The rye Mutants Identify a Role for Ssn/Srb Proteins of the RNA Polymerase II Holoenzyme During Stationary Phase Entry in Saccharomyces cerevisiae

Corresponding author: Paul K. Herman, Department of Molecular Genetics, The Ohio State University, 484 W. 12th Ave., Rm. 984, Columbus, OH 43210., herman.81{at}osu.edu (E-mail)

Communicating editor: F. WINSTON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Saccharomyces cerevisiae cells enter into a distinct resting state, known as stationary phase, in response to specific types of nutrient deprivation. We have identified a collection of mutants that exhibited a defective transcriptional response to nutrient limitation and failed to enter into a normal stationary phase. These rye mutants were isolated on the basis of defects in the regulation of YGP1 expression. In wild-type cells, YGP1 levels increased during the growth arrest caused by nutrient deprivation or inactivation of the Ras signaling pathway. In contrast, the levels of YGP1 and related genes were significantly elevated in the rye mutants during log phase growth. The rye defects were not specific to this YGP1 response as these mutants also exhibited multiple defects in stationary phase properties, including an inability to survive periods of prolonged starvation. These data indicated that the RYE genes might encode important regulators of yeast cell growth. Interestingly, three of the RYE genes encoded the Ssn/Srb proteins, Srb9p, Srb10p, and Srb11p, which are associated with the RNA polymerase II holoenzyme. Thus, the RNA polymerase II holoenzyme may be a target of the signaling pathways responsible for coordinating yeast cell growth with nutrient availability.

SACCHAROMYCES cerevisiae cell proliferation is controlled primarily by nutrient availability (PRINGLE and HARTWELL 1981 ; WERNER-WASHBURNE et al. 1993 ). Upon nutrient deprivation, yeast cells arrest division within the G₁ phase of the cell cycle and may subsequently enter into a specialized resting state known as stationary phase. The entry into this resting state is marked by a number of changes in cellular physiology that ultimately result in an elevated resistance to prolonged starvation and other environmental stresses (WERNER-WASHBURNE et al. 1993 , WERNER-WASHBURNE et al. 1996 ). Genetic and biochemical evidence suggests that stationary phase cells are not arrested within the cell cycle but instead are in a distinct, out-of-cycle phase of growth (DREBOT et al. 1987 ; WERNER-WASHBURNE et al. 1993 , WERNER-WASHBURNE et al. 1996 ; BRAUN et al. 1996 ). The overall nature of the S. cerevisiae stationary phase suggests a similarity to the G₀ phase described for mammalian cells (PARDEE 1989 ).

Many of the changes that occur to a yeast cell upon nutrient deprivation and entry into stationary phase have been well documented (reviewed in WERNER-WASHBURNE et al. 1993 , WERNER-WASHBURNE et al. 1996 ). One of the most notable is the marked decrease in the overall rate of cellular metabolism. The rate of protein translation in a stationary phase cell is 300 times less than that occurring in a dividing yeast cell (BOUCHERIE 1985 ; FUGE et al. 1994 ; WERNER-WASHBURNE et al. 1996 ). In addition, the RNA pol II-mediated transcription of most genes is greatly diminished (CHODER 1991 ). As a result, the total level of mRNA in a stationary phase cell is about 35-fold lower than the level in a log phase cell (CHODER 1991 ; CHODER and YOUNG 1993 ). Despite this general trend, several mRNAs and proteins do increase in relative abundance during stationary phase (CHODER 1991 ; WERNER-WASHBURNE et al. 1993 ; FUGE et al. 1994 ; PADILLA et al. 1998 ). These latter proteins are likely responsible for many of the properties associated with the stationary phase cell.

The best-studied example of the yeast cell response to nutrient deprivation is that which occurs following fermentative growth on glucose (LAGUNAS 1986 ). When glucose becomes limiting, the cell transiently arrests growth and then switches to a respiratory mode of energy production. This period of transition is known as the diauxic shift. During the subsequent period of respiration, the cells grow rather slowly and utilize the by-products of the previous fermentation as their primary sources of carbon. When these are finally exhausted, the cells enter the true stationary phase, when the cell number is no longer increasing (WERNER-WASHBURNE et al. 1996 ). Each of these growth transitions is accompanied by widespread changes in gene expression (WERNER-WASHBURNE et al. 1996 ; DERISI et al. 1997 ). For example, some genes are turned on transiently during the diauxic shift period, whereas others are turned on only much later, when the cells are already in stationary phase (CHODER 1991 ; WERNER-WASHBURNE et al. 1993 ; BRAUN et al. 1996 ; DERISI et al. 1997 ). The strict coordination of these expression profiles is likely important for the proper response to nutrient deprivation and hence for stationary phase entry.

