The Ins and Outs of Regulating Gln3, the Major Nitrogen‐Responsive Transcription Activator in Saccharomyces cerevisiae
Authors: Cooper Terrance G. , Rai Rajendra , Tate Jennifer J. 
Published: November 2023

Gln3 is the major transcription activator mediating nitrogen‐responsive gene expression in Saccharomyces cerevisiae. When cells are cultured in nitrogen‐replete environments (excess glutamine, asparagine or ammonia media), Gln3 is sequestered in the cytoplasm thereby minimizing nitrogen catabolite repression‐ (NCR‐) sensitive transcription. In the face of limiting nitrogen supplies (proline medium), Gln3 relocates to the nucleus and activates transcription of the NCR‐sensitive regulon whose products can transport and degrade a variety of poorly used nitrogen sources, thus expanding the cell's nitrogen acquisition capability. Four distinct regulatory pathways cumulatively participate in control of nitrogen‐responsive Gln3 localization. Each pathway responds to a specific environmental condition: (a) nitrogen limitation and short‐term nitrogen starvation, (b) long‐term nitrogen starvation, (c) glutamine starvation [methionine sulfoximine (Msx) inhibition of glutamine synthetase] and (d) rapamycin inhibition of the TorC1 serine/threonine kinase complex. Each response exhibits unique protein phosphatase requirements. Importantly, cytoplasmic sequestration of Gln3 occurs in the absence of Gtr‐Ego complex components required for TorC1 activation in amino acid excess. In agreement with these observations, Gln3 localization is unresponsive to leucine starvation or leucyl tRNA synthetase inhibitors which down‐regulate TorC1 activity. Together, these data argued that Gln3 regulation was achieved by multiple mechanisms one or more of which does not involve TorC1 regulation as it is currently understood. Therefore, our goal was to identify these additional regulatory mechanisms. Towards achieving this goal, we discovered a new mode of Gln3 regulation. Gln3 entry into the nucleus has long been known to be regulated by the cell's overall nitrogen supply, but regulated export has never been addressed. To explore nuclear Gln3 export, we substituted residues in the Gln3 GATA DNA‐binding Zn‐finger motif that abolished its binding to DNA. These amino acid substitutions do not affect Gln3 binding to or regulation by Ure2, the negative regulator responsible for Gln3's cytoplasmic sequestration. Using these mutants, we show that nuclear Gln3 can take one of two possible paths in exiting from the nucleus. The path taken is regulated specifically by glutamine itself rather than the cell's overall nitrogen supply. When glutamine levels are high, as occurs with glutamine as sole nitrogen source or glutamine analogues are added to the medium, Gln3 can readily exit from the nucleus without binding to DNA. It accumulates in the cytoplasm of the GATA zinc finger mutants. In contrast, when glutamine levels are lowered, by inhibiting glutamine synthetase or by adding a second nitrogen source (e.g. glutamate) to glutamine‐grown cells, nuclear Gln3 export is dependent on prior binding to its promoter DNA targets. If DNA binding is abolished by mutating the GATA zinc finger, Gln3 remains sequestered in the nucleus even when cells are provided with a non‐glutamine, highly repressive nitrogen source such as asparagine. The advantage of glutamine specific regulation of Gln3 export occurs during transitions from excess to adverse nitrogen conditions or vice‐versa.

Support or Funding Information

NIH Grant GM‐35642