Avihai Danon

Light regulated translational activators : identification of chloroplast gene specific mRNA binding proteins

Genetic analysis has revealed a set of nuclear‐encoded factors that regulate chloroplast mRNA translation by interacting with the 5′ leaders of chloroplastic mRNAs. We have identified and isolated proteins that bind specifically to the 5′ leader of the chloroplastic psbA mRNA, encoding the photosystem II reaction center protein D1. Binding of these proteins protects a 36 base RNA fragment containing a stem‐loop located upstream of the ribosome binding site. Binding of these proteins to the psbA mRNA correlates with the level of translation of psbA mRNA observed in light‐ and dark‐grown wild type cells and in a mutant that lacks D1 synthesis in the dark. The accumulation of at least one of these psbA mRNA‐binding proteins is dependent upon chloroplast development, while its mRNA‐binding activity appears to be light modulated in developed chloroplasts. These nuclear encoded proteins are prime candidates for regulators of chloroplast protein synthesis and may play an important role in coordinating nuclear‐chloroplast gene expression as well as provide a mechanism for regulating chloroplast gene expression during development in higher plants.

ADP-dependent phosphorylation regulates RNA-binding in vitro: implications in light-modulated translation.

Light‐regulated translation of chloroplastic mRNAs in the green alga Chlamydomonas reinhardtii requires nuclear encoded factors that interact with the 5′‐untranslated region (5′‐UTR) of specific mRNAs to enhance their translation. We have previously identified and characterized a set of proteins that bind specifically to the 5′‐UTR of the chloroplastic psbA mRNA. Accumulation of these proteins is similar in dark‐ and light‐grown cells, whereas their binding activity is enhanced during growth in the light. We have identified a serine/threonine protein phosphotransferase, associated with the psbA mRNA‐binding complex, that utilizes the beta‐phosphate of ADP to phosphorylate and inactivate psbA mRNA‐binding in vitro. The inactivation of mRNA‐binding in vitro is initiated at high ADP levels, levels that are attained in vivo only in dark‐grown chloroplasts. These data suggest that the translation of psbA mRNA is attenuated by phosphorylation of the mRNA‐binding protein complex in response to a rise in the stromal concentration of ADP upon transfer of cells to dark.

Translation of Chloroplast psbA mRNA Is Modulated in the Light by Counteracting Oxidizing and Reducing Activities

ABSTRACT Light has been proposed to stimulate the translation ofChlamydomonas reinhardtii chloroplast psbA mRNA by activating a protein complex associated with the 5′ untranslated region of this mRNA. The protein complex contains a redox-active regulatory site responsive to thioredoxin. We identified RB60, a protein disulfide isomerase-like member of the protein complex, as carrying the redox-active regulatory site composed of vicinal dithiol. We assayed in parallel the redox state of RB60 and translation of psbA mRNA in intact chloroplasts. Light activated the specific oxidation of RB60, on the one hand, and reduced RB60, probably via the ferredoxin-thioredoxin system, on the other. Higher light intensities increased the pool of reduced RB60 and the rate of psbA mRNA translation, suggesting that a counterbalanced action of reducing and oxidizing activities modulates the translation of psbA mRNA in parallel with fluctuating light intensities. In the dark, chemical reduction of the vicinal dithiol site did not activate translation. These results suggest a mechanism by which light primes redox-regulated translation by an unknown mechanism and then the rate of translation is determined by the reduction-oxidation of a sensor protein located in a complex bound to the 5′ untranslated region of the chloroplast mRNA.

Dual targeting of the protein disulfide isomerase RB60 to the chloroplast and the endoplasmic reticulum

RB60 is an atypical protein disulfide isomerase (PDI) that functions as a member of a redox regulatory protein complex controlling translation in the chloroplast of Chlamydomonas reinhardtii, but also contains a C-terminal endoplasmic reticulum (ER) retention signal, -KDEL. Here, we show by fluorescence microscopy that RB60 resides in the chloroplast but also outside of the chloroplast colocalized with BiP, an ER marker protein. RB60 accumulates in microsomes that exhibit a typical ER magnesium-shift, and cotranslationally translocates into ER microsomes. The first 50-aa leader of RB60 is sufficient for both chloroplast and ER targeting. The leader is cleaved upon translocation into the ER, whereas it remains intact after import to the chloroplast. The leader sequence also contains an acidic domain that appears necessary for the proteins association with the thylakoid membranes. Based on these and additional results, we propose that the dual localization of RB60 occurs via the two conserved transport mechanisms, to the chloroplast and to the ER, that the chloroplast RB60 most likely carries an additional function in the ER, and that its mode of transport, including the differential cleavage of its N terminus, plays an important role in its suborganellar localization and organellar-specific function.

