The primary regulator of nitrogen metabolic genes in fungi is the GATA transcription factor AreA. Subcellular localization of AreA in A. nidulans is dependent on nutrient availability. When glucose and nitrogen sources are available, AreA is found throughout the cytoplasm and the nucleus. When the cell becomes nitrogen starved, AreA accumulates in the nucleus due to blocked AreA nuclear export. We have recently found redundancy in the nuclear localization signals (NLSs) of AreA. Five conserved canonical NLSs and one conserved noncanonical arginine-based bipartite NLS within the zinc-finger DNA binding domain all function together to mediate nuclear import. Only mutation of the noncanonical bipartite NLS within the DNA binding domain had any significant effect on AreA function, completely abolishing transcriptional activity. In order to determine whether AreA DNA binding affects the intracellular localization of AreA we have HA-epitope-tagged and analyzed the two classical DNA binding mutants; an altered DNA binding specificity mutant, AreA102, and a non-binding mutant, AreA217. The AreA102HA mutant protein showed a similar pattern of subcellular localization to wildtype AreA except when transferred to uric acid (a nitrogen source the areA102 mutant cannot utilize). On uric acid AreA102HA accumulated in the nucleus as observed during nitrogen starvation. The AreA217HA nonbinding mutant protein accumulated in the nuclei of nitrogen-starved cells, demonstrating that DNA binding is not required for AreA nuclear accumulation. AreA217HA does not show nuclear accumulation when ammonium is present. In contrast to wildtype, AreA217HA accumulated in the nucleus when alternative nitrogen sources were available. These findings suggest that while nuclear accumulation of AreA is independent of DNA binding, nuclear export is dependent on AreA DNA binding function. We show using nitrogen metabolism mutants that this is likely due to the requirement of AreA for metabolic signaling of nitrogen availability.
Full conference title:
- Asperfest 14 (2017)