Stress-Responsive Gln3 Localization In S. cerevisiae Is Separable From and Can Overwhelm Nitrogen Source Regulation

Abstract

Intracellular localization of S. cerevisiae GATA-family transcription activator, Gln3, is used as a downstream readout of rapamycin-inhibited Tor1,2 control of Tap42 and Sit4 activities. Gln3 is cytoplasmic in cells provided with repressive nitrogen sources such as glutamine and nuclear in cells growing with a derepressive nitrogen source such as proline or those treated with rapamycin or methionine sulfoximine (Msx). Although gross Gln3-Myc¹³ phosphorylation levels in wild type cells do not correlate with nitrogen source-determined intracellular Gln3-Myc¹³ localization, the phosphorylation levels are markedly influenced by several environmental perturbations. Msx-treatment increases Snf1-independent Gln3-Myc¹³ phosphorylation, while carbon-starvation increases both Snf1-dependent and -independent Gln3-Myc¹³ phosphorylation. Here, we demonstrate that a broad spectrum of environmental stresses (temperature, osmotic, oxidative) increase Gln3-Myc¹³ phosphorylation. In parallel, these stresses elicit rapid (< 5 mins. for NaCl) Gln3-Myc¹³ relocalization from the nucleus to the cytoplasm. The response of Gln3-Myc¹³ localization to stressful conditions can completely overwhelm its response to nitrogen source quality or inhibitor-generated disruption of the Tor1,2 signal transduction pathway. Adding NaCl to cells cultured under conditions in which Gln3-Myc¹³ is normally nuclear, i.e., proline-grown, nitrogen-starved, Msx-, caffeine- and rapamycin-treated wild type, or ure2Δ cells, results in its prompt relocalization to the cytoplasm. Together these data identify a major new level of regulation to which Gln3 responds, and adds a new dimension to mechanistic studies of this transcription factor’s regulation.

Transcription, mediated by the GATA-family transcription activators Gln3 and Gat1/Nil1, has become an important terminal read-out in mechanistic studies of the clinically important, rapamycin-inhibitable Tor1,2 signal transduction pathway (1–4). From a physiological point of view, the regulation of Gln3 and Gat1 intracellular localization and nuclear function is responsible for selective utilization of nitrogen sources available to S. cerevisiae; this regulatory process has been designated Nitrogen Catabolite Repression or NCR (5–9). In NCR, when readily used (i.e., repressive) nitrogen sources such as glutamine or, in some strains, ammonia are available in sufficient supply, yeast cells will not utilize poorer (i.e., derepressive) nitrogen sources in their environment. This selectivity is accomplished by repressing expression of genes encoding the permeases and catabolic enzymes required for utilization of these poorer nitrogen sources. On the other hand, when the environmental nitrogen supply becomes limiting, Gln3/Gat1-dependent transcription is derepressed. As a result, production and activation of permeases and catabolic enzymes required for utilization of poor (i.e., derepressive) nitrogen sources increases. Thus the cells are equipped to scavenge otherwise unused nitrogen sources until a better nutritional environment returns.

PI3-related serine/threonine protein kinases, Tor1,2 are participants in NCR-sensitive transcriptional control (1–4, 10). Tor1,2 are the S. cerevisiae counterparts of mammalian mTor, with the human protein being a target of therapies treating multiple types of cancer and tissue rejection in transplant patients (10–17). According to the original S. cerevisiae model (Fig. 1, left panel), Tor1,2 received nutrient-responsive signals, which resulted in their activation (1–4). One such signal may be glutamine or a related metabolite, a conclusion derived from the observation that treating cells with the glutamine synthetase inhibitor, methionine sulfoximine (Msx), correlates with activation of the Tor1,2 pathway and its influence on downstream readouts (18, 19). Activated Tor1,2 phosphorylates and positively regulates the essential protein Tap42 (20–22). Tap42, in turn, interacts with and negatively regulates type-2A-related and type-2A serine/threonine protein phosphatases, Sit4, Pph3, and Pph21,22 (1, 3, 4, 20–26). For the sake of simplicity, we have not discussed Tip41 phosphorylation and its protein-protein interactions (22). Thus inhibited, these phosphatases were posited to be unable to dephosphorylate Gln3. Under such conditions of excess nitrogen, Gln3 was found in a complex with Ure2 (1, 4, 27), a multifunctional protein which is additionally a prion precursor (28, 29) and required participant in heavy metal ion and hydroperoxide detoxification (30, 31). The presence of the Gln3-Ure2 complex correlates with exclusion of Gln3 from the nucleus and repression of NCR-sensitive transcription (1,4). Existing models alternatively suggested that Gln3 phosphorylation was a requirement for Gln3-Ure2 complex formation (1) or that the complex stabilized the phosphorylated form of Gln3 (4). The differing views derived, in part, from the observation that both phosphorylated and dephosphorylated forms of Gln3 were found in Gln3-Ure2 complexes (4). In any case, complexed and/or phosphorylated Gln3 correlated with its exclusion from the nucleus.

Figure 1.

Original model (left panel) proposed for Tor1,2 regulation of Gln3 phosphorylation and localization (1, 4) as well as influence of methionine sulfoximine (Msx) on Tor pathway regulation (18). A more recent version of the Tor regulatory pathway incorporating data from Jacinto et al. (22), Loewith et al. (51) Re-inke et al. (52), Rohde et al. (25) Wang et al. (24), and Yan et al. (26). For the sake of simplicity neither model is exhaustive in detailing all of the known components and/or protein-protein interactions and protein modifications.

When cells are treated with the Tor1,2 inhibitor, rapamycin, events opposite to those described above were envisioned. Tor1,2 no longer positively regulated Tap42, which in turn, no longer bound to and inhibited Sit4 phosphatase. Thus freed from negative regulation by Tap42, Sit4 phosphatase dephosphorylated Gln3 and it was this dephosphorylated form which correlated with nuclear localization following rapamycin-treatment (1, 4). However, it was subsequently found that this correlation was observed only at early times (20–30 min.) following rapamycin-treatment; it was not observed 60 mins. after treatment (32).

Although the positive correlation of short-term rapamycin-treatment, Gln3 dephosphorylation and nuclear localization has been repeatedly confirmed, our understanding of the mechanistic steps from which these correlations derive are still evolving significantly (Fig. 1, right panel). An early study, which established a physiologically significant association between Tap42 and Sit4, concluded either that Tap42 positively regulated type-2A and type-2A-related phosphatase activities (i.e., the Tap42-phosphatase complex was active for a specific phosphatase function) rather than acting as a negative regulator, or alternatively that a Tap42 function was regulated by the phosphatase (20). More recently, the idea of positive Sit4 regulation by Tap42 has re-emerged in the demonstration that association of Tap42 with Sit4 is required for the latter to be active (Fig. 1, right panel) (24). Further, rapamycin-treatment was demonstrated to release the Tap42-Sit4 complex from its association with Tor complex 1 (TORC1), also an event required for the Tap42-Sit4 complex to be active (Fig. 1, right panel) (26).

