Rapamycin-induced Gln3 dephosphorylation is insufficient for nuclear localization: Sit4 and PP2A phosphatases are regulated and function differently
ABSTRACT Gln3, the major activator of nitrogen catabolite repression (NCR)-sensitive transcription, is often used as an assay of Tor pathway regulation in Saccharomyces cerevisiae. Gln3 is cytoplasmic in cells cultured with repressive nitrogen sources (Gln) and nuclear with derepressive ones (Pro) or after treating Gln-grown cells with the Tor inhibitor, rapamycin (Rap). In Raptreated or Pro-grown cells, Sit4 is posited to dephosphorylate Gln3, which then dissociates from a Gln3-Ure2 complex and enters the nucleus. However, in contrast with this view, Sit4-dependent Gln3 dephosphorylation is greater in Gln than Pro. Investigating this paradox, we show that PP2A (another Tor pathway phosphatase)-dependent Gln3 dephosphorylation is regulated oppositely to that of Sit4, being greatest in Pro- and least in Gln-grown cells. It thus parallels nuclear Gln3 localization and NCR-sensitive transcription. However, because PP2A is not required for nuclear Gln3 localization in Pro, PP2A-dependent Gln3 dephosphorylation and nuclear localization are likely parallel responses to derepressive nitrogen sources. In contrast, Rap-induced nuclear Gln3 localization absolutely requires all four PP2A components (Pph21/22, Tpd3, Cdc55, and Rts1). In pph21Delta22Delta, tpd3Delta, or cdc55Delta cells, however, Gln3 is dephosphorylated to the same level as in Rap-treated wild-type cells, indicating Rap-induced Gln3 dephosphorylation is insufficient to achieve nuclear localization. Finally, PP2A-dependent Gln3 dephosphorylation parallels conditions where Gln3 is mostly nuclear, while Sit4-dependent and Rap-induced dephosphorylation parallels those where Gln3 is mostly cytoplasmic, suggesting the effects of these phosphatases on Gln3 may occur in different cellular compartments.
- SourceAvailable from: Evelyne Dubois
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- "Cells were classified into one of three categories: cytoplasmic (cytoplasmic Gln3-Myc 13 fluorescence only; red bars in the histograms ), nuclear-cytoplasmic (Gln3-Myc 13 fluorescence appearing in the cytoplasm as well as co-localizing with DAPI-positive material; yellow bars), and nuclear (Gln3-Myc 13 fluorescence co-localizing only with DAPI-positive material; green bars). Representative " standard " images demonstrating the differences in these categories are shown in Figure 2 of Tate et al. 2009, along with a description of how the scoring criteria were applied. Time-course experiments of the kind presented here do not easily lend themselves to statistical analysis because even small experiment-to-experiment shifts in the overall shape of the response curves destroy the apparent precision of the individual measurements. "
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 following 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 previously envisioned. To test this idea directly, we determined whether or not Gtr1/2- and Ego1/3-dependent TorC1 activation was also 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 re-feeding glutamine to nitrogen-limited or -starved cells; GATA factor-dependent transcription was also 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 and III: Gtr and Ego complex proteins participate in nuclear Gln3-Myc(13) localization, heretofore unrecognized functions for these proteins, 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. Copyright © 2015 Author et al.G3-Genes Genomes Genetics 05/2015; 5(8). DOI:10.1534/g3.115.019307 · 3.20 Impact Factor
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- "Results were confirmed using independent cultures. Detailed examples of the three scoring categories for Gln3-Myc13 and Gat1-Myc13 as well as scoring precision (within and between experiments) appear in references (Tate et al. 2006, 2009, 2010; Tate and Cooper 2007, 2008; Georis et al. 2008). "
ABSTRACT: Nitrogen catabolite repression (NCR) is the regulatory pathway through which Saccharomyces cerevisiae responds to the available nitrogen status and selectively utilizes rich nitrogen sources in preference to poor ones. Expression of NCR-sensitive genes is mediated by two transcription activators, Gln3 and Gat1, in response to provision of a poorly used nitrogen source or following treatment with the TORC1 inhibitor, rapamycin. During nitrogen excess, the transcription activators are sequestered in the cytoplasm in a Ure2-dependent fashion. Here, we show that Vps components are required for Gln3 localization and function in response to rapamycin treatment when cells are grown in defined yeast nitrogen base but not in complex yeast peptone dextrose medium. On the other hand, Gat1 function was altered in vps mutants in all conditions tested. A significant fraction of Gat1, like Gln3, is associated with light intracellular membranes. Further, our results are consistent with the possibility that Ure2 might function downstream of the Vps components during the control of GATA factor-mediated gene expression. These observations demonstrate distinct media-dependent requirements of vesicular trafficking components for wild-type responses of GATA factor localization and function. As a result, the current model describing participation of Vps system components in events associated with translocation of Gln3 into the nucleus following rapamycin treatment or growth in nitrogen-poor medium requires modification.MicrobiologyOpen 06/2014; 3(3). DOI:10.1002/mbo3.168 · 2.21 Impact Factor
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- "For instance, induction by rapamycin of the RTG responsive gene, CIT2, is nitrogen-source dependent, occurring in ammonia or glutamine but not proline or glutamate grown cells (Tate and Cooper 2003). In addition, the pattern of Gln3 phosphorylation differs in rapamycin-treated vs. nitrogen starved or methionine sulfoximine treated cells, indicating that nitrogen deprivation and rapamycin impinge on Gln3 phosphorylation status in different ways (Tate et al. 2009). Finally, Gln3 and Gat1 both regulate NCR genes but Gln3 nuclear localization occurs in response predominantly to nitrogen limitation or methionine sulfoximine treatment rather than rapamycin treatment, whereas Gat1 nuclear localization occurs in response predominantly to rapamycin treatment and is immune to nitrogen starvation or methionine sulfoximine treatment (Tate et al. 2010). "
ABSTRACT: Availability of key nutrients, such as sugars, amino acids, and nitrogen compounds, dictates the developmental programs and the growth rates of yeast cells. A number of overlapping signaling networks-those centered on Ras/protein kinase A, AMP-activated kinase, and target of rapamycin complex I, for instance-inform cells on nutrient availability and influence the cells' transcriptional, translational, posttranslational, and metabolic profiles as well as their developmental decisions. Here I review our current understanding of the structures of the networks responsible for assessing the quantity and quality of carbon and nitrogen sources. I review how these signaling pathways impinge on transcriptional, metabolic, and developmental programs to optimize survival of cells under different environmental conditions. I highlight the profound knowledge we have gained on the structure of these signaling networks but also emphasize the limits of our current understanding of the dynamics of these signaling networks. Moreover, the conservation of these pathways has allowed us to extrapolate our finding with yeast to address issues of lifespan, cancer metabolism, and growth control in more complex organisms.Genetics 09/2012; 192(1):73-105. DOI:10.1534/genetics.111.135731 · 5.96 Impact Factor