Phosphorylated Residues of the Gln3 Ure2 Relief Sequence Abolish Nuclear Gln3 Localization and Tor1‐Gln3 Interaction

The GATA‐family proteins Gln3 and Gat1 locate to the nuclei of S. cerevisiae cells in response to adverse nitrogen conditions. There they activate the transcription of Nitrogen Catabolite Repression (NCR) sensitive genes. These genes encode proteins required for the uptake and catabolism of poorly utilized nitrogen sources (e.g., proline, allantoin, urea), interconversion of nitrogenous biosynthetic precursors and ATG14 , a subunit of the PI3‐Kinase Complex I required for phagophor assembly during autophagy. In contrast, Gln3 is sequestered in the cytoplasm of cells growing in nitrogen replete conditions (glutamine). This NCR‐ sensitive regulation of Gln3 localization is achieved through the actions of the multiple regulatory kinases, TorC1, Gcn2 and Nnk1, protein phosphatases PP2A and Sit4, the 14‐3‐3 protein Bmh1/2 proteins and a cytoplasmic prion precursor, Ure2. Tor1 interacts with C‐terminal Gln3 residues 656‐666 in a 2‐hybrid assay. Ure2, complexing with Gln3 residues 101‐150, is required for its cytoplasmic sequestration. While mapping these and other targets of these regulators on the Gln3 molecule itself, using indirect immune‐fluorescence analyses, we identified a Gln3 region that we designate as the Ure2‐Relief Sequence (URS). The URS, consisting of a predicted α‐helix situated in a disordered coiled coil region of Gln3, Gln3 247‐282 , is absolutely required for nuclear Gln3 localization when Ure2 is present but not in its absence. Our current experiments, including mass spectral analyses, demonstrate that 11 of the 17 URS serine/threonine residues are phosphorylated in nitrogen‐rich SD medium. Treating cells with rapamycin, an inhibitor of the TorC1 kinase, reduces the phosphorylation of 8 residues by more than 3 to 6‐fold and 3 of them by 10 to 15‐fold. Phosphomimetic substitutions of URS serine/threonine residues abolish nuclear Gln3 localization suggesting that phosphorylation/dephosphorylation of these residues is physiologically significant. In addition to the URS requirement for nuclear localization, these phosphomimetic substitutions also abolish Tor1‐Gln3 interaction at the URS in a 2‐hybrid assay. This N‐terminal Tor1‐Gln3 interaction functions both independently of and collaboratively with the C‐terminal Tor1‐Gln3 interaction site. Together, these data suggest that Tor1‐Gln3 interactions participate both positively and negatively in the regulation of intracellular Gln3 localization and that the action of the URS is regulated by its phosphorylation/dephosphorylation.