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Reversible Oxidation of a Conserved Methionine in the Nuclear Export Sequence Determines Subcellular Distribution and Activity of the Fungal Nitrate Regulator NirA.

Gallmetzer A, Silvestrini L, Schinko T, Gesslbauer B, Hortschansky P, Dattenböck C, Muro-Pastor MI, Kungl A, Brakhage AA, Scazzocchio C, Strauss J - PLoS Genet. (2015)

Bottom Line: Exposure of A. nidulans cells to nitrate led to rapid reduction of NirA-Metox169 to Met169; this reduction being independent from thioredoxin and classical methionine sulfoxide reductases.Co-immunoprecipitation analysis of NirA-KapK interactions and subcellular localization studies of NirA mutants lacking different parts of the protein provided evidence that Met169 oxidation leads to a change in NirA conformation.Based on these results we propose that in the presence of nitrate the activation domain is exposed, but the NES is masked by a central portion of the protein (termed nitrate responsive domain, NiRD), thus restricting active NirA molecules to the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Fungal Genetics and Genomics Unit, Department of Applied Genetics and Cell Biology, BOKU-University of Natural Resources and Life Science, Vienna, Vienna, Austria.

ABSTRACT
The assimilation of nitrate, a most important soil nitrogen source, is tightly regulated in microorganisms and plants. In Aspergillus nidulans, during the transcriptional activation process of nitrate assimilatory genes, the interaction between the pathway-specific transcription factor NirA and the exportin KapK/CRM1 is disrupted, and this leads to rapid nuclear accumulation and transcriptional activity of NirA. In this work by mass spectrometry, we found that in the absence of nitrate, when NirA is inactive and predominantly cytosolic, methionine 169 in the nuclear export sequence (NES) is oxidized to methionine sulfoxide (Metox169). This oxidation depends on FmoB, a flavin-containing monooxygenase which in vitro uses methionine and cysteine, but not glutathione, as oxidation substrates. The function of FmoB cannot be replaced by alternative Fmo proteins present in A. nidulans. Exposure of A. nidulans cells to nitrate led to rapid reduction of NirA-Metox169 to Met169; this reduction being independent from thioredoxin and classical methionine sulfoxide reductases. Replacement of Met169 by isoleucine, a sterically similar but not oxidizable residue, led to partial loss of NirA activity and insensitivity to FmoB-mediated nuclear export. In contrast, replacement of Met169 by alanine transformed the protein into a permanently nuclear and active transcription factor. Co-immunoprecipitation analysis of NirA-KapK interactions and subcellular localization studies of NirA mutants lacking different parts of the protein provided evidence that Met169 oxidation leads to a change in NirA conformation. Based on these results we propose that in the presence of nitrate the activation domain is exposed, but the NES is masked by a central portion of the protein (termed nitrate responsive domain, NiRD), thus restricting active NirA molecules to the nucleus. In the absence of nitrate, Met169 in the NES is oxidized by an FmoB-dependent process leading to loss of protection by the NiRD, NES exposure, and relocation of the inactive NirA to the cytosol.

No MeSH data available.


