INTRODUCTION

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A central goal of modern cell biology is to elucidate the molecular details of how eucaryotic cells sense their environments and transduce those signals into cellular responses. Many of these responses result in changes in the expression of genes responding to the initial signal. Saccharomyces cerevisiae can sense and selectively utilize good nitrogen sources (e.g., asparagine) in preference to poor ones (e.g., proline) through the process of nitrogen catabolite repression (NCR) (7, 8). Four GATA family transcription factors mediate NCR-sensitive gene expression, two that act as transcriptional activators (6, 37) (Gln3p and Gat1p/Nil1p) (2; see references 4 to 6 for reviews) and two (Dal80p/Uga43p and Deh1p/Nil2p/Gzf3p) that repress the action of Gln3p and Gat1p (2, 3, 10-14, 26, 34, 35, 37). All of the GATA-factor genes except GLN3 contain upstream GATA sequences and are nitrogen regulated (1, 5, 6, 34, 35). GLN3 does not appear to be highly regulated at transcription (26-28). Gln3p-dependent transcription is negatively regulated by Ure2p (9, 15) and one or more uncharacterized proteins (6).

Two reports have identified elements associated with NCR nitrogen sensing or signaling (23, 30). One of these reports contends that Mep2p (ammonia permease) is the nitrogen sensor for ammonia-grown cells and that amino acid permeases serve this function when amino acids are provided instead (23). The second, which is the focus of this work, proposes that tRNA^Gln-CUG is a key element in transducing the nitrogen signal (30).

Filamentation and sporulation are two developmental tracks taken in response to nitrogen starvation. Murray et al. (30) observed that a specific temperature-sensitive allele (sup70-65) of the essential SUP70/CDC65 gene (Gln tRNA^CUG) results in gratuitous filamentation and sporulation in nitrogen-rich medium at 30°C. Shifting the sup70-65 mutant from 23 to 30°C in rich medium also resulted in fourfold "derepression" of CAR1 (arginase) expression. This led them to conclude that tRNA^Gln-CUG was central to the regulation of all three processes (30).

That tRNA^Gln-CUG was a key element in signaling for nitrogen starvation and NCR was surprising in light of work with YVH1 (17, 31). YVH1 (which encodes a dual-specificity protein phosphatase required for efficient sporulation) expression is specifically induced by nitrogen starvation (17, 31). Murray et al.'s report leads one to expect a relation between YVH1 and NCR-sensitive gene expression. Previous data (31), however, suggested otherwise. YVH1 expression is induced by nitrogen starvation but is not NCR sensitive (31). NCR-sensitive gene expression is also not affected by disruption of YVH1 (31). This prompted us to investigate YVH1 and NCR-sensitive gene expression in the tRNA^Gln-CUG mutant generously provided by Murray and Johnston.

	MATERIALS AND METHODS

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Strains LMDWLU (Mata/MAT SUP70/SUP70 ura3-52/ura3-52 leu2-3,112/leu2-3,112 ade1-1/ADE1) and LMD65-1LU (MATa/MAT cdc65-1/sup70/cdc65-1/sup70 leu2-3,112/leu2-3,112 ura3-52/ura3-52) (30) were used. The sup70-65 mutation in the strain we used (LMD65-1LU) was confirmed via sequencing of a PCR amplification product. Plasmid pRS316HAYVH1 is a hemagglutinin-tagged allele of YVH1 cloned into plasmid pRS316; it complements a yvh1 disruption. All strains and transformants were propagated at 22°C until experiments were initiated by growing cultures (500 ml) at 30°C to an A₆₀₀ of 0.7 to 0.8 in 0.17% YNB (yeast nitrogen base [Difco]) plus 2% glucose plus 0.1% nitrogen plus leucine and uracil (120 and 20 mg/liter). Total RNA (20 µg per lane, isolated as described previously [20]) was analyzed by Northern analysis (5). Radioactive DAL5 and CAR1 probes were made by random priming of PCR products. Plasmid pC4 was the control; expression of the uncharacterized gene it contains is unaffected by nutrition (21). Poly(A)⁺ RNA (1.5 µg/lane) was analyzed similarly. The 350-bp NdeI-BglII YVH1 fragment (labeled by random priming) was the probe. Blots were washed as described previously (20). For -galactosidase assays (32), strains LMDWLU and LMD65-1LU were transformed with plasmids pJCD52 (DAL5) and pTSC572 (DAL80). Transformants were grown at 22 or 30°C in the medium described above. Cells (25 ml) were harvested and assayed, with each value representing the mean of three determinations.

	RESULTS

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Dimorphic growth and sporulation share multiple characteristics, including their regulation by Ras2p (16, 22, 29). Both processes require complete or very nearly complete nitrogen starvation in wild-type cells (16). NCR, on the other hand, depends not on starvation but on nitrogen source quality and is not cell type restricted. It could reasonably be argued that nitrogen starvation is the most extreme case of a poor nitrogen source. However, we contend that growth on a poor nitrogen source is fundamentally different from starvation. In a chemostat, wild-type cultures divide indefinitely when provided with a poor nitrogen source, albeit more slowly than with a good one. In contrast, cells starved for nitrogen arrest in the G₁ stage of the cell cycle (18, 19). The fundamental difference is progression through the cell cycle. The regulation of gene expression in response to nitrogen source quality (NCR) does not result in arrest of the cycle, whereas nitrogen starvation does.

