Correspondence to: Ted Powers, Section of Molecular and Cellular Biology, Division of Biological Sciences, University of California Davis, Davis, CA 95616. Tel:(530) 754-5052 Fax:(530) 752-3085
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Abstract |
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De novo biosynthesis of amino acids uses intermediates provided by the TCA cycle that must be replenished by anaplerotic reactions to maintain the respiratory competency of the cell. Genome-wide expression analyses in Saccharomyces cerevisiae reveal that many of the genes involved in these reactions are repressed in the presence of the preferred nitrogen sources glutamine or glutamate. Expression of these genes in media containing urea or ammonia as a sole nitrogen source requires the heterodimeric bZip transcription factors Rtg1 and Rtg3 and correlates with a redistribution of the Rtg1p/Rtg3 complex from a predominantly cytoplasmic to a predominantly nuclear location. Nuclear import of the complex requires the cytoplasmic protein Rtg2, a previously identified upstream regulator of Rtg1 and Rtg3, whereas export requires the importin-ß-family member Msn5. Remarkably, nuclear accumulation of Rtg1/Rtg3, as well as expression of their target genes, is induced by addition of rapamycin, a specific inhibitor of the target of rapamycin (TOR) kinases. We demonstrate further that Rtg3 is a phosphoprotein and that its phosphorylation state changes after rapamycin treatment. Taken together, these results demonstrate that target of rapamycin signaling regulates specific anaplerotic reactions by coupling nitrogen quality to the activity and subcellular localization of distinct transcription factors.
Key Words: gene expression, metabolism, phosphorylation, rapamycin, signal transduction
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Introduction |
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Normal cell growth requires that cells adjust their metabolic activity according to nutrient availability and other environmental cues. Specialized signal transduction mechanisms exist that enable cells to perceive and integrate these cues in order to establish and/or maintain appropriate patterns of gene expression. Understanding how these pathways function is thus important for understanding both normal cellular behavior and the underlying basis of many human diseases, including cancer. One important signaling pathway used by all eukaryotic cells is the target of rapamycin (TOR)1 kinase pathway. This pathway was discovered through the action of the antibiotic rapamycin, a potent inhibitor of T cell proliferation, which combines with the small immunophilin FKBP and targets the large, evolutionarily conserved TOR kinase (
Recent studies have shown that TOR kinase activity is essential for the transcription of ribosomal RNA and ribosomal protein genes, as well as for the modulation of r-protein gene expression in response to changes in nutrient sources (
One of the most striking sets of genes affected by rapamycin treatment is composed of genes involved in the use of different sources of assimilable nitrogen (
The molecular mechanism by which TOR controls the expression of several genes involved in nitrogen metabolism, including GLN1, MEP2, and GAP1, has been shown recently to involve regulated changes in the subcellular localization of the Gln3 transcription factor (
In addition to permeases and degradative enzymes required for the use of specific nitrogen sources, distinct pathways involved in carbon metabolism are also responsive to nitrogen availability. For example, it has been observed that several genes encoding enzymes involved in the TCA and glyoxylate cycles are required for glutamate prototrophy (-ketoglutarate, the primary precursor to glutamate (
Studies of both prokaryotic and eukaryotic cells emphasize glutamate and glutamine as important regulators of nitrogen metabolism (reviewed by -ketoglutarate for use in the TCA cycle. Glutamine is also an immediate precursor for the biosynthesis of nucleotides and other nitrogen containing molecules, including NAD+, and thus represents a primary means by which nitrogen is assimilated into cellular material. Not surprisingly, cells have evolved elaborate mechanisms to sense the intracellular levels of these amino acids and to use this information to regulate their uptake and/or synthesis. Studies of enteric bacteria have revealed a complex signaling pathway involving a two-component regulatory system that couples intracellular levels of glutamine to changes in gene expression (reviewed by
We are interested in understanding further both the scope and the mechanisms by which gene expression is modulated according to nitrogen availability in yeast. Toward this end, we have explored a novel use of genome-wide expression analysis by identifying genes that are expressed differentially when yeast cells are grown in the presence of two defined nitrogen sources, the primary source glutamine versus an alternative source, urea. We find that a surprisingly small number of genes (<40) show significant differences in their levels of expression under these two conditions, where each identified gene is either induced or repressed by glutamine. In addition to Gln3-dependent target genes, one of the most concise sets of genes subject to glutamine-mediated repression includes metabolic genes regulated by the transcription factors Rtg1 and Rtg3. We demonstrate that, like Gln3, Rtg1 and Rtg3 are regulated by changes in their subcellular localization according to available nitrogen and, moreover, that the TOR kinase pathway plays an essential role in this regulation. Our data further suggest that glutamine-responsive transcriptional modulation defines a distinct branch of TOR signaling in yeast.