Similarly significant changes have occurred in our understanding of the relationship between Gln3-Myc¹³ phosphorylation and intracellular localization. Although rapamycin-treatment and growth of cells in poor nitrogen sources bring about the same outcome, i.e., nuclear Gln3-Myc¹³ localization, the gross Gln3-Myc¹³ phosphorylation profiles do not similarly correlate (19, 32). Further, Sit4 has recently been shown to be active irrespective of the nitrogen source provided to the cells, i.e., with both good and poor nitrogen sources (33). Finally, when SIT4 is deleted, Gln3-Myc¹³ phosphorylation responds to the nature of the nitrogen source provided, i.e., an influence of the nitrogen source on Gln3-Myc¹³ phosphorylation is unmasked (33). These observations led Tate et al. to suggest the nitrogen source is probably more directly connected to the regulation of one or more protein kinase activities that phosphorylate Gln3-Myc¹³ than it is to Sit4 phosphatase that continuously dephosphorylates it irrespective of the nitrogen source (33).

Finally, protein kinase Npr1 was reported to be a negative regulator of Gln3, because deletion of NPR1 resulted in constitutive nuclear localization of Gln3 and NCR-sensitive transcription in ammonia-grown cells (34). Most recently, it has been independently shown by two groups that the npr1Δ phenotype derives from loss of the normal ammonia permease activities in the mutant rather than direct negative regulation of Gln3 (35, 36).

The central role of Tap42 in the control of Sit4 and its subsequent regulation of downstream Tor1,2 targets, prompted us to investigate the effects generated by loss of Tap42 on intracellular Gln3-myc¹³ localization and phosphorylation. The mutation we elected to analyze was the temperature sensitive tap42-11 allele because it was one of the mutations initially used to demonstrate many of the Tap42 functions in general (20) and Tap42 involvement in Gln3 regulation in particular (1). Although the tap42-11 mutant is rapamycin resistant at its permissive temperature of 24° C, the growth inhibitory and other phenotypes it possesses require a non-permissive temperature of 37° C (20). Therefore, before proceeding further, we performed several control experiments, including one that assessed whether the non-permissive temperature for tap42-11 (37° C) would affect Gln3 regulation.

These experiments led us to the surprising discovery of a new form of Gln3 regulation: (i) Gln3-Myc¹³ relocates from the nucleus to the cytoplasm when proline-grown, wild type cells are shifted from 24° C to 37° C and (ii) Gln3-Myc¹³ phosphorylation levels increase in parallel with its relocalization. We further showed that Gln3-Myc¹³ phosphorylation and localization responded similarly to multiple stress conditions, i.e., temperature, osmotic and oxidative stress. Such a broad range of responses is characteristic of a generalized stress response, a form of regulation with which Gln3 has not previously been associated. Equally surprising, the response of Gln3-Myc¹³ to environmental stress can overwhelm previously studied forms of Gln3 regulation, rapamycin- caffeine-, or Msx-treatment, nitrogen-starvation, limiting nitrogen supply with poor nitrogen sources such as proline, and deletion of URE2. Experiments described below broaden our view of Gln3 regulation from a uni-dimensional response to nitrogen supply to a multi-variant response accounting for other environmental conditions representing potentially greater and more immediate threats to a cell’s well-being.

METHODS

Strains And Culture Conditions

Saccharomyces cerevisiae strains used in this work were TB123 (MATa, leu2-3,112, ura3-52, rme1, trp1, his4, GAL⁺, HMLa, GLN3-Myc¹³[KanMX]), TB136-2a, (MATa, leu2-3,112, ura3-52, rme1, trp1, his4, GAL⁺, HMLa, GLN3-Myc¹³[KanMX], sit4::kanMX), TB138-1a (Mata, leu2-3,112, ura3-52, rme1, trp1, his4, GAL⁺, HMLa, GLN3-Myc¹³[KanMX], ure2:URA3), and TB123-hog1 (MATa, leu2-3,112, ura3-52, rme1, trp1, his4, GAL⁺, HMLa, GLN3-Myc¹³[KanMX], hog1::natMX).

Strains were grown at 30°C (unless otherwise indicated in the figure legend) to mid-log phase (A_{600 nm} = 0.5) in Difco yeast nitrogen base (YNB) (without amino acids and ammonium sulfate) medium, containing 2% glucose, required auxotrophic supplements (120 μg/ml leucine, 20 μg/ml uracil, 20 μg/ml histidine, 20 μg/ml tryptophan, 20 μg/ml arginine), and the nitrogen source indicated (0.1% final concentration unless otherwise indicated). Rapamycin (Sigma-Aldrich) (dissolved in 10% Tween 20 + 90% ethanol) was added to the cultures, where indicated, to a final concentration of 0.2 μg/ml. Caffeine (20 mM final concentration, added as a solid) or methionine sulfoximine (Msx, 2 mM final concentration, dissolved in water) were added where indicated. The duration of treatment which occurred prior to cell harvest is indicated in the pertinent figure legends.

Cell Collection and Protein Preparation For Western Blot Analysis

Experiments in which yeast were transferred from one medium to another were performed as follows. An exponentially growing culture (A_600nm = 0.4–0.5), or half of it in the case of a split culture, was quickly collected by filtration (at room temperature), and resuspended in an equal volume of the same, fresh, pre-warmed (to the specified temperature), pre-aerated medium; transfer was completed in 35–45 seconds. Following incubation for the indicated times (see figure legends), cells were collected using the filtration method of Tate et al. (19) and protein extracts prepared as described by Cox et al. (32). In the case of treated cultures (rapamycin, Msx, caffeine, sorbitol, sodium chloride, or hydrogen peroxide), a pre-treatment (zero time) sample was collected as described above. The remainder of the culture was then treated with the indicated perturbant at the concentration and for the times indicated in the figure legends. Samples of these treated cultures were collected and processed as described above.

Indirect Immunofluorescence Microscopy

Cell collection and fixation for indirect immunofluorescence was performed using the method of Cox et al. (37) as modified by Tate et al. (33, 35). Gln3-Myc¹³ localization was visualized using 9E10(c-myc) monoclonal antibody (Covance MMS-150P, at a dilution of 1:1000) as the primary antibody and Alexa Fluor 594 goat anti-mouse IgG antibody (Molecular Probes, at a dilution of 1:200) as secondary antibody. DNA was visualized using 4′6′-diamino-2-phenyl-indole (DAPI) contained in the mounting medium (Sigma) (37).

Cells were imaged using a Zeiss Axioplan 2 imaging microscope with a 100x Plan-Apochromat 1.40 oil objective at room temperature. Images were acquired using a Zeiss Axio camera and AxioVision 3.0 (Zeiss) software. Images were processed with Adobe Photo-shop and Illustrator programs. Gamma settings were altered where necessary to avoid any change in cellular detail during processing; changes were applied uniformly to the image.