Based on these arguments, we reexamined tRNA^CUG regulation of NCR-sensitive gene expression using the sup70-65 mutant of Murray et al. (30). DAL5 (encoding allantoate permease)-lacZ transformants were grown in minimal glucose-proline or -asparagine medium at 22 or 30°C (33). NCR sensitivity of DAL5 expression was the same in both strains, though reporter gene activity was 30 to 40% higher in the wild type (Fig. 1A). Similarly, the NCR sensitivities of DAL80-lacZ expression in wild-type and mutant strains were not qualitatively different at 22 or 30°C (Fig. 1B). Small, gene-specific differences in overall expression were, however, noted.

View larger version (31K): [in this window] [in a new window]   FIG. 1.   DAL5-lacZ (plasmid pJCD52) (A) and DAL80-lacZ (plasmid pTSC572) (B) in wild-type (LMDWLU) and sup70-65 (LMD65-1LU) strains growing at 22 and 30°C with proline (open bars) or asparagine (filled bars) as the nitrogen source. Units are those of Miller but are based on 25 ml of culture.

Since the lacZ fusion studies demonstrated no effect on NCR in sup70-65 strains, we assayed the NCR sensitivity of CAR1 expression by Northern analysis because this was the gene used by Murray et al. (30). We prepared total RNA from wild-type and sup70-65 mutant strains growing at 30°C with proline or asparagine as the nitrogen source. The critical difference between our experiment and that of Murray et al. was the way NCR sensitivity was assessed. Murray et al. assayed CAR1 mRNA in mutant cells containing a plasmid vector or a wild-type SUP70 gene growing in rich medium at 23°C or after being shifted to 30°C for 1 h (30). CAR1 mRNA increased in mutant cells at 30°C relative to that of the control at 23°C, an increase not seen when the wild-type gene was present. We assayed NCR sensitivity in wild-type and mutant cells in balanced growth at 30°C (in contrast to a 1-h shift) with proline or asparagine as the sole nitrogen source. We observed significantly more CAR1 mRNA in wild-type and sup70-65 mutant cells growing in proline than in asparagine medium (Fig. 2). DAL5 expression was used as a control.

View larger version (90K): [in this window] [in a new window]   FIG. 2.   (Upper panels) Effects of sup70-65 mutation on steady-state levels of CAR1 and DAL5 mRNA (total RNA analyzed) in cultures growing at 30°C. Wild-type (W.T.) (LMDWLU, lanes A, B, E, and F) and sup70-65 strains (LMD65-1LU, lanes C, D, G, and H) were grown at 30°C in the presence of either 0.1% proline (PRO) or asparagine (ASN) as described in Materials and Methods. These data were generated with a phosphorimager and then quantitated. There was 20- and 12-fold more DAL5 mRNA in proline-grown wild-type cells and sup70-65 mutant cells, respectively, than in similar cultures provided with asparagine. The analogous values for CAR1 mRNA were 54- and 225-fold, respectively. (Lower panel) Effects of sup70-65 mutation on steady-state levels of YVH1 and DAL5 mRNA in cultures growing as described above except that the poly(A)+ fraction was used in place of total RNA. This enrichment step was added because of the very low concentrations of YVH1 mRNA that exist under all conditions.

Because SUP70 transmitted a nitrogen-starvation-induced signal, we were prompted to look at YVH1 (17, 31). YVH1 expression [poly(A)⁺ RNA] was unaffected by the sup70-65 mutation and, as expected, YVH1 expression was unaffected by nitrogen source quality. These results suggest that the SUP70 molecule functions independently or downstream of YVH1p.

We confirm the reported pseudohyphal and temperature-sensitive phenotypes of the sup70-65 mutation (30) (data not shown). We further investigated the pseudohyphal phenotype of strains LMDWLU and LMD65-1U differently from Murray et al. (30), i.e., by starving wild-type and sup70-65 mutant cells for nitrogen at 22 and 30°C. Neither wild-type nor sup70-65 mutant strains formed filaments after 120 h at 22°C on SLAHD starvation plates (16) (Fig. 3). At 30°C, the sup70-65 mutant formed pseudohyphae by 48 h on starvation medium (Fig. 3B and F). The wild type did not form pseudohyphae on starvation medium (Fig. 3) at 30°C. In other words, the mutant strain growing at 30°C was the only strain to filament and did so on both yeast extract-peptone-dextrose (data not shown) and starvation medium; the wild type did not filament at any temperature when bona fide nitrogen starvation was imposed.