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Materials and Methods |
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Strains, Media, and General Methods
All strains of S. cerevisiae used in this study are listed in Table 1. The following culture media was used: YPD (1% yeast extract, 2% peptone, 2% dextrose); minimal dextrose (MD) (0.8% yeast nitrogen base without amino acids and ammonium sulfate, pH 5.5, 2% dextrose); synthetic complete dextrose (SCD) (0.7% yeast nitrogen base without amino acids, pH 5.5, 2% dextrose). MD media contained in addition one or more of the following nitrogen sources: glutamine, glutamate, ammonia, or urea, as indicated in the text, each at 0.2% final concentration. To supplement the auxotrophic requirements of strains used for the fluorescence microscopy experiment presented in Fig 4 (below), required amino acids, adenine, and uracil were added to MD media at concentrations described by
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Gene Expression Analysis Using cDNA Microarrays
Strain S288c was grown with vigorous shaking to 0.5 OD600/ml in 1 liter of MD media containing appropriate nitrogen sources, as indicated in the text. Cells were immediately harvested by centrifugation, flash frozen in liquid nitrogen, and stored at -80°C. Relative mRNA levels were determined by hybridizing fluorescently labeled cDNAs to microarrays containing cDNAs representing virtually every yeast open reading frame (
Northern Blots
Northern-blot analysis was performed as described previously (
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Plasmid Construction
Green fluorescent protein (GFP)tagged plasmids were constructed by PCR amplification of the promoter and ORF of RTG1, RTG2, and RTG3. Primers were designed such that 500 base pairs of upstream promoter region and the entire ORF of each gene were amplified. Each 5' upstream primer contained an XhoI restriction endonuclease site and each 3' downstream primer contained an EcoRI restriction endonuclease site immediately following the stop codon. After digestion with XhoI and EcoRI, each fragment was introduced in the XhoI and EcoRI sites of pRS316-GFP, which contains GFPS65T (
Construction of Yeast Strains
Strains derived from DBY7286 that were deleted for RTG1 or RTG3 were constructed using standard gene replacement techniques (
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Strains derived from DBY8943 that produced versions of Rtg1-Rtg3 tagged at their COOH termini with three copies of the hemagglutinin (HA) epitope were constructed using the PCR-based method described by
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Strains derived from K699 that were deleted for RTG1, RTG2, or RTG3 were made using the same PCR-based gene disruption technique described above, using the TRP1 gene of Candida glabrata as a selectable marker (
MSN5 was deleted from K699 using a HIS3-marked disruption vector, as described previously ( msn5
strain was made by mating the rtg2
and msn5
strains described above and selecting TRP+ HIS+ segregants after sporulation and tetrad dissection. The resulting msn5
and rtg2
msn5
strains, EY0736 and EY0744, respectively, were used for experiments presented in Fig 8 (below).
Strains containing the TOR1-1 allele combined with either rtg1/pRTG1-GFP or rtg3
/pRTG3-GFP were constructed using the following approach. Strains EY0733 and EY0735 were transformed with pRTG1-GFP or pRTG3-GFP, respectively. The resulting transformants were mated to strain JH11-1c and diploids were selected by their ability to grow on SCD agar plates lacking both adenine and uracil. After sporulation, TRP+, URA+ segregants were isolated and tested for their ability to grow on plates containing 0.2 µg/ml rapamycin. The resulting selected strains, PLY079 and PLY083, were used for the experiment shown in Fig 5 B (below).