Determination Of Intracellular Gln3-Myc¹³ Distribution

To provide a more representative and complete view of Gln3-Myc¹³ behavior than can be obtained from a single image of four to eight cells, we scored Gln3-Myc¹³ localization in 200 or more cells that appeared in multiple, randomly chosen microscopic fields of each experimental sample, including the ones from which the images we present were taken. These cells were placed in one of three categories: cytoplasmic (cytoplasmic fluorescence only), nuclear-cytoplasmic (fluorescence appeared in the cytoplasm as well as co-localizing with DAPI-positive material), and nuclear (co-localizing only with DAPI-positive material). The scoring method’s limitations and reproducibility are described in Tate et al. (33, 35). It is important to emphasize again here, as described in detail earlier, that the nuclear-cytoplasmic category is, of necessity, arbitrary. Placing cells in that category is based on subjective visual observation by the individual scoring the cells; it is not an objective instrument-based measurement. When Gln3-Myc¹³ is not restricted to a single cellular compartment, scoring depends upon repeated decisions that conclude whether Gln3-Myc¹³ in a given cell is localized in the center nuclear-cytoplasmic category or a category flanking it. While intracellular Gln3-Myc¹³ distribution was scored as carefully and consistently as possible, our interpretations and conclusions are based on straightforwardly detected changes in overall distribution patterns that are apparent in the microscopic images. Histograms of cell scoring results that accompany the images provide supplemental descriptions of our observations. They will undoubtedly differ in detail, however, from those of another observer, who sets their localization category dividing lines differently. Experiments similar to those described were repeated two or more times with similar results. Experiment to experiment variation can be ascertained by comparing similar experimental conditions within the present work, as well as between similar experimental conditions in present and previous work (33, 35). Finally, while we have used the appearance of Gln3-Myc¹³ fluorescence in both cellular compartments as a descriptor of our observations, we do not a priori assume this appearance represents a separate and unique physiological state.

In vitro Akaline phosphatase treatment of cell extracts

Alkaline phosphatase-treatment of crude cell extracts was performed as described earlier (19).

RESULTS

Intracellular Localization Of Gln3-Myc¹³ Responds To Increased Temperature

This investigation was originally initiated to study the effects of altering Tap42 activity on intracellular Gln3-myc¹³ localization and phosphorylation. The mutant we elected to analyze contained the temperature sensitive tap42-11 allele. Our study began with several control experiments, the most important of which tested the prerequisite condition that increased temperature, required to alter some Tap42 activities, did not otherwise affect Gln3-Myc¹³ behavior in wild type cells. As expected, Gln3-Myc¹³ was cytoplasmic in ammonia-grown cells whether cultured at 24°, 30° C or shifted from 24° to 37° C for 45 min. (Fig. 2A, Am.). To provide a more complete picture of what we observed microscopically, we categorized Gln3-Myc¹³ localization (see Methods) in multiple fields of the same slide from which the images in Fig. 2A were taken (Fig. 2B, left histogram). In proline-grown cells, Gln3-Myc¹³ was, as also expected, nuclear in most cells cultured at 24° C or 30° C (Figs. 2A, Pro and 2B, right histogram). Unexpectedly, however, Gln3-Myc¹³ was cytoplasmic in nearly all proline-grown cells transferred from 24° C to 37° C (Figs. 2A, Pro and 2B, right histogram).

Figure 2.

Temperature stress or transfer of cells from one medium to another elicits Gln3-Myc¹³ relocalization from the nucleus to cytoplasm of proline-grown cells. Panels A and B. TB123 cells were cultured at 24° C (24°) or 30° C (30°) in YNB-ammonia (⁺NH₄) or –proline (PRO) medium. Alternatively, they were cultured at 24° C and then transferred to fresh pre-aerated medium at 37° C for 45 mins. (37° C). Cells were prepared, microscopic images obtained, processed, and intracellular Gln3-Myc¹³ distribution scored as described in Materials and Methods; cytoplasmic (red bars), nuclear-cytoplasmic (nucl.-cyto., yellow bars), or nuclear (green bars). Panels C and D. TB123 was cultured to mid-log phase at 30° C in YNB-proline. At zero time a sample was collected for assay (0′ Control). The remaining culture was divided into two portions. One portion was harvested by filtration, transferred to an equal volume of pre-warmed, pre-aerated medium (Fresh Medium) identical to that in which they were cultured. The second portion was similarly harvested and then resuspended in the same medium from which they had just been harvested (Spent Medium). Samples were then taken for assay at the times (in minutes, mins.) indicated. Panels E and F. Three cultures of TB123 were grown in YNB-proline medium, two at 24° C and a third at 30° C. The temperature of one 24° C culture (50 ml in a 125 ml flask) was increased by transferring it to a 1 l flask pre-warmed to 37°C. After 10 mins. in that flask, the cells were returned to a 125 ml flask pre-warmed to 37° C and incubated for an additional 50 mins. (60 total mins. at 37° C) (designated 37°). The 30° C culture (30°) was used as an untreated control. As an additional control, the second 24° C culture was transferred to a 1 l flask equilibrated at 24° C. After 10 min. in the 1 l flask, the culture was returned to a 125 ml flask equilibrated at 24° C and incubated for 10 mins. After the incubations, samples of each culture were collected and processed for assay. In panels A, C and E, images of DAPI-positive material (blue) are presented below corresponding Gln3-Myc¹³ images (red).

Although Gln3-Myc¹³ localization appeared to respond markedly to increased temperature, it was possible the effect did not derive from increasing the temperature to 37° C, but from transferring the cells to fresh, pre-aerated, 37° C medium, i.e., it was the fresh medium rather than increased temperature that caused Gln3-Myc¹³ relocalization. Testing this possibility, we observed that transferring proline-grown cells cultured at 30° C to fresh 30° C proline medium resulted in transient relocation of Gln3-Myc¹³ from the nucleus to the cytoplasm, coinciding with an earlier observation (Fig. 2C, and 2D, left histogram) (38). Relocation derived from the fresh medium rather than the transfer process itself, because it did not occur when cells were harvested by filtration and returned to the same spent medium (Fig. 2C, spent medium and 2D, right histogram). Although we haven’t identified the source of this perturbation, it does not derive from changes in pH that occur during growth. The same results occurred whether or not the pH of the fresh medium was adjusted to that of the spent medium prior to cell transfer (data not shown). To eliminate the medium shift variable all together, we increased the temperature of a culture (50 ml) grown at 24° C to 37° C by transferring it to a 1 l flask that had been pre-equilibrated at 37°C. The large surface to volume ratio of the 1 l flask, acted as an effective heat sink, rapidly increasing the culture temperature without the need of fresh medium. After 10 min. incubation in the 1 l flask, the culture was returned to a 125 ml flask (at 37° C), similar to the one in which it had been initially grown (the same surface to volume ratio), for an additional 50 mins. This change in protocol did not significantly alter the results, as Gln3-Myc¹³ relocated to the cytoplasm of most cells when the temperature was increased (Figs. 2E and 2F). These data suggested that Gln3-Myc¹³ localization was sensitive to temperature shock.

