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.
Highlights
The Target of Rapamycin Complex 1 (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
Do the Gtr1/Gtr2 and Ego1/Ego3 complexes, required for TorC1 activation and downstream Sch9 phosphorylation in nitrogen-replete medium, participate in cytoplasmic Gln3 sequestration during nitrogen excess and/or nuclear Gln3 localization and subsequently Gln3-dependent gene expression during nitrogen limitation/starvation? To frame these questions in a context that permitted overall comparisons of present Gln3 localization data with that reported for growth recovery and Sch9 phosphorylation (Binda et al 2009), we used an experimental format similar to the one originally employed to establish that Gtr1, Gtr2, Ego1, and Ego3 were required for recovery from rapamycin-induced growth arrest and TorC1 activation
GATA factor-mediated, NCRsensitive DAL5 and GDH2 transcription closely correlate with Gln3 localization, further supporting the conclusion
Summary
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. Increasing evidence supports the idea that TorC1 activation may not be as central to nitrogen-responsive intracellular Gln localization as envisioned previously To test this idea directly, we determined whether Gtr1/2- and Ego1/3-dependent TorC1 activation was required for cytoplasmic Gln sequestration and repressed GATA factor-mediated transcription by abolishing the Gtr-Ego complex proteins. When the supply of readily used nitrogen sources (e.g., yeast extract, peptone, dextrose; glutamine; or ammonia) become exhausted or unavailable, Gln relocates to the nucleus, where Gln3-activated transcription increases dramatically so that cells are able to scavenge a broader range of nitrogenous compounds from their environments This nitrogenresponsive regulation has long been referred to as nitrogen catabolite repression (NCR) (Cooper 1982)
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