even under mild light and growth conditions. Thus, it is not surprising that redox regulation is used to dynamically adjust and coordinate the stromal and thylakoid compartments. While oxidation of regulatory proteins is necessary for the regulation, the identity and the mechanism of action of the oxidizing pathway are still unresolved. Here, we studied the oxidation of a thylakoid-associated atypical thioredoxin-type protein, ACHT1, in the Arabidopsis thaliana chloroplast. We found that after a brief period of net reduction in plants illuminated with moderate light intensity, a significant oxidation reaction of ACHT1 arises and counterbalances its reduction. Interestingly, ACHT1 oxidation is driven by 2-Cys peroxiredoxin (Prx), which in turn eliminates peroxides. The ACHT1 and 2-Cys Prx reaction characteristics in plants further indicated that ACHT1 oxidation is linked with changes in the photosynthetic production of peroxides. Our findings that plants with altered redox poise of the ACHT1 and 2-Cys Prx pathway show higher nonphotochemical quenching and lower photosynthetic electron transport infer a feedback regulatory role for this pathway.

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Section: Introductionmentioning

confidence: 99%

A Chloroplast Light-Regulated Oxidative Sensor for Moderate Light Intensity in Arabidopsis

et al. 2012

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“…Of the three precursor proteins, only the two with internal His tags were recognized without or with thrombin cleavage (compare lanes 1 and 2 with 5/9 and 6/10, respectively). We found that the signal from the internally tagged proteins was completely lost after incubation with chloroplast lysate, with or without thrombin cleavage (lanes 7,8,11,12). This suggested that the 47 kDa species was created by excising sequences within or upstream of the Sph His tag, and within or downstream of the Bsp His tag, and that the higher molecular weight bands in the splicing reactions all were species lacking the NCL, most likely aggregates with different degrees of resistance to SDS denaturation.…”

Section: Resultsmentioning

confidence: 94%

“…RB47 had been discovered previously in Chlamydomonas chloroplasts through affinity purification of proteins binding to the psbA mRNA 5Ј-UTR (7,8). Based on a variety of correlative experiments, RB47 was proposed to regulate psbA translation in response to light (9 -11) and redox poise (12). RB47, for which the isolated cDNA encoded a protein of 68 kDa (80 kDa by SDS-PAGE), could be converted during in vitro chloroplast import to the 47 kDa size found in chloroplasts (7), a difference far in excess of what can be accounted for by removal of the short N-terminal transit peptide (8).…”

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confidence: 99%

Activation of an Endoribonuclease by Non-intein Protein Splicing

Campbell

Stern

2016

Journal of Biological Chemistry

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The Chlamydomonas reinhardtii chloroplast-localized poly(A)-binding protein RB47 is predicted to contain a nonconserved linker (NCL) sequence flanked by highly conserved N-and C-terminal sequences, based on the corresponding cDNA. RB47 was purified from chloroplasts in association with an endoribonuclease activity; however, protein sequencing failed to detect the NCL. Furthermore, while recombinant RB47 including the NCL did not display endoribonuclease activity in vitro, versions lacking the NCL displayed strong activity. Both full-length and shorter forms of RB47 could be detected in chloroplasts, with conversion to the shorter form occurring in chloroplasts isolated from cells grown in the light. This conversion could be replicated in vitro in chloroplast extracts in a light-dependent manner, where epitope tags and protein sequencing showed that the NCL was excised from a full-length recombinant substrate, together with splicing of the flanking sequences. The requirement for endogenous factors and light differentiates this protein splicing from autocatalytic inteins, and may allow the chloroplast to regulate the activation of RB47 endoribonuclease activity. We speculate that this protein splicing activity arose to post-translationally repair proteins that had been inactivated by deleterious insertions or extensions.The chloroplast transcriptome is highly dependent on RNA processing, because pervasive transcription has to be balanced with activities that discard nonfunctional or deleterious transcripts (1). Among the most prominent of these activities are endo-and exoribonucleases that exert RNA quality control and create mature 5Ј and 3Ј termini (2-4). We previously examined the mechanism of 3Ј end maturation for the Chlamydomonas reinhardtii chloroplast atpB mRNA, whose 3Ј terminus is defined by a stem-loop structure. The atpB 3Ј-extended precursor undergoes two-step maturation, in which an endonuclease cleaves at a specific site called the endonuclease cleavage site (ECS) 2 ϳ10 nucleotides (nt) distal to the stem-loop, followed by exonucleolytic trimming (5). Of particular interest was that the sequence downstream of the ECS was extremely rapidly degraded both in vivo and in vitro, even in ectopic contexts (6). We sought to purify this novel ribonuclease activity from Chlamydomonas chloroplasts, with the purification assay based on endoribonucleolytic cleavage near the ECS.During the course of purification, we identified an endoribonuclease activity that appeared to cleave the RNA substrate near the ECS. As shown below, the endoribonuclease we isolated turned out to be RB47, a member of the polyadenylatebinding protein family (PABP). RB47 had been discovered previously in Chlamydomonas chloroplasts through affinity purification of proteins binding to the psbA mRNA 5Ј-UTR (7,8). Based on a variety of correlative experiments, RB47 was proposed to regulate psbA translation in response to light (9 -11) and redox poise (12). RB47, for which the isolated cDNA encoded a protein of 68 kDa (80 kDa by SDS-PAGE), coul...