Intracellular Gln3-Myc¹³ Localization Is Sensitive to Osmotic Stress

These data prompted us to query whether changes in Gln3-Myc¹³ distribution derived from a specific or more generalized stress response. Addition of either 1M NaCl or 1M sorbitol elicited relocalization of Gln3-Myc¹³ from the nuclei to cytoplasm of proline-grown cells (Figs. 3A, None, NaCl and Sorb. and 3B), indicating sensitivity to osmotic stress. The response, however, was not influenced by loss of the osmotic stress-responsive Hog1 protein kinase, i.e., similar results were observed in wild type and a hog1Δ (Figs. 3C and 3D).

Figure 3.

Multiple environmental stresses elicit re-location of Gln3-Myc¹³ from the nucleus to cytoplasm of proline-grown cells. Wild type (Panels A–D) or hog1Δ (Panels C and D) cells were cultured at 30° C in the absence (None) or presence of 0.3 mM hydrogen peroxide (H₂O₂) for 30 mins., 1M sodium chloride for 45 mins. (NaCl), 1M sorbitol for 15 mins. (Sorb.). Proline was used as the nitrogen source in Panels A and B, and ammonia or proline, as indicated, in Panels C and D.

Intracellular Gln3-Myc¹³ Localization Is Sensitive To Oxidative Stress

The third environmental condition we assayed was oxidative stress by adding a low concentration of hydrogen peroxide (0.3 mM) to a proline-grown culture. Gln3-Myc¹³ was localized to the nuclei of the majority of untreated cells (Figs. 3A and Fig. 3B, None). In contrast, Gln3-Myc¹³ was cytoplasmic or nuclear-cytoplasmic in the majority of cells following the addition of hydrogen peroxide (Figs. 3A, H₂O₂ and 3B, H₂O₂). The response to hydrogen peroxide addition was less drastic than observed with NaCl or sorbitol, i.e., more nuclear staining remained following treatment with hydrogen peroxide than with NaCl or sorbitol (Figs. 3A, H₂O₂, NaCl and Sorb. and 3B, H₂O₂, NaCl and Sorb.).

Together, the above data suggested intracellular Gln3-Myc¹³ localization responded to multiple environmental stresses. It is important to note that experiments measuring intracellular Gln3-Myc¹³ localization could not be performed with ammonia or glutamine as nitrogen sources, because Gln3-Myc¹³ is already localized to the cytoplasm of nearly all cells under these growth conditions (Figs. 2A, Am. and 2B, Figs. 6A and 6C, and Fig. 7A> in ref. 33). Finally, deletion of SIT4 in proline-grown cells had no demonstrable effect on stress-induced Gln3-Myc¹³ intracellular localization (data not shown); Gln3-Myc¹³ was cytoplasmic in unstressed, proline-grown sit4Δ in this strain background.

Gln3-Myc¹³ Phosphorylation Increases In Response To Temperature-Induced Stress

Dramatic intracellular relocation of Gln3-Myc¹³ observed in response to environmental stresses raised the possibility that its phosphorylation state might be affected in parallel. Therefore, proline-grown wild type cells were maintained at 24° C or transferred from 24° C to 37° C, using the 1 l heat-sink flask method described above. Crude cell extracts were then prepared and Gln3-Myc¹³ phosphorylation assayed by western blot analyses. Gln3-Myc¹³ mobility decreased in extracts derived from cells shifted to 37° C relative to those at 24° C (Fig. 4A, lanes A and C vs. B and D), behavior characteristic of increased phosphorylation (Fig. 4 of reference 32). To determine whether decreased Gln3-Myc¹³ mobility at the higher temperature was associated with proline as the nitrogen source, or was a general characteristic of temperature stress per se, we performed a further experiment using ammonia-grown cells. Gln3-Myc¹³ mobility detectably decreased within fifteen mins. of transferring ammonia- grown cells from 24° C to 37° C (Fig. 4B, lanes A and B), reaching minimal levels by 30 mins. post-transfer (Fig. 4B, lane C). The fastest migrating species in lane A had disappeared in lane C and a slower mobility species appeared (lower and upper black dots, respectively).

Figure 4.

Temperature stress increases Gln3-Myc¹³ phosphorylation. Panel A. Wild type (TB123) cells were cultured in YNB-proline medium at 24° C (24°) and then shifted to 37° C (37°) using the 1 l heat-sink flask method of temperature shift as described in Figs. 2E and 2F. After 60 mins. incubation at 37° C, cells were harvested for western blot analyses as described in Methods. Panel B. Cells were cultured in YNB-ammonia medium at 24° C, transferred to fresh pre-warmed, pre-aerated ammonia medium at 37° C and samples harvested and processed for western-blot assay at the indicated times (in mins.) thereafter. Panel C. Cells were cultured to mid- log phase in YNB-ammonia medium at 24° or 30° C (lanes A, B, E, and F). At that time they were harvested (lanes A and B) or treated for 30 mins. with Msx and then harvested (lanes E and F). The samples in lanes C and D. were cultured at 24° C to mid-log phase and transferred to pre-aerated, pre-warmed (37° C) medium for 60 mins. The sample in lane C was harvested at that time, while the one in lane D was treated with Msx (2 mM final concentration) for an additional 30 mins. before harvest. Panel D. Cells were cultured as in Panel A and extracts prepared as described in Materials and Methods. Extracts were treated with calf intestional alkaline phosphatase (CIP) with and without sodium pyrophosphate (Na₄P₂O₇).

Decreased Gln3-Myc¹³ mobility following temperature shift was similar to that which occurred after Msx-treatment, though the relative distribution among the various resolved species was not identical (Fig. 4C, compare lanes A, C and D). Following Msx-treatment at 37° C, i.e., the slowest mobility species of Gln3-Myc¹³ was predominant (Fig. 4C, lane D), whereas a more even distribution among the three principal Gln3-Myc¹³ species was observed in extracts of cells subjected to temperature shift in the absence of Msx (Fig. 4C, lane C).

If decreased Gln3-Myc¹³ mobility observed above derived from increased phosphorylation, it should be sensitive to in vitro treatment with alkaline phosphatase (1, 4, 19, 32). Therefore, extracts were prepared from cells incubated at 37° C for 60 mins. and treated with calf intestine alkaline phosphatase. As shown, in Fig. 4D, alkaline phosphatase-treatment markedly increased Gln3-Myc¹³ mobility (lanes B vs. C). Increased Gln3-Myc¹³ mobility occurred to a much lesser extent when sodium pyrophosphate, a phosphatase inhibitor, was also included in the reaction mixture (lane E). This indicated that transferring cells to higher temperature increased Gln3-Myc¹³ phosphorylation.

