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

Abstract

Gln3, a transcription activator mediating nitrogen-responsive gene expression in Saccharomyces cerevisiae, is sequestered in the cytoplasm, thereby minimizing nitrogen catabolite repression (NCR)-sensitive transcription when cells are grown in nitrogen-rich environments. In the face of adverse nitrogen supplies, Gln3 relocates to the nucleus and activates transcription of the NCR-sensitive regulon whose products transport and degrade a variety of poorly used nitrogen sources, thus expanding the cell's nitrogen-acquisition capability. Rapamycin also elicits nuclear Gln3 localization, implicating Target-of-rapamycin Complex 1 (TorC1) in nitrogen-responsive Gln3 regulation. However, we long ago established that TorC1 was not the sole regulatory system through which nitrogen-responsive regulation is achieved. Here we demonstrate two different ways in which intracellular Gln3 localization is regulated. Nuclear Gln3 entry is regulated by the cell's overall nitrogen supply, i.e., by NCR, as long accepted. However, once within the nucleus, Gln3 can follow one of two courses depending on the glutamine levels themselves or a metabolite directly related to glutamine. When glutamine levels are high, e.g., glutamine or ammonia as the sole nitrogen source or addition of glutamine analogues, Gln3 can exit from the nucleus without binding to DNA. In contrast, when glutamine levels are lowered, e.g., adding additional nitrogen sources to glutamine-grown cells or providing repressive nonglutamine nitrogen sources, Gln3 export does not occur in the absence of DNA binding. We also demonstrate that Gln3 residues 64-73 are required for nuclear Gln3 export.

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

“…Therefore, these strains possessed two copies of the GLN3 gene which accounted for the higher expression level in glutamine-grown cells. Note that wild-type GDH2 expression in Figure 6B was the same as that observed in Rai et al (2015).…”

“…Importantly, the gcn2D phenotype exhibited high specificity. Gln3-Myc 13 responses to rapamycin or Msx treatment, which have been shown to occur via mTorC1 and glutaminemediated intranuclear regulation, respectively, were unaffected; they remained the same as wild type (Beck and Hall 1999;Cardenas et al 1999;Hardwick et al 1999; Rai et al 2015). The responses to abolishing Gcn2 were restricted to nitrogen sources whose uptake and/or metabolism depended on NCR-sensitive gene expression.…”

“…The data obtained demonstrated that once Gln3 enters the nucleus, its intranuclear path is regulated by glutamine levels themselves or by those of a glutamine metabolite able to be mimicked by glutamine analogs (Rai et al 2015). In high glutamine, Gln3 can exit from the nucleus in the absence of binding to its promoter GATA targets.…”

“…Extracts for Western blots were prepared and Western analyses were performed as previously described (Cox et al 2004a;Rai et al 2015).…”

“…Quantitative RT-PCR (qRT-PCR) analyses were performed as described in Rai et al (2015). GDH2 and TBP1 primer sequences were as previously described Georis et al (2008Georis et al ( , 2011.…”