Gln3 phosphorylation and intracellular localization in nutrient limitation and starvation differ from those generated by rapamycin inhibition of Tor1/2 in Saccharomyces cerevisiae.

Ajit Kulkarni,Jennifer J. Tate,Kathleen H. Cox,Terrance G. Cooper

doi:10.1074/jbc.m312023200

Ajit Kulkarni, Jennifer J. Tate + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m312023200

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Abstract

The ability of the cell to sense environmental conditions and alter gene expression in response to them is critical to its survival. In Saccharomyces cerevisiae, the Tor1/2 serine/threonine kinases are global regulators situated at the top of a signal cascade reported to receive and transmit nutritional signals associated with the nitrogen supply of the cell. At the other end of that cascade is Gln3, one of two transcriptional activators responsible for most nitrogen catabolic gene expression. When nitrogen is in excess, Tor1/2 are active, and Gln3 is phosphorylated and localizes to the cytoplasm. If Tor1/2 are inhibited by rapamycin or mutation, Gln3 becomes dephosphorylated, accumulates in the nucleus, and mediates nitrogen catabolite repression (NCR)-sensitive transcription. The observations that Gln3 also accumulates in the nuclei of cells provided with poor nitrogen sources or during nitrogen starvation has led to the conclusion that Tor1/2 control intracellular Gln3 localization and NCR-sensitive transcription by regulating Gln3 phosphorylation/dephosphorylation. To test this model, we compared Gln3 phosphorylation states and intracellular localizations under a variety of physiological conditions known to elicit different levels of NCR-sensitive transcription. Our data indicate that: (i) observable Gln3 phosphorylation levels do not correlate in a consistent way with the quality or quantity of the nitrogen source provided, the intracellular localization of Gln3, or the capacity to support NCR-sensitive transcription. (ii) Gln3-Myc(13) is hyperphosphorylated during nitrogen and carbon starvation, but this uniform response does not correlate with Gln3 intracellular localization. (iii) Gln3-Myc(13) dephosphorylation and nuclear localization correlate with one another at early but not late times after rapamycin treatment. These data suggest that rapamycin treatment and growth with poor nitrogen sources bring about nuclear accumulation of Gln3 but likely do so by different mechanisms or by a common mechanism involving molecules other than Gln3 and/or other than the levels of Gln3-Myc(13) phosphorylation thus far detected by others and ourselves.

Highlights

Our data indicate that: (i) observable Gln3 phosphorylation levels do not correlate in a consistent way with the quality or quantity of the nitrogen source provided, the intracellular localization of Gln3, or the capacity to support nitrogen catabolite repression (NCR)-sensitive transcription. (ii) Gln3-Myc13 is hyperphosphorylated during nitrogen and carbon starvation, but this uniform response does not correlate with Gln3 intracellular localization. (iii) Gln3-Myc13 dephosphorylation and nuclear localization correlate with one another at early but not late times after rapamycin treatment
Gln3-Myc13 Phosphorylation Profiles Generated in Response to Nitrogen Limitation Differ from Those Generated by Rapamycin Treatment—A critical correlation required by the current model describing Tor1/2 regulation of NCR-sensitive transcription is that Gln3 be dephosphorylated in cells provided with a poor nitrogen source and phosphorylated when cells are growing in rich medium
Because these results are identical to those observed by multiple laboratories after rapamycin treatment of cells provided with rich nitrogen sources, we used these conditions in all subsequent experiments to establish positive and negative electrophoretic mobility standards

Summary

Introduction

To cope with these demands, cells have evolved sophisticated signal transduction pathways that sense important components of their environment, such as nitrogen and carbon supplies. These transduction pathways culminate in the activation of transcription factors that adjust gene expression to maximize the potential for survival, growth, and reproduction. By rapamycin prevents Tor1/2-mediated phosphorylation reactions and thereby generates the opposite outcomes, i.e. Gln dephosphorylation, dissociation of the Gln31⁄7Ure complex, nuclear localization of Gln, and derepressed NCR-sensitive gene expression. (i) Inactivation of Tor1/2 by rapamycin treatment results in derepressed expression of many NCR-sensitive genes, nuclear localization of Gln, dissociation of Gln from its negative regulator Ure, and dephosphorylation of Gln (10 –14, 19). The above model is based on several correlations. (i) Inactivation of Tor1/2 by rapamycin treatment results in derepressed expression of many NCR-sensitive genes, nuclear localization of Gln, dissociation of Gln from its negative regulator Ure, and dephosphorylation of Gln (10 –14, 19). (ii) Many of the wide ranging cellular activities altered markedly by inactivation of Tor1/2 are impacted by nutrient starvation [1,2,3,4,5,6,7,8,9]

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