Gln3-Myc¹³ Phosphorylation Increases In Response To Osmotic Stress

Next, we evaluated the effects of osmotic stress on Gln3-Myc¹³ phosphorylation. Addition of 1M NaCl to proline, ammonia- or glutamine-grown cultures, strongly decreased Gln3-Myc¹³ mobility (Fig. 5A–C lanes A vs. B). With proline, a slower migrating Gln3-Myc¹³ species appeared in the extract from NaCl-treated cells (Fig. 5A, lanes A and B, black dot). With ammonia and glutamine, the fastest migrating Gln3-Myc¹³ species observed in the untreated extract disappeared altogether (Figs. 5A, and 5B, lane A, bottom black dot) and the relative amounts of the remaining species shifted towards the slowest migrating species (lanes A and B, top black dot). We tested whether the decreased Gln3-Myc¹³ mobilities noted above derived from increased phosphorylation by assaying their sensitivity to alkaline phosphatase. Alkaline phosphatase sensitivity similar to that observed with increased temperature occurred here as well (Figs. 5D and 5E). Therefore, osmotic stress increased Gln3-Myc¹³ phosphorylation. Also, as reported earlier, sodium pyrophosphate addition sometimes inexplicably results in a lower protein signal (19, 32).

Figure 5.

Osmotic stress increases Gln3-Myc¹³ phosphorylation. Wild type (TB123) cells were cultured in YNB medium containing proline (PRO), ammonia (⁺NH₄), or glutamine (GLN) as the nitrogen source. 1M NaCl or sorbitol was added and the cultures incubated thereafter for the time indicated (in mins.). Where times are not indicated, the cultures were incubated with NaCl or sorbitol for 45 mins. Where indicated, the cultures were incubated with Msx for 30 mins. Cultures were then harvested and extracts prepared for western-blot analyses. Alkaline phosphatase influence on Gln3-Myc¹³ mobility (Panels D and E) was assayed as described in Fig. 4D and Materials and Methods.

As occurred with increased temperature, the most highly phosphorylated Gln3-Myc¹³ species in extracts from NaCl-treated ammonia- or glutamine-grown cells were similar to that observed with Msx-treated, ammonia-grown cells (Figs. 5B and C, lanes B and C). With proline, the Gln3-Myc¹³ phosphorylation level did not reach that observed with extracts of the Msx-treated cells (Fig. 5A, lanes B and C). The data in Figs. 4 and 5 also demonstrated that stress-generated Gln3-Myc¹³ phosphorylation was independent of the nitrogen source provided, since phosphorylation occurred with proline-, ammonia- or glutamine-medium.

To further test the hypothesis that osmotic stress increased Gln3-Myc¹³ phosphorylation in NaCl-treated cells, we assayed cells treated with a different osmotic agent, sorbitol. As shown in Fig. 5F, Gln3-Myc¹³ phosphorylation increased within 5 mins. of sorbitol addition (lanes A and B). The effect began to moderate by 30 mins. and was less noticeable when extracts from ammonia-grown cells were analyzed following 45 mins. of treatment (Fig. 5G, lanes A and B). Gln3-Myc¹³ phosphorylation profiles observed in glutamine-grown cells (Fig. 5H) were similar to those seen with ammonia (Fig. 5G). Irrespective of the nitrogen source, the level of Gln3-Myc¹³ phosphorylation attained was similar, but not identical, to that observed with Msx-treated, ammonia-grown cells (Fig. 5F, lanes E and F, Fig. 5G, lanes B and C, and Fig. 5H, lanes C and D). Together, the above data demonstrate Gln3-Myc¹³ phosphorylation increases following the onset of osmotic stress.

Gln3-Myc¹³ Phosphorylation Increases In Response To Oxidative Stress

The final stress condition we evaluated was that caused by hydrogen peroxide. In a manner similar to other stress conditions, 0.3 mM hydrogen peroxide (30 min.) increased Gln3-Myc¹³ phosphorylation in extracts from glutamine-grown cells (Fig. 6A, lanes A and B). The fastest migrating Gln3-Myc¹³ species was absent (Fig. 6A, lanes A and B, bottom black dot), and the slower migrating species were much more prominent (top black dot). Similar results occurred in ammonia-grown cells (Fig. 6B). The extent of phosphorylation, however, did not occur to the extent observed following Msx-treatment (Figs. 6A and 6B, lanes B and D). It is pertinent, in this regard, to note that we used a relatively low concentration of hydrogen peroxide (0.3 mM) compared with those we had used in studies of Ure2 function (2 or 3 mM, 30, 31). This was done to avoid cell injury and possible secondary effects derived from it. The somewhat diminished phosphorylation response correlated with the fact that Gln3-Myc¹³ relocalization to the cytoplasm following hydrogen peroxide addition appeared less strong than observed with NaCl (Fig. 3A and B).

Figure 6.

Oxidative stress increases Gln3-Myc¹³ phosphorylation in a nitrogen source-dependent manner. Wild type (TB123) cells were cultured in YNB medium with ammonia (⁺NH₄) or glutamine (Gln) (indicated by +) as nitrogen source and incubated with 0.3 mM hydrogen peroxide (H₂O₂) for the times indicated (in mins.). Msx (2 mM) was present for 30 mins. where indicted. Cells were then harvested for western-blot analyses.

Effects Of Caffeine-Treatment On Gln3-Myc¹³ Phos-phorylation And Intracellular Localization Are Similar To Those Elicited by Rapamycin

Kuranda et al., investigating the mechanisms of caffeine toxicity, reported results of a transcriptome experiment in which treating cells with caffeine elicited increased expression of many NCR-sensitive genes (39). The transcription profile of caffeine-treated cells resembled that observed following rapamycin-treatment (39, 40). These observations prompted us to determine whether caffeine-induced Gln3-Myc¹³ phosphorylation and localization paralleled the profile seen in rapamycin-, or alternatively, Msx-treated cells. Both inhibitors induce increased NCR-sensitive transcription and nuclear Gln3-Myc¹³ localization (1–4, 18, 19), but in our hands, oppositely affect its phosphorylation. Rapamycin decreases Gln3-Myc¹³ phosphorylation, whereas Msx, increases it (19). As shown in Figs. 7A and 7B, caffeine addition like rapamycin-treatment, elicited Gln3-Myc¹³ relocalization from the cytoplasm to nucleus of glutamine-grown cells. Neither inhibitor elicited nuclear Gln3-Myc¹³ localization in a sit4Δ (Figs. 7A and 7B).

Figure 7.

Effect of caffeine and rapamycin on intracellular Gln3-Myc¹³ localization in wild type (TB123) and sit4Δ (TB136) strains. Cells were cultured in YNB medium containing the nitrogen source indicated at the top of each panel. The cultures were divided into three portions. One portion received no further additions (None), while the other two received 200 ng/ml rapamycin (Rap; for 20 mins.) or 20 mM caffeine (Caff.; for 20 mins.). Cells were then processed, imaged, and scored as described in Fig. 2. The nitrogen sources provided for each experiment are indicated above the microscopic images.

The effects of caffeine-addition on Gln3-Myc¹³ localization extended to, and were more easily observed with, ammonia-grown cells. Gln3-Myc¹³ was highly nuclear in nearly all caffeine-treated cells, compared to only about half of those treated with rapamycin (Figs. 7C and 7D). Additionally, as we observed with Msx (19), the Sit4 requirement for nuclear localization of Gln3-Myc¹³ in ammonia-grown cells was weaker for caffeine- than rapamycin-treatment (Figs. 7C and 7D).