All wild-type parental strains used for construction of the above strains were examined by Northern blot analysis to confirm that expression of the RTG-dependent target genes CIT2 and DLD3 was (a) repressed by preferred nitrogen sources, and (b) induced by rapamycin treatment.
Fluorescence Microscopy
For all microscopy experiments, cells were freshly transformed with plasmids that expressed appropriate GFP-fusion proteins. Cells were first grown overnight in SCD media that lacked uracil to select for plasmid maintenance. To lower the background auto fluorescence of the parent strain, additional adenine and tryptophan were added to a final concentration of 0.005%. Cells were then diluted to 0.005 OD600/ml in media appropriate for each experiment, as indicated in the text, and were examined directly by fluorescence microscopy when they reached 0.5 OD600/ml. Rapamycin was added to a final concentration of 1.0 µg/ml for microscopy experiments presented in the figures. Identical results were also obtained at the lower rapamycin concentration of 0.2 µg/ml. All images documenting GFP localization were collected on an microscope with a 100x objective (BX60; Olympus) and recorded with a CCD camera (Photometrics) using identical settings for each experiment and an average exposure time of 1.01.5 s.
Preparation of Cell Extracts and Western Blot Analysis
For detection of Rtg1-Rtg3 by immunoblotting, cells were grown in appropriate media, as described in the text, to 0.5 OD600/ml, treated with rapamycin where indicated, and harvested directly. Extracts were prepared as described previously (
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Results |
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Identification of Nitrogen-regulated Genes
We used genome-wide gene expression analysis to compare mRNA levels following the growth of yeast cells in the presence of two distinct sources of assimilable nitrogen, glutamine and urea. Whereas glutamine is used directly in the biosynthesis of several nitrogen-containing compounds, urea must first be degraded, via a bifunctional enzyme encoded by the DUR1,2 gene, to ammonia and carbon dioxide before the ammonium ion can be incorporated into glutamate and glutamine (110 min in minimal dextrose media containing either glutamine (MD-glutamine) or urea (MD-urea) as a sole source of nitrogen. This was in contrast to several other alternative nitrogen sources that we tested, including arginine and proline, which resulted in reduced growth rates (data not shown). Thus we reasoned that any observed differences in gene expression would be restricted to the use of glutamine and urea as nitrogen sources, rather than secondary effects due to differences in growth rate. Accordingly, S288c was grown to mid-log phase in MD-glutamine or MD-urea and PolyA mRNA was isolated. Fluorescently labeled cDNAs were then prepared and applied to DNA microarrays that contained nearly every yeast open reading frame (
Of the more than 6,200 genes examined, a surprisingly small number (<40) displayed differences in expression threefold or greater under these two nitrogen conditions; 12 were expressed preferentially in MD-glutamine and 24 were expressed preferentially in MD-urea (Table 2). We confirmed these results for a representative number of genes directly by Northern blot analysis (Fig 1). The majority of genes that were expressed better in MD-glutamine encoded permeases specific for amino acids associated with rich nutrient conditions, including GNP1 and TAT2, which encode high affinity permeases specific for glutamine and tryptophan, respectively (
To extend the above results, we determined the relative expression of a representative number of genes following the growth of cells in media containing one of two other preferred nitrogen sources, glutamate and ammonia. In general, the pattern of expression produced by cells in MD-glutamate was similar to that produced in MD-glutamine, particularly for the genes involved in the TCA and glyoxylate cycles (Fig 1 B, compare lanes 1 and 2). Several differences were also observed, however. For example, GLN1, which encodes glutamine synthetase, was expressed in MD-glutamate but not in MD-glutamine (Fig 1 C, compare lanes 1 and 2). This result demonstrates the extreme selectivity in gene expression that exists according to the precise nitrogen source provided (
Very few differences in gene expression were observed when ammonia and urea were compared, one of the most notable being DUR3 (Fig 1 C, compare lanes 3 and 4). Indeed, microarray analysis revealed that <10 genes were expressed differently when cells were grown in media containing each of these two nitrogen sources (T. Powers, unpublished results). These results were somewhat surprising given that ammonia is known to display many of the same regulatory properties as glutamine and glutamate (
Demonstration of Glutamine as a Global Regulator of Gene Activity
The results of the above microarray experiment revealed the scope of genes whose expression differed significantly in the presence of glutamine versus urea. This experiment could not distinguish, however, whether these differences resulted from the stimulation or repression of gene activity by either nitrogen source. We reasoned that we could address this issue using microarrays by pair-wise comparisons of cultures grown in the presence of one versus both nitrogen sources. The logic here was that a given gene should display the same level of expression (i.e., have a ratio 1.0) if the activating (or repressing) nitrogen source was present in the two samples being compared. Accordingly, we compared mRNA levels from cells grown in the following media: MD-glutamine, MD-urea, and MD-glutamine+urea. The results of this experiment are shown in the form of scatter plots in Fig 2.