View larger version (114K): [in this window] [in a new window]   FIG. 3.   The effect of nitrogen starvation on pseudohyphal growth in SUP70 and sup70-65 strains. Colonies of strain LMDWLU and LMD65-1LU that were actively growing at 22°C on a yeast extract-peptone-dextrose plate were transferred to SLAHD (plus L-leucine and uracil to provide for auxotrophic requirements) starvation plates and incubated for the indicated times at 22 or 30°C. The medium used was that described by Gimeno et al. (16) except that 2% agarose was used in place of 2% agar because its clarity yielded higher quality photographs. This change in support had no effect on the level of pseudohyphal development (data not shown). After being photographed, the plates were returned to the appropriate temperature for further incubation. (A) Strain LMDWLU after 48 h at 30°C; (B) strain LMD65-1LU after 48 h at 30°C; (C) strain LMDWLU after 120 h at 22°C; (D) strain LMD65-1LU after 120 h at 22°C; (E) strain LMDWLU after 120 h at 30°C; (F) strain LMD65-1LU after 120 h at 30°C.

	DISCUSSION

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This work demonstrates that pseudohyphal growth and NCR are genetically separable. We confirm the report of Murray et al. that pseudohyphal growth occurs gratuitously in sup70-65 mutants (30). In contrast with their results, however, we observed that the regulation of gene expression by NCR was not affected in the sup70-65 mutant, i.e., SUP70/CDC65 is not required to distinguish between high- and low-quality nitrogen sources. Although our data do not permit the conclusion that regulatory circuits controlling cellular responses to nitrogen starvation and NCR share no step in common, they do indicate that at least two steps associated with nitrogen starvation, i.e., those mediated by Yvh1p and tRNA^Gln-CUG, are not involved in the NCR regulatory pathway.

How then does one explain the fourfold increase in CAR1 mRNA observed by Murray et al. (30)? It may derive from the particular gene used to assay NCR. Although CAR1 is NCR sensitive, it is also induced by arginine (39). This is important because arginine is also a major nitrogen reserve sequestered in the cell vacuole (36). Not only is nitrogen starvation a prerequisite for pseudohyphal growth and sporulation, it also results in cell cycle arrest at G₁ (36). Cells caught outside of G₁ are more sensitive to hostile environments than are those in G₁ (19). As a defense against becoming stranded midcycle by the exhaustion of environmental nitrogen sources, S. cerevisiae cells contain a reservoir of nitrogen in their vacuoles (36). A condition such as starvation that brings about G₁ arrest also mobilizes these nitrogen reserves, induces production of the enzymes to degrade arginine and allantoin, and thereby provides a short-term supply of nitrogen for an otherwise stranded cell (24, 25, 36). If the cellular signal that distinguishes the presence of nitrogen from nitrogen starvation were destroyed by the sup70-65 mutation, it is reasonable to expect that sequestered arginine would be gratuitously mobilized when the sup70-65 mutant was shifted to 30°C. This, in turn, might account for the increased CAR1 expression observed by Murray et al. (30). Increased expression would occur, by this reasoning, not from the loss of the signal needed for NCR but as a result of internal induction mediated by the mobilized arginine. Although plausible, this explanation should be viewed cautiously until it is experimentally verified.

Although the sup70-65 mutation results in gratuitous sporulation and pseudohyphal growth, it does not induce the expression of YVH1, a gene whose transcription is specifically induced by nitrogen starvation (17, 31). These results may be interpreted in two ways, which we cannot presently distinguish. (i) The differing responses of YVH1 expression and pseudohyphal growth to yvh1 and sup70-65 mutations derive from further branching in the nitrogen starvation regulatory circuit. (ii) They are a result of the tRNA^Gln-CUG molecule being situated farther downstream than Yvh1p in the signal transduction pathway.

Since the sup70-65 mutation causes gratuitous nitrogen starvation signaling, all else being equal, one would expect wild-type and sup70-65 cells to exhibit similar phenotypes at 22°C when a bona fide nitrogen starvation signal is imposed. In contrast to that expectation, our results show that nitrogen starvation of wild-type cells at 22 or 30°C or sup70-65 cells at 22°C does not result in the level of pseudohyphal growth seen in sup70-65 cells at 30°C (Fig. 3). Stated in another way, wild-type strain LMDWLU is unable to filament during nitrogen starvation. The sup70-65 mutation confers the ability to filament at 30°C and to do so constitutively in nitrogen excess as well as starvation.

If increased CAR1 expression in sup70-65 cells at 30°C largely derives from the mobilization of sequestered arginine (25, 36), then vacuole dumping and pseudohyphal growth may be coregulated in diploid cells because both processes would be triggered by nitrogen starvation and occur when the sup70-65 mutant is shifted to 30°C on nitrogen-rich medium. Pseudohyphal growth is enhanced in cells containing an activated RAS/cAMP pathway (16). Further, intracellular amino acid levels, in general, and vacuolar levels of basic amino acids, in particular, are significantly reduced in RAS^Val19 mutant cells relative to levels in the wild type (24). The Ras/cAMP pathway is constitutively activated in this mutant (24). We suggest that inability to accumulate nitrogen reserves in the RAS^Val19 mutant may derive from vacuolar nitrogen reserves being constitutively mobilized in these cells. If so, it would indicate that cAMP enhances both pseudohyphal growth and vacuolar dumping.