Responses to caffeine- and rapamycin-treatment were indistinguishable in wild type cells provided with proline as nitrogen source (Figs. 7E and 7F). The two inhibitors also elicited localization profiles that were indistinguishable from one another in a proline-grown sit4Δ. However, Gln3-Myc¹³ was less nuclear in caffeine-treated sit4Δ cells than wild type and than observed earlier when sit4Δ cells were treated with Msx (compare Figs. 7E and 7F with Fig. 6D of reference 33).

The similarity of Gln3-Myc¹³ intracellular localization in caffeine- and rapamycin-treated cells (Fig. 8A) suggested the transcription factor might also be dephosphorylated in caffeine-treated cells. As shown in Fig. 8B, Gln3-Myc¹³ was dephosphorylated (higher electrophoretic mobility) within five mins. of adding caffeine to ammonia-grown cells. The phosphorylation profile was indistinguishable from that observed following rapamycin-treatment (Fig. 8B, lanes D and E). Also like rapamycin-treatment (19, 32), Gln3-Myc¹³ dephosphorylation did not reverse even after extended treatment (Fig. 8C, lanes B and E).

Figure 8.

Effects of rapamycin, caffeine, and Msx on Gln3-Myc¹³ localization (Panel A) and phosphorylation (Panels B and C) in TB123. Ammonia was used as the nitrogen source throughout. Panel A. 0.2 μg/ml Rapamycin (+Rap), 20 mM caffeine (+ Caff.), or 2 mM methionine sulfoximine (+Msx) were added for 20, 20, and 30 mins., respectively, before cells were harvested. Panels B and C, 20 mM caffeine was added for the indicated times. Rapamycin (Rap) or Msx was added where indicated (+) for 20 or 30 mins., respectively.

Environmental Stress Predominates Over Nitrogen-Dependent And Tor1,2 Regulation Of Gln3-Myc¹³

The above results demonstrated Gln3-Myc¹³ localization to be strongly responsive to environmental stresses. The fact that Gln3-Myc¹³ responses to stress were the opposite of those elicited by nitrogen-limitation prompted us to investigate which response predominated when the two perturbations were applied together. We addressed this question by following Gln3-Myc¹³ localization in ammonia-grown wild type (TB123) cells initially starved for nitrogen and then subjected to osmotic stress via addition of 1M NaCl. Gln3-Myc¹³ remained cytoplasmic throughout the 2 hr experiment in unstarved control cells, and as expected, relocated to the nuclei of most nitrogen-starved cells within 30 min (Figs. 9A and 9B). In contrast, irrespective of whether NaCl was added following 30 or 60 mins. of nitrogen starvation, Gln3-Myc¹³ became cytoplasmic (Figs. 9A and 9B).

Figure 9.

NaCl reverses the effect of nitrogen starvation on Gln3-Myc¹³ intracellular localization. Wild type (TB123) was grown in YNB-ammonia to mid-log phase. The culture was then split into two portions: one portion received no further treatment (left histogram), while the other was nitrogen starved for the times indicated in the middle histogram. Samples were removed after 30 and 60 mins. of starvation, and then treated with 1 M NaCl for an additional 30 or 60 mins. Samples were then harvested for assay as described in Fig. 2.

These results argued that when cells were subjected to both nitrogen starvation and osmotic stress, it was to stress that Gln3-Myc¹³ appreared to respond. Since the stress response (NaCl-treatment) also predominated over the response to limited nitrogen (Proline, None vs. NaCl in Figs. 3A, 3B, and 3C and 3D wild type), we inquired whether the response to NaCl-treatment predominated over that elicited by the inhibitors we had been using to alter NCR-sensitive transcription and Gln3-Myc¹³ localization. To this end, we treated ammonia-grown mid-log phase cells, in which Gln3-Myc¹³ is cytoplasmic, with rapamycin (Figs. 10A and 10B), caffeine (Figs. 10C and 10D), or Msx (Figs. 10E and 10F). Within seven to ten mins. of inhibitor addition, Gln3-Myc¹³ became nuclear-cytoplasmic or nuclear in most cells. After being treated for twenty mins. with these inhibitors, NaCl (1 M) was added to each culture. Within five mins., Gln3-Myc¹³ had relocalized to the cytoplasm irrespective of the initial inhibitor treatment (Figs. 10A–F).

Figure 10.

NaCl reverses the effects of rapamycin- (Panels A and B), caffeine- (Panels C and D), or Msx-(Panels E and F) induced nuclear Gln3-Myc¹³ localization. TB123 cultures were grown in YNB-ammonia medium. The indicated inhibitor was added immediately after the zero time point sample (0 or Untreated) was taken. Additional samples were taken as indicated for the next 20 mins. for assay. After the 20 min points were taken, NaCl (1M) was added to each culture and it was sampled every five mins. for the next 20 mins.

The uniform ability of environmental stress to predominate over the cell’s response to normal environmentally generated nutritional signals, i.e., nitrogen-starvation and -limitation (growth with proline as nitrogen source) or those elicited by inhibitors (rapamycin, caffeine, or Msx), argued that either (i) stress completely altered, neutralized or overwhelmed the nutrient signals, or (ii) generated an additional signal to which Gln3-Myc¹³ preferentially responded. To determine whether the stress response was operating through alteration of the NCR-sensitive regulatory pathway, we eliminated the most Gln3-proximal regulatory element known to participate in nitrogen-responsive regulation, Ure2 (27). NaCl was added to a glutamine-grown ure2Δ. Gln3-Myc¹³ accumulated in the nuclei of most untreated ure2Δ cells (Figs 11A and 11B, ure2Δ Glutamine 0 min.), just as it had in the proline-grown wild type (Figs. 11A and 11B, Wild Type Proline 0 min.). However, within five mins. of adding NaCl to the culture, Gln3-Myc¹³ was cytoplasmic in both wild type and the ure2Δ (Figs. 11A and 11B, +NaCl 5 min.). Between 15 and 30 mins. post NaCl-treatment, Gln3-Myc¹³ was nuclear-cytoplasmic in a small fraction of the cells (Figs. 11A and 11B, +NaCl 15 min.).

Figure 11.

Panel A. NaCl reverses the effects of a ure2Δ on Gln3-Myc¹³ localization. Wild type and ure2Δ cultures were grown in YNB-proline and -glutamine media, respectively. NaCl (1 M) was added immediately after the zero time point sample was taken. Cultures were then sampled every five or ten mins. thereafter and assayed as described in Fig. 2. A similar experiment was performed for Panel B except that its duration which was much longer.

Relationship Between Stress-Induced Gln3-Myc¹³ Regulation And The GATA-Factor Requirement Of Ena1-Mediated Cation Tolerance

When considered together, the above data and several earlier reports describing control of ENA1 expression and cation tolerance appeared paradoxical, and therefore, required further investigation. There are three S. cerevisiae ENA genes, ENA1, ENA2, and ENA5, whose coding sequences are nearly identical as are the upstream regions of ENA2 and ENA5. All three upstream regions contain multiple GATAA sequences, though their locations relative to the ATG are different in ENA1 than in ENA2 and ENA5. ENA1 expression is activated by Crz1, which must be dephosphorylated by calcineurin before entering the nucleus (41–44). ENA1 transcription is also repressed via Mig1 and CRE (Sko1) sites regulated by the glucose repression and Hog1 osmotic shock signaling pathways (44–46). A third level of ENA1 regulation, which generated the paradox, is mediated by the GATA-factors Gln3 and Gat1.