Related in: MedlinePlus

Methionine oxidation in the NirA-NES defines subcellular localization and activity.(A) NirA-GFP sub-cellular localization in different strains under non-induced (NI, 3 mM arginine), non-induced plus n-octylamine (NIo, 3 mM arginine plus 10 mM n-octylamine), induced (IND, 10 mM nitrate) and induced plus n-octylamine (INDo, 10 mM nitrate plus 10 mM n-octylamine) conditions. Pictures were taken two minutes after addition of the respective compounds. All strains harbour the Leptomycin B (LMB) sensitive allele kapK1. Leptomycin B sensitivity enables testing if loss of nuclear accumulation is related to/dependent on exportin (KapK) function. Except strain kapK1 NirAM169A, which carries a Met to Ala substitution within the NES sequence, all strains carry a NirA-GFP wild type construct. Scale bars refer to 5 μm. (B) Effect of the fmoB deletion on niaD transcription. Strains were grown under standard conditions and transferred for 20 minutes to the media as indicated and described in panel A. Signal intensities were calculated with ImageQuant (Molecular Dynamics) and niaD expression was normalized to 18S rRNA. (C) Effect of nitrogen source and n-octylamine on NirA protein stability. Whole cell extracts of a FLAG-tagged NirA strain were analysed by Western blot. The strain was grown for 16 hours under non induced conditions with 0.2% fructose and 3 mM arginine as nitrogen source; NI. The cultures where subsequently treated with 2 mM n-octylamine, NIo; 10 mM nitrate, IND; and 10 mM nitrate plus 2 mM n-octylamine, INDo. Specific signals were normalized against actin (ActA).
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pgen.1005297.g002: Methionine oxidation in the NirA-NES defines subcellular localization and activity.(A) NirA-GFP sub-cellular localization in different strains under non-induced (NI, 3 mM arginine), non-induced plus n-octylamine (NIo, 3 mM arginine plus 10 mM n-octylamine), induced (IND, 10 mM nitrate) and induced plus n-octylamine (INDo, 10 mM nitrate plus 10 mM n-octylamine) conditions. Pictures were taken two minutes after addition of the respective compounds. All strains harbour the Leptomycin B (LMB) sensitive allele kapK1. Leptomycin B sensitivity enables testing if loss of nuclear accumulation is related to/dependent on exportin (KapK) function. Except strain kapK1 NirAM169A, which carries a Met to Ala substitution within the NES sequence, all strains carry a NirA-GFP wild type construct. Scale bars refer to 5 μm. (B) Effect of the fmoB deletion on niaD transcription. Strains were grown under standard conditions and transferred for 20 minutes to the media as indicated and described in panel A. Signal intensities were calculated with ImageQuant (Molecular Dynamics) and niaD expression was normalized to 18S rRNA. (C) Effect of nitrogen source and n-octylamine on NirA protein stability. Whole cell extracts of a FLAG-tagged NirA strain were analysed by Western blot. The strain was grown for 16 hours under non induced conditions with 0.2% fructose and 3 mM arginine as nitrogen source; NI. The cultures where subsequently treated with 2 mM n-octylamine, NIo; 10 mM nitrate, IND; and 10 mM nitrate plus 2 mM n-octylamine, INDo. Specific signals were normalized against actin (ActA).

Mentions: As methionine oxidation could generally affect NirA protein stability under non-induced conditions, we tested the amounts of FLAG-NirA in crude extracts obtained from arginine-grown (NI) or NO3-grown (IND) cells. We found that NirA levels were basically equal under both conditions and this result indicates that overall stability of NirA is not influenced by the Met169 oxidation state (Fig 2C).


Reversible Oxidation of a Conserved Methionine in the Nuclear Export Sequence Determines Subcellular Distribution and Activity of the Fungal Nitrate Regulator NirA.

Gallmetzer A, Silvestrini L, Schinko T, Gesslbauer B, Hortschansky P, Dattenböck C, Muro-Pastor MI, Kungl A, Brakhage AA, Scazzocchio C, Strauss J - PLoS Genet. (2015)

Methionine oxidation in the NirA-NES defines subcellular localization and activity.(A) NirA-GFP sub-cellular localization in different strains under non-induced (NI, 3 mM arginine), non-induced plus n-octylamine (NIo, 3 mM arginine plus 10 mM n-octylamine), induced (IND, 10 mM nitrate) and induced plus n-octylamine (INDo, 10 mM nitrate plus 10 mM n-octylamine) conditions. Pictures were taken two minutes after addition of the respective compounds. All strains harbour the Leptomycin B (LMB) sensitive allele kapK1. Leptomycin B sensitivity enables testing if loss of nuclear accumulation is related to/dependent on exportin (KapK) function. Except strain kapK1 NirAM169A, which carries a Met to Ala substitution within the NES sequence, all strains carry a NirA-GFP wild type construct. Scale bars refer to 5 μm. (B) Effect of the fmoB deletion on niaD transcription. Strains were grown under standard conditions and transferred for 20 minutes to the media as indicated and described in panel A. Signal intensities were calculated with ImageQuant (Molecular Dynamics) and niaD expression was normalized to 18S rRNA. (C) Effect of nitrogen source and n-octylamine on NirA protein stability. Whole cell extracts of a FLAG-tagged NirA strain were analysed by Western blot. The strain was grown for 16 hours under non induced conditions with 0.2% fructose and 3 mM arginine as nitrogen source; NI. The cultures where subsequently treated with 2 mM n-octylamine, NIo; 10 mM nitrate, IND; and 10 mM nitrate plus 2 mM n-octylamine, INDo. Specific signals were normalized against actin (ActA).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4488483&req=5

