New targets of Arabidopsis thioredoxins revealed by proteomic analysis

Marchand, Christophe; Maréchal, Pierre Le; Meyer, Yves; Miginiac‐Maslow, Myroslawa; Issakidis‐Bourguet, Emmanuelle; Decottignies, Paulette

doi:10.1002/pmic.200400805

Cited by 183 publications

(183 citation statements)

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“…Interactions occur in vivo, which distinguishes this approach from other protein-targeting methods, and can be performed between two proteins from different organisms. Unfortunately, when we first used commercial Y2H systems to search for TRX targets using DNA libraries, using yeast or plant TRXs as bait, very few targets were isolated, in contrast to the numerous putative targets isolated by using in vitro proteomics (19,20,48). Even Y2H binary interactions involving TRX targets isolated by another in vivo method failed in classical Y2H yeast strains (26).…”

Section: Discussionmentioning

confidence: 99%

A yeast two-hybrid knockout strain to explore thioredoxin-interacting proteinsin vivo

Vignols

Bréhélin

Surdin-Kerjan

et al. 2005

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

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All organisms contain thioredoxin (TRX), a regulatory thiol:disulfide protein that reduces disulfide bonds in target proteins. Unlike animals and yeast, plants contain numerous TRXs for which no function has been assigned in vivo. Recent in vitro proteomic approaches have opened the way to the identification of >100 TRX putative targets, but of which none of the numerous plant TRXs can be specifically associated. In contrast, in vivo methodologies, including classical yeast two-hybrid (Y2H) systems, failed to reveal the expected high number of TRX targets. Here, we developed a yeast strain named CY306 designed to identify TRX targets in vivo by a Y2H approach. CY306 contains a GAL4 reporter system but also carries deletions of endogenous genes encoding cytosolic TRXs (TRX1 and TRX2) that presumably compete with TRXs introduced as bait. We demonstrate here that, in the CY306 strain, yeast TRX1 and TRX2, as well as Arabidopsis TRX introduced as bait, interact with known TRX targets or putative partners such as yeast peroxiredoxins AHP1 and TSA1, whereas the same interactions cannot be detected in classical Y2H strains. Thanks to CY306, we also show that TRXs interact with the phosphoadenosine-5-phosphosulfate (PAPS) reductase MET16 through a conserved cysteine. Moreover, interactions visualized in CY306 are highly specific depending on the TRX and targets tested. CY306 constitutes a relevant genetic system to explore the TRX interactome in vivo and with high specificity, and opens new perspectives in the search for new TRX-interacting proteins by Y2H library screening in organisms with multiple TRXs. affinity chromatography ͉ gene disruption ͉ thioredoxin targets ͉ two-hybrid system ͉ redox T hioredoxins (TRXs) are small heat-stable oxidoreductases containing two redox-active half-cysteine residues in an active site with a conserved amino acid sequence CXXC (where X indicates various amino acids) (1). TRX was originally identified as hydrogen donor for the reduction of methionine sulfoxide (MetSO) (2) and of sulfate (3) in yeast and received its name when it was characterized as a small protein dithiol hydrogen donor to Escherichia coli ribonucleotide reductase (4). TRX is a hydrogen donor to peroxiredoxins (5) and is also required for a number of metabolic enzymes as part of their catalytic cycle. TRX is implicated in many cellular processes, including protein folding and regulation of transcription factors (6), protein repair after damage by oxidation, sulfur metabolism (7, 8), reduction of dehydroascorbate (9), germination, or reduction of the Calvin cycle and stromal enzymes in plants (10,11).Occurrence of TRXs in all genomes is highly variable and somewhat complex, depending on the organisms concerned. Most organisms, such as E. coli, the yeast Saccharomyces cerevisiae, and mammals (12) contain a limited number of TRX or TRX-like genes (usually fewer than five). In contrast, plants possess numerous TRX isoforms (13,14) or TRX-like proteins (11,(13)(14)(15). During the latest inventories in the Arabidopsis gen...

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Section: Discussionmentioning

confidence: 99%

A yeast two-hybrid knockout strain to explore thioredoxin-interacting proteinsin vivo

Vignols

Bréhélin

Surdin-Kerjan

et al. 2005

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

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“…13 The proteins sensitive to redox potential, termed potential thioredoxin targets (PTTs), have been intensively investigated. 13 Here, we screened possible PTTs from the differentially expressed proteins (DEPs) by use of a known PTT database 13,[28][29][30][31][32][33] (Supplemental Table S3). About two-thirds of the DEPs matched known PTTs.…”

Section: Identification Of Differentially Expressed Proteins Ms and mentioning

confidence: 99%

Integrated Proteomic and Cytological Study of Rice Endosperms at the Storage Phase