Three earlier observations framed our thinking about Gln3/Gat1 participation in ENA1 regulation. (i) Withee et al. reported that mutation of URE2 increased Na⁺ tolerance (1.2 M) of cna1cna2 mutants, lacking Ca²⁺/calmodulin-dependent calcineurin, an outcome that required both Gln3 and cation extrusion ATPase, Ena1 (41). ENA1 expression, increased 20- and 10-fold (30 mins. after 0.8M NaCl addition) in wild type and cna1cna2 strains, respectively. It did not, however, detectably increase in a ure2 mutant whether or not Na⁺ was present (41). (ii) Masuda et al. reported Li⁺, Na⁺, and K⁺ induced SIT4 expression 2–3-fold (47). However, SIT4 over-expression did not increase Na⁺ or K⁺ tolerance (only Li⁺ tolerance increased), or ENA1 expression, nor was Na⁺- or K⁺-induced ENA1 expression diminished in a sit4 mutant (47). (iii) Crespo et al. reported rapamycin-induced ENA1-lacZ mRNA production, and showed that 0.4M NaCl induced ENA1-lacZ-mediated β-galactosidase production 4-fold (48). This induced level decreased 60–70% in a gln3gat1 double mutant (48). Together, these observations showed Gln3, Gat1 and Crz1 were responsible, in varying degrees, for high level ENA1 expression, but that cells could get by, albeit less well, with only one of the activation systems operating.

The above observations, and conclusions they support, raised the obvious question of how Gln3 could activate NaCl-induced ENA1 expression in a ure2Δ when Gln3-Myc¹³ exited from the nucleus when we treated wild type, and more importantly, ure2Δ cells with 1 M NaCl (Figs. 3, 9, 10, and 11A and B). Furthermore, the previous experiments cited above were performed in YPD medium where Gln3 is localized to the cytoplasm (Fig. 6B in reference 33 and earlier observations from multiple laboratories, e.g., 1).

The consistent characteristics of these previous ENA1 observations are: (i) the observed effects are generated by relatively low (<2-fold) to undetectable changes in NaCl-induced, Gln3-dependent levels of ENA1 mRNA, and (ii) the Gln3 contribution to NaCl tolerance was most convincingly observed when normal negative regulation of Gln3 was genetically eliminated by deleting URE2.

Of the many experiments performed throughout this work, Gln3-Myc¹³ was cytoplasmic in nearly all NaCl-treated cells except on two occasions: (i) a small number of cells with nuclear-cytoplasmic Gln3-Myc¹³ were observed 20 mins. after NaCl was added to wild type cells previously treated with Msx (Figs. 10E, NaCl 20 min. and 10F, right histogram), and (ii) 15 or more mins after NaCl was added to a glutamine-grown ure2Δ (Figs. 11A, + NaCl 15 min. and 11B). These data suggested that Gln3-Myc¹³ might be re-entering the nuclei of NaCl-treated cells, which was not unreasonable given that we had seen signs, in the earlier phosphorylation experiments, that the stress response was not sustained long-term (Figs. 5F, 5G and 6A). Also, cells continued to grow following addition of NaCl to the medium, albeit more slowly.

We investigated whether Gln3-Myc¹³ could re-enter the nucleus of NaCl-treated ure2Δ cells by extending the time of sampling to six hrs. As shown in Figs. 11C and 11D, adding NaCl to the ure2Δ resulted in Gln3-Myc¹³ quickly (5–10 min.) relocating from the nucleus to the cytoplasm. With increasing time, however, Gln3-Myc¹³ began to reappear in the nuclei of NaCl-treated cells. Between 20 and 60 mins., Gln3-Myc¹³ was cytoplasmic in about half the cells and nuclear-cytoplasmic or nuclear in the other half. Thereafter, Gln3-Myc¹³ was again predominantly cytoplasmic (Figs. 11C, 10, 45 and 120 min. and 11D). We anecdotally noted, both here and in other experiments, that the area of DAPI-positive material appeared to shrink transiently in cells treated with NaCl compared to those that were untreated or in those after extended treatment (Fig 11A, +NaCl 5min. and Fig. 11C, 10 min.).

Finally, we investigated whether treating wild type cells with NaCl elicited detectable changes in NCR-sensitive gene expression. Wild type TB123 was grown in YNB-proline medium and treated with NaCl. Northern blot analyses were unable to detect changes in DAL5 mRNA levels following NaCl-treatment (data not shown). This was not a wholly unexpected result because: (i) Whithee et al. did not observe detectable differences in ENA1 expression between wild type and ure2 mutant strains either in the presence or absence of NaCl under conditions that required Gln3 for NaCl tolerance (41), (ii) we had already observed instances in which the levels of NCR-sensitive gene expression did not closely correlate with intracellular Gln3-Myc¹³ localization, i.e., the level of a gene’s expression derives from the combined actions of transcription factors bound to all of the functional cis-acting regulatory elements in its promoter (35), and (iii) the effects of NaCl-treatment on Gln3-Myc¹³ localization were transient (Figs. 5 and 11).

DISCUSSION

Gln3-Myc¹³ Phosphorylation And Localization Respond In Parallel To Environmental Stress

Data presented above demonstrate that multiple environmental stresses (temperature, osmotic, oxidative) increase Gln3-Myc¹³ phosphorylation. In parallel, Gln3-Myc¹³ rapidly relocalizes from the nucleus to the cytoplasm of environmentally stressed cells under conditions that it would normally be highly nuclear, i.e., proline-grown, nitrogen-starved, Msx-, caffeine- and rapamycin-treated wild type cells. The stress responses reported here join our earlier observation that centrifugation, a potentially stressful condition that increases Snf1 phosphorylation (49), also increases the Gln3-Myc¹³ phosphorylation levels (19). This represents a major new type of regulation to which Gln3 is subjected and opens new insight into the regulation of intracellular Gln3 localization. Ever since the discovery of Gln3 and its requirement for NCR-sensitive gene expression, investigations have focused on elucidating how a cell’s nitrogen supply regulates Gln3 function and thereby the wide array of genes whose expression depends upon it. As important as that is, we would be unable to adequately understand Gln3 regulation without recognizing that other environmental inputs can at times predominate over nitrogen supply, if only transiently.