When mRNA levels from cells grown in MD-glutamine and MD-urea were compared, most genes appeared as points along a diagonal with a slope of 1.0 and corresponded to genes expressed similarly under the two conditions (Fig 2 A). A characteristic number of points fell both above and below this diagonal and corresponded to genes (reported in Table 2) that were preferentially expressed in glutamine or urea, respectively (Fig 2 A). This pattern of expression was remarkably similar when cells grown in MD-urea and MD-glutamine+urea were compared, indicating that the presence of urea in both cultures did not significantly change the relative expression of any gene (Fig 2, compare A and B; note that the plot in B appears as the reciprocal of the plot in A due to the arrangement of axes). In dramatic contrast, when MD-glutamine and MD-glutamine+urea samples were compared, essentially all points collapsed onto the diagonal, demonstrating the dramatic effect by glutamine on gene expression (Fig 1 C). From these results, we conclude that essentially all differences in gene expression observed in these experiments result from glutamine acting as both an activator and a repressor of gene activity. This conclusion was confirmed for a number of representative genes by Northern blotting (Fig 1, lanes 57) and is consistent with the demonstrated role of glutamine as an important regulator of nitrogen-dependent gene expression (
RTG-dependent Gene Expression: An Interface between Carbon and Nitrogen Metabolism
Many of the differences in gene expression observed in the preceding experiments were likely to reflect altered metabolic needs as cells use distinct nitrogen sources. For example, de novo biosynthesis of amino acids requires intermediates provided by the TCA cycle, primarily oxaloacetate and -ketoglutarate, that must be replaced through anaplerotic reactions to maintain the respiratory competency of the cell (
-ketoglutarate, namely ACO1, IDH1, and IDH2, that we found to be repressed by glutamine (or glutamate) (Fig 1 B and 3 A, and data not shown). Thus, one physiological response of cells growing in the absence of these nitrogen sources was increased expression of genes involved in these anaplerotic reactions. We decided to explore this regulation in greater detail.
