GATA Factor Regulation in Excess Nitrogen Occurs Independently of Gtr-Ego Complex-Dependent TorC1 Activation

Abstract

The TorC1 protein kinase complex is a central component in a eukaryotic cell’s response to varying nitrogen availability, with kinase activity being stimulated in nitrogen excess by increased intracellular leucine. This leucine-dependent TorC1 activation requires functional Gtr1/2 and Ego1/3 complexes. Rapamycin inhibition of TorC1 elicits nuclear localization of Gln3, a GATA-family transcription activator responsible for the expression of genes encoding proteins required to transport and degrade poor nitrogen sources, e.g., proline. In nitrogen-replete conditions, Gln3 is cytoplasmic and Gln3-mediated transcription minimal, whereas in nitrogen limiting or starvation conditions, or after rapamycin treatment, Gln3 is nuclear and transcription greatly increased. Increasing evidence supports the idea that TorC1 activation may not be as central to nitrogen-responsive intracellular Gln3 localization as envisioned previously. To test this idea directly, we determined whether Gtr1/2- and Ego1/3-dependent TorC1 activation also was required for cytoplasmic Gln3 sequestration and repressed GATA factor-mediated transcription by abolishing the Gtr-Ego complex proteins. We show that Gln3 is sequestered in the cytoplasm of gtr1Δ, gtr2Δ, ego1Δ, and ego3Δ strains either long term in logarithmically glutamine-grown cells or short term after refeeding glutamine to nitrogen-limited or -starved cells; GATA factor−dependent transcription also was minimal. However, in all but a gtr1Δ, nuclear Gln3 localization in response to nitrogen limitation or starvation was adversely affected. Our data demonstrate: (i) Gtr-Ego-dependent TorC1 activation is not required for cytoplasmic Gln3 sequestration in nitrogen-rich conditions; (ii) a novel Gtr-Ego-TorC1 activation-independent mechanism sequesters Gln3 in the cytoplasm; (iii) Gtr and Ego complex proteins participate in nuclear Gln3-Myc13 localization, heretofore unrecognized functions for these proteins; and (iv) the importance of searching for new mechanisms associated with TorC1 activation and/or the regulation of Gln3 localization/function in response to changes in the cells’ nitrogen environment.

Nuclear Gln3 Import Is Regulated by Nitrogen Catabolite Repression Whereas Export Is Specifically Regulated by Glutamine

“…First, gln3 substitution mutants lacking NES function accumulate Gln3 in the nucleus and hence should exhibit rapamycin hypersensitivity. This phenotype should a priori be similar to the one reported for a ure2D mutation, which also results in constitutively nuclear Gln3 and Gat1 localization (Bertram et al 2000;Carvalho and Zheng 2003;Feller et al 2013, Tate et al 2015. Second, in contrast, gln3 substitution mutants with a damaged DNA binding domain should be unable to effectively activate NCR-sensitive transcription and hence should exhibit rapamycin resistance, a phenotype similar to that of a gln3D mutant (Beck and Hall 1999;Bertram et al 2000;Carvalho and Zheng 2003).…”

“…Sch9 is a protein kinase that regulates protein synthesis and other cellular processes (Powers 2007;Urban et al 2007). Further, cytoplasmic sequestration of Gln3 in a nitrogen-rich repressive medium, a condition that activates TorC1 kinase, does not require any of the components of the Gtr-Ego complexes required to activate TorC1 (Tate et al 2015).…”

More than One Way in: Three Gln3 Sequences Required To Relieve Negative Ure2 Regulation and Support Nuclear Gln3 Import in Saccharomyces cerevisiae

“…Cell collection and Gln3-Myc 13 visualization by indirect immunofluorescence microscopy were performed as described (Feller et al 2013). Intracellular EGFP-Gln3 peptide localization was determined and scored as described in Tate et al (2010Tate et al ( , 2015. As described earlier, only two-category, rather than three-category, scoring is possible when EGFP is used as the fluorescent tag.…”

“…In addition to the TorC1 and Gcn2 kinases, two phosphatases play equally important roles in NCR (DiComo and Arndt 1996;Beck and Hall 1999;Jiang and Broach 1999;Bertram et al 2000;Wang et al 2003;Cox et al 2004;Tate et al 2006Tate et al , 2009Tate et al , 2010Tate et al , 2015Yan et al 2006;Georis et al 2011b;Tate and Cooper 2013). A Gln3 response to rapamycin requires both PP2A and Sit4 phosphatases, whereas short-term nitrogen starvation, or growth under nitrogen-limiting conditions requires only Sit4 .…”

General Amino Acid Control and 14-3-3 Proteins Bmh1/2 Are Required for Nitrogen Catabolite Repression-Sensitive Regulation of Gln3 and Gat1 Localization

“…On the other hand, long-term nitrogen starvation (correlating with G1 arrest) or nuclear Gln3 localization in response to the glutamine synthetase inhibitor, methionine sulfoximine (Msx), required neither phosphatase. Most telling, Gln3 intracellular localization was immune to leucine starvation, leucyl tRNA synthetase inhibitors, or abolishment of the Gtr-Ego complex components needed to activate mTorC1 Tate et al 2015a).…”