pgen.1005297.g002: Methionine oxidation in the NirA-NES defines subcellular localization and activity.(A) NirA-GFP sub-cellular localization in different strains under non-induced (NI, 3 mM arginine), non-induced plus n-octylamine (NIo, 3 mM arginine plus 10 mM n-octylamine), induced (IND, 10 mM nitrate) and induced plus n-octylamine (INDo, 10 mM nitrate plus 10 mM n-octylamine) conditions. Pictures were taken two minutes after addition of the respective compounds. All strains harbour the Leptomycin B (LMB) sensitive allele kapK1. Leptomycin B sensitivity enables testing if loss of nuclear accumulation is related to/dependent on exportin (KapK) function. Except strain kapK1 NirAM169A, which carries a Met to Ala substitution within the NES sequence, all strains carry a NirA-GFP wild type construct. Scale bars refer to 5 μm. (B) Effect of the fmoB deletion on niaD transcription. Strains were grown under standard conditions and transferred for 20 minutes to the media as indicated and described in panel A. Signal intensities were calculated with ImageQuant (Molecular Dynamics) and niaD expression was normalized to 18S rRNA. (C) Effect of nitrogen source and n-octylamine on NirA protein stability. Whole cell extracts of a FLAG-tagged NirA strain were analysed by Western blot. The strain was grown for 16 hours under non induced conditions with 0.2% fructose and 3 mM arginine as nitrogen source; NI. The cultures where subsequently treated with 2 mM n-octylamine, NIo; 10 mM nitrate, IND; and 10 mM nitrate plus 2 mM n-octylamine, INDo. Specific signals were normalized against actin (ActA).
Mentions: As methionine oxidation could generally affect NirA protein stability under non-induced conditions, we tested the amounts of FLAG-NirA in crude extracts obtained from arginine-grown (NI) or NO3-grown (IND) cells. We found that NirA levels were basically equal under both conditions and this result indicates that overall stability of NirA is not influenced by the Met169 oxidation state (Fig 2C).

Bottom Line: Exposure of A. nidulans cells to nitrate led to rapid reduction of NirA-Metox169 to Met169; this reduction being independent from thioredoxin and classical methionine sulfoxide reductases.Co-immunoprecipitation analysis of NirA-KapK interactions and subcellular localization studies of NirA mutants lacking different parts of the protein provided evidence that Met169 oxidation leads to a change in NirA conformation.Based on these results we propose that in the presence of nitrate the activation domain is exposed, but the NES is masked by a central portion of the protein (termed nitrate responsive domain, NiRD), thus restricting active NirA molecules to the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Fungal Genetics and Genomics Unit, Department of Applied Genetics and Cell Biology, BOKU-University of Natural Resources and Life Science, Vienna, Vienna, Austria.

ABSTRACT
The assimilation of nitrate, a most important soil nitrogen source, is tightly regulated in microorganisms and plants. In Aspergillus nidulans, during the transcriptional activation process of nitrate assimilatory genes, the interaction between the pathway-specific transcription factor NirA and the exportin KapK/CRM1 is disrupted, and this leads to rapid nuclear accumulation and transcriptional activity of NirA. In this work by mass spectrometry, we found that in the absence of nitrate, when NirA is inactive and predominantly cytosolic, methionine 169 in the nuclear export sequence (NES) is oxidized to methionine sulfoxide (Metox169). This oxidation depends on FmoB, a flavin-containing monooxygenase which in vitro uses methionine and cysteine, but not glutathione, as oxidation substrates. The function of FmoB cannot be replaced by alternative Fmo proteins present in A. nidulans. Exposure of A. nidulans cells to nitrate led to rapid reduction of NirA-Metox169 to Met169; this reduction being independent from thioredoxin and classical methionine sulfoxide reductases. Replacement of Met169 by isoleucine, a sterically similar but not oxidizable residue, led to partial loss of NirA activity and insensitivity to FmoB-mediated nuclear export. In contrast, replacement of Met169 by alanine transformed the protein into a permanently nuclear and active transcription factor. Co-immunoprecipitation analysis of NirA-KapK interactions and subcellular localization studies of NirA mutants lacking different parts of the protein provided evidence that Met169 oxidation leads to a change in NirA conformation. Based on these results we propose that in the presence of nitrate the activation domain is exposed, but the NES is masked by a central portion of the protein (termed nitrate responsive domain, NiRD), thus restricting active NirA molecules to the nucleus. In the absence of nitrate, Met169 in the NES is oxidized by an FmoB-dependent process leading to loss of protection by the NiRD, NES exposure, and relocation of the inactive NirA to the cytosol.

No MeSH data available.


Related in: MedlinePlus