Two distinct transcriptional regulatory complexes, namely HAP and RTG, have been demonstrated to regulate expression of ACO1, IDH1, and IDH2 (
To determine directly whether the RTG transcription factors were required for regulated expression of the above metabolic genes under our experimental conditions, we performed the following nutrient-shift experiment. Wild type, rtg1, or rtg3
cells were grown in MD-glutamine to early log phase, and were then transferred either to fresh MD-glutamine (as a control) or to MD-urea. Total RNA was isolated and mRNA levels of a representative number of these genes were analyzed by Northern blotting and normalized to actin mRNA levels (Fig 3 B). As expected, each gene examined displayed increased expression, relative to actin, when wild-type cells were transferred to MD-urea but not to MD-glutamine (Fig 3 B, compare lanes 1 and 2 with 3). In contrast, no increased expression of ACO1, IDH1, IDH2, CIT2, or DLD3 was observed upon transfer of either rtg1
or rtg3
cells to MD-urea (Fig 3 B, lanes 6 and 9). Interestingly, PYC1 expression was increased in each mutant strain in MD-urea by about half the extent observed in wild-type cells (Fig 3 B, compare lane 3 with 6 and 9). In addition, similar levels of expression were observed for MLS1 in both wild-type and each rtg mutant strains in MD-urea (data not shown). These latter results demonstrate that factors in addition to the RTG genes are likely to be involved in the regulated expression of these two metabolic genes under these conditions and is consistent with previous biochemical and molecular genetic analyses of RTG-dependent control of PYC1 expression (
Nitrogen-dependent Changes in the Subcellular Localization of Rtg1 and Rtg3
We wanted to understand the mechanism by which Rtg1 and Rtg3 activity is regulated. No significant differences were observed in the steady state levels of either RTG1 or RTG3 mRNAs, nor of Rtg1 or Rtg3 proteins in MD-glutamine versus MD-urea, suggesting that their activity was regulated post-translationally (data not shown; see below). Recently, a number of nutrient-responsive transcription factors have been shown to be regulated at the level of nuclear transport (reviewed in
Both Rtg1-GFP and Rtg3-GFP appeared predominantly cytoplasmic when cells were grown in MD-glutamine (Fig 4, left). Similar results were obtained when glutamate was used instead as a nitrogen source (data not shown). In contrast, both proteins were concentrated in the nucleus when cells were grown in MD-urea (Fig 4, right). Also, in close agreement with the transcriptional responses described above, both Rtg1-GFP and Rtg3-GFP remained cytoplasmic when cells were grown in media that contained both glutamine and urea (data not shown). From these results, we conclude that RTG-dependent gene activation involves changes in the subcellular distribution of the Rtg1/Rtg3 complex.
Rtg1 and Rtg3 Activity and Subcellular Localization Is Regulated by the TOR Pathway
We wished to identify the signaling pathway(s) that linked nitrogen quality to the localization of the Rtg1/Rtg3 complex. Here a clue was provided by the fact that many RTG-dependent target genes become induced when cells are treated with rapamycin, a specific inhibitor of the TOR kinases (
A prediction of the above results was that inhibiting the TOR pathway might be sufficient to result in RTG-dependent gene activation. To test this directly, we performed a time course of rapamycin treatment of wild-type, rtg1, and rtg3
cells grown in MD-glutamine, and then analyzed mRNA levels of several RTG-dependent targets by Northern blotting. In wild-type cells, each target gene examined showed increased expression within 15 min after addition of rapamycin (Fig 6, compare lanes 1 and 2). The expression levels of these genes peaked at
30 min and were comparable with the levels observed in cells grown in MD-urea (Fig 6, lane 3; compare with Fig 3 B, lane 3).
In striking contrast, no induction was observed for any of these genes when rapamycin was added to rtg1 and rtg3
cells (Fig 6 B, lanes 512). The sole exception was PYC1, which showed a level of induction in each mutant strain of about half that observed in wild-type cells. This latter result is thus reminiscent of the behavior of PYC1 in the nutrient shift experiment described above (Fig 4 B) and indicates that an additional rapamycin-sensitive regulatory factor(s) is involved in the expression of this gene. As a control, we observed similar induction of two Gln3-dependent targets, DUR3 and DAL5, in both wild-type and the rtg deletion strains, demonstrating that the loss of induction in the rtg mutants is specific for RTG-dependent targets. The specificity of these results was also confirmed by an observed decrease in RPL32 mRNA levels in all strains after rapamycin treatment, as reported previously (
Interestingly, deletion of the genes encoding the GATA transcriptional regulators Gln3 and Gat1, whose nucleocytoplasmic transport is similarly regulated by TOR, confers weak resistance to rapamycin ( or rtg3
cells to this drug, in comparison to wild-type cells (data not shown). Thus, we conclude that rapamycin-induced expression of RTG-dependent target genes does not contribute to the toxic effects of this drug on yeast cells.
