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14‐3‐3 Protein‐Dependent GATA Transcription Factor Control in Saccharomyces cerevisiae

14‐3‐3 proteins, Bmh1/2, are major participants in the control of most nutrient‐ and stress‐sensitive regulons in Saccharomyces cerevisiae. However, the Nitrogen Catabolite Repression (NCR)‐sensitive regulon and its Gln3 and Gat1 GATA transcription activators have not been reported to be Bmh1/2‐regulated. Observations from proteomic studies identify Gln3 as a candidate Bmh1/2 interacting protein (Biochem 46:7781; Genomics 94:287), and NCR‐sensitive GAP1 expression is about 1.5‐fold higher in bmh2 than bmh1 mutants treated with TorC1 inhibitor, rapamycin (Biosci. Rep. 34;art:e0009). Therefore, we determined whether Bmh1/2 participates directly in Gln3 and Gat1 control or more indirectly via Bmh1/2‐dependent TorC1 regulation. The question of Bmh1/2‐dependent GATA factor regulation can only be effectively investigated in a ∑1278b background because in most other strains the double mutant is lethal. In poor nitrogen conditions, Gln3 and Gat1 are nuclear and support high level NCR‐sensitive transcription in wild type cells. In contrast, when abundant nitrogen is available, Gln3 and Gat1 relocate to the cytoplasm and GATA factor‐dependent transcription is repressed. This wild type regulation is achieved via post‐translational Gln3 regulation by the TorC1 and Gcn2 kinases, Sit4 and PP2A phosphatases and downstream negative regulator Ure2. In a bmh1Δbmh2Δ double mutant, we find that: (i) NCR‐sensitive transcription of three reporters, GDH2, DAL80 and DAL5, is almost completely abolished. (ii) Gln3 and Gat1‐mediated transcriptional responses to rapamycin‐treatment are detectable but severely diminished. (iii) Loss of Bmh1/2 results in Gln3 dephosphorylation similar to the levels observed following rapamycin treatment. (iv) The response of Gln3 and Gat1 nuclear localization parallels that observed for GATA‐factor mediated transcription. (v) Cells form pseudohyphae‐like chains when provided with nitrogen sources whose utilization requires NCR‐sensitive transcription. Prior to the point when bmh1Δbmh2Δ mother and daughter cells separate, only one contains detectable GFP‐Gln3. Either Bmh1 or Bmh2 will fulfill the 14‐3‐3 requirement as nuclear Gln3 localization is normal in single bmh1 mutants. Using bmh1Δbmh2Δ and bmh1Δbmh2Δure2Δ mutants, we demonstrate that a bmh1Δbmh2Δ double deletion is epistatic to an ure2 deletion. We conclude Bmh1/2 are absolutely required for nuclear Gln3 localization and likely function downstream of Ure2, heretofore the most downstream regulator of the nitrogen‐sensitive regulon.