Translation of chloroplast psbA mRNA is regulated by signals initiated by both photosystems II and I.

Light controls the translation of several mRNAs in fully developed chloroplasts via at least two regulatory pathways. In the first, the light signal is transduced as a thiol-mediated signal that modulates translation in parallel to light intensity. The second light-controlled pathway, termed priming, is a prerequisite to the thiol-mediated regulatory pathway. Light regulation is rapid and requires intrachloroplast photoreceptor(s). To delineate the signaling pathways controlling each of these regulatory events, we assayed the effect of photosynthetic inhibitors and electron donors on the translation of chloroplastic psbA mRNA. We show that the thiol-mediated signal is generated by photosystem I and transduced by vicinal dithiol-containing proteins. We also found that the priming signal probably initiates on reduction of plastoquinone. These findings suggest that translation of chloroplast psbA mRNA is controlled by both linear photosynthetic electron transport, exerted by the reduction of the ferredoxin–thioredoxin system, and the relative activities of photosystems I and II, signaled by the redox state of the plastoquinone pool. These data underscore the function of the light-capturing reactions of photosynthesis as chloroplast photoreceptors.

Translational Regulation in the Chloroplast

TRANSLATIONAL REGULATION IN THE CHLOROPLAST Gene expression is potentially amenable to regulation at different points along the linear path from a gene to a functional protein. To control gene expression the regulatory event has to occur at a limiting rate. Chloroplast gene expression has been shown to be primarily regulated by posttranscriptional events, including transcript stability, translation, protein turnover, and protein activity. An increasing body of data has demonstrated the significant role

The protein disulfide isomerase-like RB60 is partitioned between stroma and thylakoids in Chlamydomonas reinhardtii chloroplasts.

Translation of psbA mRNA inChlamydomonas reinhardtii chloroplasts is regulated by a redox signal(s). RB60 is a member of a protein complex that binds with high affinity to the 5′-untranslated region of psbAmRNA. RB60 has been suggested to act as a redox-sensor subunit of the protein complex regulating translation of chloroplastpsbA mRNA. Surprisingly, cloning of RB60 identified high homology to the endoplasmic reticulum-localized protein disulfide isomerase, including an endoplasmic reticulum-retention signal at its carboxyl terminus. Here we show, by in vitro import studies, that the recombinant RB60 is imported into isolated chloroplasts of C. reinhardtii and pea in a transit peptide-dependent manner. Subfractionation of C. reinhardtii chloroplasts revealed that the native RB60 is partitioned between the stroma and the thylakoids. The nature of association of native RB60, and imported recombinant RB60, with thylakoids is similar and suggests that RB60 is tightly bound to thylakoids. The targeting characteristics of RB60 and the potential implications of the association of RB60 with thylakoids are discussed.

Redox reactions of regulatory proteins: do kinetics promote specificity?

Signaling by redox state regulates the transcriptional and post-transcriptional events that control gene expression. To elucidate redox signaling in vivo, the effects of the reductive intracellular redox environment on regulatory redox events must be taken into account. This article focuses on proteins that contain regulatory disulfides, considering whether regulatory proteins can be oxidized and how the redox state of regulatory proteins can be uniquely controlled to allow redox signaling via specific pathways. It is possible that the favored kinetics of the redox reactions of regulatory proteins are important for attaining specificity in redox signaling.

Translation and translational regulation in chloroplasts

The translation mechanism of chloroplast mRNAs originated as prokaryotic-type, but has sinceevolved considerably. Chloroplast translation became, in large part, uncoupled from transcription,and turned into a highly regulated process. Concomitantly, chloroplast ribosomes, general translationfactors, and transcripts changed substantially from their prokaryotic counterparts. A multitudeof nucleus encoded regulatory proteins evolved that interact in a specific manner with elementsin mRNAs to allow translation regulation in response to environmental and developmental cues. Inthis chapter, we sum up the current knowledge regarding the translation machinery in the chloroplastusing examples of mechanisms utilized for chloroplast translation regulation.

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

This study identifies a regulatory oxidative pathway, comprised of thioredoxin and peroxiredoxin, in Arabidopsis thaliana chloroplasts. It shows that the pathway is used to sense photosynthetic peroxide formation under low to moderate light intensity and proposes that the oxidative signal adjusts the photosynthetic linear electron flow to fluctuating environmental conditions. 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.