Regulated Nucleocytoplasmic Transport of Rtg1 and Rtg3 Requires All Three RTG Genes
The third member of the RTG gene family, RTG2, encodes a cytoplasmic protein that contains an HSP70-like ATP binding domain and displays homology to certain bacterial polyphosphatases and phosphatases ( cells, in both the absence and presence of rapamycin. As in wild-type cells, both proteins were localized to the cytoplasm in the absence of drug (Fig 7 A, left). In contrast, no nuclear accumulation of either protein was observed after addition of rapamycin (Fig 7 A, right). Additional experiments demonstrated that Rtg2 was itself a cytoplasmic protein and that its localization did not change after rapamycin treatment (data not shown). Thus, these results demonstrate that Rtg2 is essential for rapamycin-induced nuclear accumulation of the Rtg1/Rtg3 complex and that it carries out this function in the cytoplasm.
We next determined whether both Rtg1 and Rtg3 were required for TOR-regulated nuclear transport of the Rtg1/Rtg3 complex by monitoring the localization of Rtg1-GFP in rtg3 cells or, alternatively, Rtg3-GFP in rtg1
cells, both before and after rapamycin treatment. We observed that Rtg1-GFP remained exclusively cytoplasmic in rtg3
cells, even after rapamycin addition (Fig 7 B, top, and data not shown). Since Rtg1 is a relatively small protein of 177 amino acids, we wanted to exclude the possibility that the constitutive presence of Rtg1-GFP in the cytoplasm in rtg3
cells was not due simply to the diffusion of this protein out of the nucleus after rapamycin treatment. Toward this end, we fused three tandem copies of the coding region of GFP to the 3' end of the RTG1 gene to create a much larger protein, termed Rtg1-GFP3. Control experiments confirmed that this fusion protein was functional (data not shown). We observed that Rtg1-GFP3 also remained in the cytoplasm in rapamycin-treated cells, indicating that Rtg1 cannot accumulate in the nucleus in the absence of Rtg3 (data not shown).
In striking contrast, we observed that Rtg3-GFP was localized exclusively in the nucleus in rtg1 cells, in both the presence and absence of rapamycin (Fig 7 B, bottom, and data not shown). Thus, regulated transport of the Rtg1/Rtg3 complex requires that both proteins be present together. One potential explanation to account for the constitutive localization of these two proteins in different subcellular compartments is that Rtg1 contains a nuclear export signal, whereas Rtg3 contains the nuclear import signal (NLS) for the heterodimer. Consistent with this interpretation, a recent study has confirmed that the basic domain of the bHLH motif of Rtg3 contains a functional NLS (
Export of Rtg1 and Rtg3 from the Nucleus Requires the ß-Importin Homologue Msn5
Constitutive localization of Rtg3 in the nucleus in rtg1 cells suggested that export of the Rtg1/Rtg3 complex from the nucleus might play a role in the regulation of these transcription factors. Previous studies of another nutrient-responsive transcription factor, Pho4, has demonstrated that its export from the nucleus depends on the activity of Msn5, a member of the ß-importin family of nuclear receptors (
cells, demonstrating that this factor is required, either directly or indirectly, for export of the Rtg1/Rtg3 complex from the nucleus (Fig 8 A, left). Interestingly, when we examined the localization of Rtg1-GFP and Rtg3-GFP in msn5
rtg2
cells, both proteins remained in the cytoplasm, consistent with the above observation that Rtg2 is absolutely required for nuclear entry of the Rtg1/Rtg3 complex (Fig 8 A, right). Additional control experiments demonstrated that Rtg2-GFP remained in the cytoplasm in msn5
cells (data not shown).
If regulated access to the nucleus represents the primary mechanism by which the activity of the Rtg1/Rtg3 complex is controlled, then we expected to observe constitutive activation of their target genes in msn5 cells in the absence of rapamycin. However, no such increased expression of RTG-target genes was observed in msn5
cells compared with wild type (Fig 8 B, compare lanes 1 and 3). This observation is consistent with studies of other regulated transcription factors; namely, that constitutive nuclear localization does not necessarily result in gene activation and that other regulatory mechanisms are involved (
cells after addition of rapamycin (Fig 8 B, compare lanes 3 and 4). This latter result demonstrates that despite its steady state nuclear localization in msn5
cells, the Rtg1/Rtg3 complex remains responsive to changes in TOR signaling.