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“…Oxidized cysteines represent the off state of the repressor, whereas reduced cysteines represent the on state. It has already been shown that RRM containing proteins from plant organisms involved in translational regulation of photosynthetic genes can be activity-regulated via cysteine modification (13). However, these proteins were shown to be located in the plastid.…”

Section: Discussionmentioning

confidence: 99%

“…Plasmid pGDNG1 was constructed by inserting the Wt NAB1 gene into the NdeI and EcoRI cloning sites of plasmid pGenD (13). The plasmids pGDNG1/Cys(181/226Ser), pGDNG1/ Cys(181Ser), and pGDNG1/Cys(226Ser) were generated by site-specific replacement of thymine by adenine at positions 541 and 676 of the NAB1 gene in plasmid pGDNG1 (QuikChange Site-Directed Mutagenesis Kit; Stratagene.…”

Section: Methodsmentioning

confidence: 99%

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

Wobbe

Blifernez

Schwarz

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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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 RNAbinding 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.Chlamydomonas reinhardtii ͉ light harvesting antenna ͉ redox control ͉ translation control T o compensate for changes in light intensity or spectral quality, plants have developed several short-term and longterm mechanisms to regulate the amount of light that is captured by each photosystem (1). One important long-term adaptation strategy of plant organisms involves the complex expression regulation of various nuclear-encoded light harvesting complex (Lhcb) genes (1). All levels of LHCII gene expression are targeted by regulation mechanisms (2-5) which rely on a complex retrograde and anterograde communication between plastid, nucleus, and cytosol (6). The cytosolic translation repressor NAB1, which was identified in a Chlamydomonas reinhardtii light acclimation mutant (4), is the center of interest within this work. NAB1 harbors 2 RNA-binding motifs and 1 of these motifs, located at the N terminus, belongs to the highly conserved family of CSD (cold shock domain) domains. Proteins containing a CSD motif are referred to as Y-box proteins and eukaryotic members of this large family generally contain a second auxiliary RNA-binding domain, which modulates the RNA affinity of the protein but can be dispensable for selective RNA recognition (7). In the case of NAB1, the CSD motif is combined with a C-terminal RRM (RNA recognition motif) domain, which was demonstrated not to be essential for selective RNA recognition (4). It was shown that NAB1 binds to the mRNA of LHCBM (major light-harvesting complex of photosystem II) genes, thereby preventi...

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The Chloroplast Protein Disulfide Isomerase RB60 Reacts with a Regulatory Disulfide of the RNA-binding Protein RB47

Cited by 40 publications

References 32 publications

A Chloroplast Light-Regulated Oxidative Sensor for Moderate Light Intensity in Arabidopsis

A Chloroplast Light-Regulated Oxidative Sensor for Moderate Light Intensity in Arabidopsis

Activation of an Endoribonuclease by Non-intein Protein Splicing

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

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