Yan

et al. 2010

J. Proteome Res.

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The endosperm at the storage phase undergoes a series of coordinated cellular and metabolic events, including starchy endosperm cell death, starch synthesis, and starch granule packaging, which leads to efficient accumulation of starch. However, the mechanism underlying the interconnections remains unknown. We used integrated proteomic and cytological approaches to probe the interconnections in rice (Oryza sativa) endosperm at the storage phase from 12 to 18 days after flowering (DAF). Starch granule packaging was completed first in the inner part of endosperm at 15 DAF and spread to almost the entire endosperm at 18 DAF. Programmed starchy endosperm cell death occurred after the starch granule packaging. Endogenous H 2 O 2 was detectable in the inner part of endosperm at 12 DAF and the region beyond the inner part at 15 DAF, with an H 2 O 2 burst at 15 DAF. Proteomics analysis with 2-D fluorescent difference gel electrophoresis and matrix-assisted laser-desorption ionization time-of-flight/ time-of-flight mass spectrometry revealed 317 proteins, including almost all known antioxidants, differentially expressed throughout the 3 stages of the developmental phase. More than two-thirds of the 317 proteins were potential thioredoxin targets, with a preferential skew toward central carbon metabolism, alcoholic fermentation, starch metabolism, amino acid metabolism, and protein synthesis or folding. These proteins implicated in starch synthesis and gluconeogenesis were upregulated, whereas those involved in anabolism of biomacromolecules such as proteins, lipids, and cell wall components were downregulated, with upregulated expression of proteins involved in catabolism of these biomacromolecules, which suggests remobilization of nutrients for starch synthesis. These data suggested important roles of the H 2 O 2 -antioxidant interface in coordinating starch accumulation, programmed cell death of starchy endosperm, and remobilization of nutrients during the cell death.

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“…Recent approaches make it possible to identify proteins linked to Trx by combining proteomics with affinity chromatography (1) or thiol probes (2)(3)(4). These capabilities have defined an extended role of Trx in chloroplasts (1,5) and helped elucidate its function in other plant systems: mitochondria (6), seeds (2,3,7,8), and seedlings (4,9). Currently almost 200 proteins appear to be linked to Trx in plants (10).…”

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confidence: 99%

A complete ferredoxin/thioredoxin system regulates fundamental processes in amyloplasts

Balmer

Vensel

Cai

et al. 2006

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

161

137

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A growing number of processes throughout biology are regulated by redox via thiol-disulfide exchange. This mechanism is particularly widespread in plants, where almost 200 proteins have been linked to thioredoxin (Trx), a widely distributed small regulatory disulfide protein. The current study extends regulation by Trx to amyloplasts, organelles prevalent in heterotrophic plant tissues that, among other biosynthetic activities, catalyze the synthesis and storage of copious amounts of starch. Using proteomics and immunological methods, we identified the components of the ferredoxin͞Trx system (ferredoxin, ferredoxin-Trx reductase, and Trx), originally described for chloroplasts, in amyloplasts isolated from wheat starchy endosperm. Ferredoxin is reduced not by light, as in chloroplasts, but by metabolically generated NADPH via ferredoxin-NADP reductase. However, once reduced, ferredoxin appears to act as established for chloroplasts, i.e., via ferredoxinTrx reductase and a Trx (m-type). A proteomics approach in combination with affinity chromatography and a fluorescent thiol probe led to the identification of 42 potential Trx target proteins, 13 not previously recognized, including a major membrane transporter (Brittle-1 or ADP-glucose transporter). The proteins function in a range of processes in addition to starch metabolism: biosynthesis of lipids, amino acids, and nucleotides; protein folding; and several miscellaneous reactions. The results suggest a mechanism whereby light is initially recognized as a thiol signal in chloroplasts, then as a sugar during transit to the sink, where it is converted again to a thiol signal. In this way, amyloplast reactions in the grain can be coordinated with photosynthesis taking place in leaves.redox regulation ͉ target proteins ͉ ferredoxin-thioredoxin reductase O ur understanding of the function of the regulatory disulfide protein thioredoxin (Trx) has increased dramatically in the past few years, with the advent of new methodologies. Recent approaches make it possible to identify proteins linked to Trx by combining proteomics with affinity chromatography (1) or thiol probes (2-4). These capabilities have defined an extended role of Trx in chloroplasts (1, 5) and helped elucidate its function in other plant systems: mitochondria (6), seeds (2,3,7,8), and seedlings (4, 9). Currently almost 200 proteins appear to be linked to Trx in plants (10). One major organelle that has been neglected, however, is the amyloplast, a plastid of heterotrophic tissues that performs a wide range of biosynthetic reactions, including the synthesis and storage of abundant quantities of starch.To help fill this gap, we have applied proteomic and immunological approaches to investigate the occurrence and function of Trx in amyloplasts. We now report evidence that amyloplasts isolated from wheat starchy endosperm resemble chloroplasts in containing a complete ferredoxin͞Trx system composed of ferredoxin, ferredoxin-Trx reductase (FTR), and Trx (m-type). Application of affinity chromatography and fl...

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New targets of Arabidopsis thioredoxins revealed by proteomic analysis

Cited by 183 publications

References 49 publications

A yeast two-hybrid knockout strain to explore thioredoxin-interacting proteinsin vivo

A yeast two-hybrid knockout strain to explore thioredoxin-interacting proteinsin vivo

Integrated Proteomic and Cytological Study of Rice Endosperms at the Storage Phase

A complete ferredoxin/thioredoxin system regulates fundamental processes in amyloplasts

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