Stress and Nutrient-Induced Gln3-Myc¹³ Regulation Are Separable With The Stress-Induced Control Being Dominant

The insight gained from the experiments in this work concerns the overall mechanisms through which intracellular Gln3 localization is regulated. Heretofore, regulated Gln3 localization was thought to depend upon Ure2. Two alternative models suggested nuclear exclusion of Gln3 was due to (i) phosphorylation-promoted complex formation with Ure2 or (ii) Ure2-dependent stabilization of phosphorylated Gln3. However, our experiments clearly demonstrate stress-induced, Ure2-independent nuclear Gln3 exclusion. Therefore, stress-induced Gln3-Myc¹³ exit from the nucleus derives from a regulatory pathway that (i) operates in parallel with Ure2-dependent regulation, but impinges on Gln3 localization downstream of Ure2, or (ii) is capable of overwhelming responses elicited by limiting nutrients, and inhibition of Tor1,2 and/or other inhibitor-sensitive regulatory molecules. These possibilities are supported by the observations that: (i) nuclear Gln3-Myc¹³ exit occurred when environmental stress was imposed after the onset of all five conditions previously reported to result in nuclear Gln3-Myc¹³ localization, i.e., nitrogen-starvation or -limitation, or disruption of Tor1,2 signal transduction pathway activity, i.e., Msx-, rapamycin-, or caffeine-treatment. (ii) Stress-induced Gln3-Myc¹³ phosphorylation occurred irrespective of the nitrogen source provided, i.e., similarly in proline-, ammonia- and glutamine-grown cells, and (iii) stress-induced re-localization of Gln3-Myc¹³ occurred even when the most downstream regulatory event known to affect Gln3-localization, and suggested to be regulated by Tor1,2, was eliminated, i.e., loss of Ure2 in a ure2Δ..

Although we are in the initial stages of investigating the response of Gln3-Myc¹³ phosphorylation and localization to multiple environmental insults, it is most straightforwardly characteristic of increased protein kinase activity. The absence of effect when HOG1 was deleted suggests that Gln3-Myc¹³ phosphorylation is not occurring through that pathway and increases the likelihood of it occurring via a generalized stress response pathway. Further, the failure of osmotic stress-induced Gln3 regulation to be affected in ure2Δ and sit4Δ strains is consistent with the possibility of the pathway being mechanistically distinct from Tor1,2 influenced regulation. In agreement with this contention, SIT4 over-expression does not increase Na⁺ or K⁺ tolerance (only Li⁺ tolerance increased), or ENA1 expression, nor is Na⁺- or K⁺-induced ENA1 expression diminished in a sit4 mutant (47). Alternatively, the results we observed would be those expected if Ure2 functioned to stabilize the phosphorylated form of Gln3 and it was this form that was excluded from the nucleus. On the other hand, it must be remembered that Msx-treatment increased gross Gln3-Myc¹³ phosphorylation as well as nuclear localization.

Caffeine-Induced Gln3-Myc¹³ Dephosphorylation and Nuclear Localization

Data from experiments involving caffeine complement previously reported genome-wide transcription analyses that showed caffeine-treatment increases expression of many NCR-sensitive genes (39). Those data led to the conclusion that the effects of caffeine-treatment were intimately related to Tor1,2 regulation because caffeine and rapamycin-treatments generated such similar outcomes. We extended those transcriptional correlations here by demonstrating that caffeine-treatment induces Gln3-Myc¹³ dephosphorylation and nuclear localization. They do not, however, permit us to conclude that rapamycin and caffeine operate by the same biochemical mechanisms, only that their outcomes correlate with one another. They also do not exclude the possibility that caffeine possesses more than one site of action. Multiple sites of caffeine action have been reported, the most recent of which is at TORC1 (50).

Relationship Between Stress-Induced Gln3-Myc¹³ Regulation And The GATA-Factor Requirement of Ena1-Mediated Ion Tolerance

Initially, past experiments describing Gln3 participation in ENA1 expression (41, 48) appeared inconsistent with present results. We argued above that relatively low levels of nuclear Gln3 were sufficient to support the requirements of ENA1 expression required for NaCl tolerance in wild type cells and that increased tolerance occurred when Gln3 regulation was abrogated. Several observations justify this contention: (i) Pertinent earlier experiments were performed in YPD medium where Gln3 is cytoplasmic in wild type cells and hence little would be available in the nucleus to support ENA1 transcription (41, 48). This is probably why Gln3-mediated suppression of the cna1cna2 growth phenotype occurred only in a ure2 mutant where NCR-sensitive Gln3 regulation and effects of growth with a rich nitrogen source, e.g., YPD, were abrogated (41). In other words, elimination of Ure2 was only required when the ENA1 transcriptional activator, Crz1, was unable to function because it couldn’t be dephosphorylated in the cna1cna2 mutant. (ii) NaCl tolerance was similar in YPD-grown wild type, gln3gat1 or gln3 cells, and only a little greater in a gat1 or ure2 mutant (Fig. 3A, right panel in ref. 48). Much more important, significantly greater NaCl tolerance was seen in either ure2gln3 or ure2gat1 double mutants than any of those just mentioned (Fig. 3A, right panel in ref 48). In our view, the additional necessity of inactivating GLN3 or GAT1 in a ure2 mutant suggests NaCl tolerance observed by Crespo et al. required not only increased nuclear access of Gln3 or Gat1 but also elimination of another form of negative regulation deriving from gene expression or some other process that depends upon both Gln3 and Gat1. (iii) The 30 min. NaCl induction experiment of Crespo et al. was performed with lower NaCl (0.4 M) than used in the growth experiments. This condition would generate less osmotic stress and quicker recovery. Reasoning that Gln3 normally plays a limited role in ENA1 expression, and the immediate response to NaCl-generated osmotic stress, coupled with the observation that NaCl-induced relocation of Gln3-Myc¹³ from the nucleus to the cytoplasm is transient may rectify past and present observations.

The relationship of Gln3 to LiCl tolerance possesses, with one important exception, many of the same characteristics as that of for NaCl tolerance (48). In one report, sit4 single and gln3,gat1 double mutants decreased NaCl-induced ENA1-lacZ-mediated β-galactosidase to the same degree (48). We did not pursue rectifying this observation with those of Masuda et al. that Li⁺, Na⁺, and K⁺ induce SIT4 expression 2–3-fold (47). However, SIT4 over-expression does not increase Na⁺ or K⁺ tolerance (only Li⁺ tolerance increased), or ENA1 expression, nor is Na⁺- or K⁺-induced ENA1 expression diminished in a sit4 mutant (47).

Together, these past and present data have highlighted the complex relationship that exists between NaCl tolerance and NCR-regulated Gln3. They also point to an important, but unanswered, question: why do we and others fail to observe steady state mRNA levels and intracellular Gln3-Myc¹³ localization fluctuating in parallel under conditions where Gln3 can be convincingly shown to participate in stress-induced processes? Clearly, much more remains to be learned before sufficient information exists to extend current mechanistic models of these regulatory pathways. However, knowing that stress-responsive Gln3 regulation exists is the important and successful first step required to achieve that goal.

Influence Of Stress-Induced Gln3-Myc¹³ Regulation On Experiments Employing Temperature Sensitive Mutations

Finally, our experiments were initiated to investigate a temperature sensitive mutant. However, their outcome argue that measuring the effects of temperature-sensitive mutant phenotypes on Gln3 regulation cannot be straightforwardly interpreted, at least over short time frames, because the experiments contain two variables, i.e., possible effects of the mutation itself and those generated by the increased temperature required to alter the mutant protein.