TOR-dependent Changes in the Phosphorylation State of Rtg3
Previous studies have demonstrated that changes in phosphorylation of a transcription factor can be important for regulating its activity as well as concentration in the nucleus (
We wanted to confirm that Rtg3 was a phosphoprotein by treating purified Rtg3 with phosphatases in vitro; however, the HA epitope-tagged form of this protein was not efficiently immunoprecipitated from cell extracts. We therefore used a form of Rtg3 that was fused at its COOH terminus to two z domains from Protein A, termed Rtg3-zz, which could be immunoprecipitated quantitatively from extracts using immobilized-IgG (data not shown). The results confirmed that rapamycin treatment resulted in the conversion of a portion of Rtg3-zz to a more slowly migrating form (Fig 9 B, compare lanes 1 and 2). Moreover, this slower form was abolished after treatment with phosphatase, confirming that the rapamycin-induced change in electrophoretic mobility of Rtg3-zz is due to changes in phosphorylation (Fig 9 B, compare lanes 2 and 4; arrowhead, slower migrating form). Interestingly, the rapamycin-induced shift in Rtg3 mobility was also observed in rtg2 cells, suggesting that TOR influences Rtg3 phosphorylation independently from Rtg2 function (Fig 9 B, compare lanes 5 and 6).
While the above results demonstrate that TOR influences the phosphorylation state of Rtg3, several other results indicate that phosphorylation-dependent regulation of this protein is likely to be complex. First, we observed that Rtg3-zz was phosphorylated in the absence of rapamycin treatment, a conclusion based on its increased mobility after phosphatase treatment, suggesting that Rtg3 is likely to be phosphorylated on multiple sites (Fig 9 B, compare lanes 1 and 3). The existence of multiple sites of phosphorylation could account for the relatively poor resolution of Rtg3 on SDS-PAGE gels in the absence of phosphatase treatment. Second, we were unable to detect a difference in the mobility of Rtg3-zz in cells grown in MD-glutamine versus MD-urea (Fig 9 C, lanes 1 and 2). This latter result suggests either that rapamycin-induced changes are transient or that additional changes in the phosphorylation state of this protein occur during steady state growth of cells in media lacking glutamine. Finally, multiple levels of regulation, possibly mediated by distinct changes in the phosphorylation state of Rtg3, would be consistent with our above observation that the Rtg1/Rtg3 complex is concentrated in the nucleus in msn5 cells, yet their target genes remain uninduced. Identifying the residue(s) of Rtg3 that are phosphorylated under these different conditions will be required to resolve these issues.
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Discussion |
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Genome-wide expression analysis represents a powerful approach for exploring the scope of transcriptional regulation associated with different biological processes. The appeal of this method is most often attributed to the enormous amount of information that it can yield, as exemplified by studies in yeast of the diauxic shift, sporulation, and the cell cycle, where each of these processes involves sequential changes in the expression of groups of coregulated genes that number well into the hundreds (
Specifically, we have demonstrated that a surprisingly small number of genes are differentially expressed when yeast cells use glutamine versus urea as their sole source of nitrogen (Table 2). All of these genes are either induced or repressed by the presence of glutamine, a fact that is consistent with its established importance as a major regulator of nitrogen metabolism (
A second prominent group of glutamine-repressed genes encodes enzymes involved in the TCA and glyoxylate cycles and whose expression in urea-containing media requires the heterodimeric transcription factors Rtg1 and Rtg3. Differential expression of these metabolic genes is consistent with the proposal by -ketoglutarate for use in the de novo biosynthesis of glutamate, which in turn is required for the synthesis of glutamine. In addition, our results extend involvement of RTG-dependent regulation to include PYC1, which provides an alternative route for the synthesis of oxaloacetate for use in both the TCA cycle and in amino acid biosynthesis (Fig 3). These results thus highlight the intimate relationship that exists between nitrogen and carbon metabolism as well as its importance to normal cell growth.
