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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

A conserved methionine in the NES of NirA is oxidized in the absence of nitrate.(A) Example of an MS/MS spectrum of NirA NES peptides derived from wild-type FLAG-NirA purified from cells grown under inducing (IND) and non-inducing (NI) conditions. For both conditions, cells were initially grown under NI conditions on 3 mM arginine for 14 hours and for induction, 10 mM NO3- was added to a subset of cultures and incubation proceeded for 5 minutes prior to harvesting. The difference between methionine sulfoxide (Mox) and methionine (M) is indicated by the 16 Da shift of the B ion series in the MS/MS spectra. An overview of MS/MS data obtained from PTM analyses of NirA-NES methionine 169 (Met169/Mox169) in the wild type and different mutant strains is given in Table 1. Detailed MS/MS spectra are shown in S6 Fig AA, amino acids. (B) Alignment of known or putative NES sequences comprising methionine residues in their motifs. NirA homologous genes from fungi as well as plant proteins with known or proposed function in the nitrate response were selected. Fungal species harbouring NirA homologues are aligned as follows: Aspergillus nidulans, Aspergillus fumigatus, Magnaporthe grisea, Neurospora crassa, Tolypocladium inflatum, Botrytis cinerea, Chaetomium globosum, Sclerotinia sclerotiorum, Stagonospora nodorum, Histoplasma capsulatum, Fusarium graminearum, Hansenula polymorpha.
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pgen.1005297.g001: A conserved methionine in the NES of NirA is oxidized in the absence of nitrate.(A) Example of an MS/MS spectrum of NirA NES peptides derived from wild-type FLAG-NirA purified from cells grown under inducing (IND) and non-inducing (NI) conditions. For both conditions, cells were initially grown under NI conditions on 3 mM arginine for 14 hours and for induction, 10 mM NO3- was added to a subset of cultures and incubation proceeded for 5 minutes prior to harvesting. The difference between methionine sulfoxide (Mox) and methionine (M) is indicated by the 16 Da shift of the B ion series in the MS/MS spectra. An overview of MS/MS data obtained from PTM analyses of NirA-NES methionine 169 (Met169/Mox169) in the wild type and different mutant strains is given in Table 1. Detailed MS/MS spectra are shown in S6 Fig AA, amino acids. (B) Alignment of known or putative NES sequences comprising methionine residues in their motifs. NirA homologous genes from fungi as well as plant proteins with known or proposed function in the nitrate response were selected. Fungal species harbouring NirA homologues are aligned as follows: Aspergillus nidulans, Aspergillus fumigatus, Magnaporthe grisea, Neurospora crassa, Tolypocladium inflatum, Botrytis cinerea, Chaetomium globosum, Sclerotinia sclerotiorum, Stagonospora nodorum, Histoplasma capsulatum, Fusarium graminearum, Hansenula polymorpha.

Mentions: We analysed FLAG-tagged NirA obtained by DNA affinity-purification from cells grown on non-inducing (NI, 3 mM arginine) or nitrate inducing (IND, 10mM NaNO3) conditions by tandem mass spectrometry. In the absence of NO3-, the NES of NirA is modified by oxidation of the conserved methionine (Met169) to methionine sulfoxide (Metox169). When cells were exposed to nitrate for five minutes Metox169 could not be detected any longer (Fig 1 and Table 1). This rapid response of A. nidulans cells to the inducer coincides with the swift nuclear accumulation of NirA (S1 Video). No Met169 oxidation could be detected in the NirAc1 protein (Table 1 and S6 Fig). These MS results strongly suggested a functional link between the oxidation status of M169 and intracellular localisation of NirA.


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)

A conserved methionine in the NES of NirA is oxidized in the absence of nitrate.(A) Example of an MS/MS spectrum of NirA NES peptides derived from wild-type FLAG-NirA purified from cells grown under inducing (IND) and non-inducing (NI) conditions. For both conditions, cells were initially grown under NI conditions on 3 mM arginine for 14 hours and for induction, 10 mM NO3- was added to a subset of cultures and incubation proceeded for 5 minutes prior to harvesting. The difference between methionine sulfoxide (Mox) and methionine (M) is indicated by the 16 Da shift of the B ion series in the MS/MS spectra. An overview of MS/MS data obtained from PTM analyses of NirA-NES methionine 169 (Met169/Mox169) in the wild type and different mutant strains is given in Table 1. Detailed MS/MS spectra are shown in S6 Fig AA, amino acids. (B) Alignment of known or putative NES sequences comprising methionine residues in their motifs. NirA homologous genes from fungi as well as plant proteins with known or proposed function in the nitrate response were selected. Fungal species harbouring NirA homologues are aligned as follows: Aspergillus nidulans, Aspergillus fumigatus, Magnaporthe grisea, Neurospora crassa, Tolypocladium inflatum, Botrytis cinerea, Chaetomium globosum, Sclerotinia sclerotiorum, Stagonospora nodorum, Histoplasma capsulatum, Fusarium graminearum, Hansenula polymorpha.
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Related In: Results  -  Collection

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pgen.1005297.g001: A conserved methionine in the NES of NirA is oxidized in the absence of nitrate.(A) Example of an MS/MS spectrum of NirA NES peptides derived from wild-type FLAG-NirA purified from cells grown under inducing (IND) and non-inducing (NI) conditions. For both conditions, cells were initially grown under NI conditions on 3 mM arginine for 14 hours and for induction, 10 mM NO3- was added to a subset of cultures and incubation proceeded for 5 minutes prior to harvesting. The difference between methionine sulfoxide (Mox) and methionine (M) is indicated by the 16 Da shift of the B ion series in the MS/MS spectra. An overview of MS/MS data obtained from PTM analyses of NirA-NES methionine 169 (Met169/Mox169) in the wild type and different mutant strains is given in Table 1. Detailed MS/MS spectra are shown in S6 Fig AA, amino acids. (B) Alignment of known or putative NES sequences comprising methionine residues in their motifs. NirA homologous genes from fungi as well as plant proteins with known or proposed function in the nitrate response were selected. Fungal species harbouring NirA homologues are aligned as follows: Aspergillus nidulans, Aspergillus fumigatus, Magnaporthe grisea, Neurospora crassa, Tolypocladium inflatum, Botrytis cinerea, Chaetomium globosum, Sclerotinia sclerotiorum, Stagonospora nodorum, Histoplasma capsulatum, Fusarium graminearum, Hansenula polymorpha.
Mentions: We analysed FLAG-tagged NirA obtained by DNA affinity-purification from cells grown on non-inducing (NI, 3 mM arginine) or nitrate inducing (IND, 10mM NaNO3) conditions by tandem mass spectrometry. In the absence of NO3-, the NES of NirA is modified by oxidation of the conserved methionine (Met169) to methionine sulfoxide (Metox169). When cells were exposed to nitrate for five minutes Metox169 could not be detected any longer (Fig 1 and Table 1). This rapid response of A. nidulans cells to the inducer coincides with the swift nuclear accumulation of NirA (S1 Video). No Met169 oxidation could be detected in the NirAc1 protein (Table 1 and S6 Fig). These MS results strongly suggested a functional link between the oxidation status of M169 and intracellular localisation of NirA.

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