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

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