Remarkably, we find that TOR signaling also regulates RTG-dependent gene activity. Specifically, inhibition of the TOR kinases by rapamycin results in both rapid nuclear accumulation of the Rtg1/Rtg3 complex as well as induction of their target genes. In addition, rapamycin treatment correlates with changes in the phosphorylation state of Rtg3, indicating that nucleocytoplasmic transport of the complex is likely to be regulated by differential phosphorylation of Rtg3. These results thus contribute to a growing body of evidence demonstrating that one important role of TOR is to control the activity of specific nutrient-responsive transcription factors. Moreover, our findings suggest that TOR signaling may provide an important mechanism by which carbon and nitrogen use are linked. Whether Rtg3 phosphorylation is influenced by the activity of the Tap42-Sit4 phosphatase complex, as has been shown recently for Gln3 (
Our results also shed light on the functional role of Rtg2 in regulating RTG-dependent gene expression, a previously identified upstream positive regulator of Rtg1 and Rtg3 ( mutant cells (Fig 8 B). Furthermore, our examination of Rtg3 localization in rtg1
, rtg2
, and rtg1
rtg2
mutant cells suggests that Rtg2 may interact primarily with Rtg1 to regulate nuclear entry of the Rtg1/Rtg3 complex (Fig 7, and data not shown).
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In contrast to the role played by Rtg2, we find that Msn5, a member of the importin-ß family of nuclear transport receptors, is required for nuclear export of the Rtg1/Rtg3 complex (Fig 10). Thus, in msn5 cells, both Rtg1 and Rtg3 are constitutively localized to the nucleus. We do not know yet whether Msn5 interacts directly with Rtg1 and/or Rtg3 to facilitate their export from the nucleus, as has been demonstrated for Pho4 (Kaffman et al., 1998). It is possible that Msn5 is instead required for the proper localization of another protein(s) that is involved directly in the export of the Rtg1/Rtg3 complex.
While nucleocytoplasmic transport of the Rtg1/Rtg3 complex is essential for regulated RTG-dependent gene activation, our results indicate that additional control mechanisms are involved. This conclusion is based on our analyses of msn5 cells, where both Rtg1 and Rtg3 are concentrated in the nucleus yet their target genes remain uninduced (Fig 9). As these genes can nevertheless be induced rapidly in msn5
cells following rapamycin treatment, these results raise the intriguing possibility that TOR regulates Rtg1/Rtg3 activity in both the cytoplasm as well as in the nucleus.
RTG1-RTG3 were originally identified as genes required for increased expression of CIT2 under conditions where mitochondrial respiratory function is impaired, as in rho0 petite mutants that lack mitochondrial DNA (
At odds with this conclusion, however, is the finding that nuclear accumulation of the Rtg1/Rtg3 complex in rho0 cells appears to correlate with a substantial decrease in phosphorylation of Rtg3 (
Inhibition of the TOR kinases by rapamycin affects the expression of a large number of genes, including those regulated by nitrogen and glucose-sensitive signaling pathways, glycolytic genes, genes expressed during the diauxic shift, and r-protein and rRNA genes (
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Footnotes |
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1 Abbreviations used in this paper: GFP, green fluorescent protein; HA, hemagglutinin; MD, minimal dextrose; ORF, open reading frame; SCD, synthetic complete dextrose; TOR, target of rapamycin.
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Acknowledgements |
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We are grateful to J. Derisi for his expertise and leadership and to P. Walter and other members of the Department of Biophysics and Biochemistry for their help in establishing DNA microarray technology at UC San Francisco. We thank H. Bennett for her expert instruction in preparing fluorescently labeled cDNAs. We thank D. Botstein for strains and C. Kao for help with data analysis and for sharing her unpublished results. We thank K. Burtis for help with microarray development at UC Davis. We also thank the members of the Powers lab for their assistance during the course of this work as well as J. Derisi for valuable discussions. Finally, we thank S. Burgess, W. Heyer, M. Niwa, and M. Singer for comments on the manuscript.
This work was supported by laboratory start up funds as well as a faculty research grant from U.C. Davis (T. Powers) and by grant GM 59034 from the National Institutes of Health (E.K. O'Shea). A. Komeili is a Howard Hughes Medical Institute predoctoral fellow.
Submitted: 16 August 2000
Revised: 20 September 2000
Accepted: 22 September 2000
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References |
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