Although a transcriptional response is part of the process of entering stationary phase, it is still not known how the yeast cell senses different types of nutrient limitation and how this initial signal is passed on to the transcription apparatus. In S. cerevisiae, as in other eukaryotes, RNA pol II is present as a large multi-protein complex, known as the RNA pol II holoenzyme (KIM et al. 1994 ; KOLESKE and YOUNG 1994 , KOLESKE and YOUNG 1995 ). This holoenzyme is likely to be the functional form of pol II that is actively recruited to promoters by DNA-bound trans-activators (PTASHNE and GANN 1997 ). In this model, the specificity of a transcriptional response is achieved by the accessory proteins associated with RNA pol II, which by interacting with these activators direct the polymerase to specific promoters (BARBERIS et al. 1995 ; KEAVENEY and STRUHL 1998 ; KOH et al. 1998 ; HAN et al. 1999 ). Therefore, there are two a priori targets for the nutritional signal that mediates entry into stationary phase: the trans-activators at each of the responding promoters or these regulatory proteins within the holoenzyme itself.

We are interested in understanding the genetic requirements for the entry into, and the maintenance within, stationary phase in S. cerevisiae. This report describes a set of mutants that were defective for stationary phase entry following nutrient deprivation. These rye mutants (defective for the regulation of YGP1 expression) were identified on the basis of defects in the expression pattern of YGP1, a gene normally induced upon nutrient deprivation (DESTRUELLE et al. 1994 ; RIOU et al. 1997 ). Interestingly, several of the RYE genes encoded Ssn/Srb proteins, which are associated with the RNA pol II holoenzyme. These Ssn/Srb proteins regulate gene expression by modulating the activity of RNA pol II toward a subset of yeast promoters (CARLSON 1997 ). Our data indicated that these Ssn/Srb proteins were also required for the proper entry into stationary phase. Altogether, our observations suggested that the RNA pol II holoenzyme may be a target of the signaling pathways responsible for coordinating cell growth with nutrient availability.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Growth media:
Standard Escherichia coli growth conditions and media were used throughout this study (MILLER 1972 ). Yeast rich (YPAD) and synthetic complete (SC) growth media were as described (KAISER et al. 1994 ). YM-glucose medium refers to a yeast minimal medium containing 0.67% yeast nitrogen base (Difco, Detroit), 2% glucose, and only those growth supplements required for cell proliferation. The bromcresol purple (BCP)-sucrose selection medium was as described, except that 75 µg of antimycin A₁ was top-spread onto the plates immediately before use (ROBINSON et al. 1988 ). For most stationary phase experiments, yeast cells were grown in a YM-glucose minimal medium at 30°. The cultures were typically started at a density of 0.1 OD₆₀₀ units per milliliter. Under these conditions, the cells appeared to undergo the diauxic shift after 1 day of growth and entered into stationary phase after about 4 days of growth.

Plasmid constructions:
The YGP1-SUC2 fusion plasmid, pYGP1-SUC2 (originally called pGPI354-308), containing the partial YGP1 promoter was described previously (DESTRUELLE et al. 1994 ). This reporter plasmid encodes a Ygp1p-Suc2p protein fusion that contains the entire Ygp1p open reading frame fused to amino acid 22 of the Suc2 protein. The expression of this reporter is driven by YGP1 promoter sequences present within the 350 bp immediately upstream of the Ygp1p initiation codon.

Yeast strain constructions:
The strains used in this study are listed in Table 1. Unless otherwise noted, the strains were from our lab collections or were derived during the course of this work. Standard yeast genetic methods were used throughout this study (KAISER et al. 1994 ). The strains PHY1220 and PHY1222 are derivatives of SEY6210 and SEY6211, respectively (ROBINSON et al. 1988 ). The strains PHY1025 and PHY1682 are isogenic with the W303 genetic background. PCR-mediated deletions of the SUC2 locus were performed with PHY1682 and PHY1025 to construct PHY1106 and PHY1108, respectively (BAUDIN et al. 1993 ). The cdc28-4 (PHY1348) and cdc25-1 (PHY1452) strains were constructed by backcrossing the original isolates (JT249 and JRY0890, respectively) to the W303 background five times and then transforming the resulting strains with pYGP1-SUC2.

Isolation and genetic characterization of rye mutants:
The isolation of rye mutants was carried out in two MAT strains (PHY1184 and PHY1185) and in two MATa strains (PHY1354 and PHY1355). To select for Rye^- mutants, 3 x 10⁷ cells were plated onto BCP-sucrose plates and incubated at 30° for 3–4 days. Colonies formed on these plates were streaked to BCP-sucrose medium and incubated again at 30°. The candidates that formed single colonies on this second set of plates were potential rye mutants. The recessive or dominant nature of each allele was determined by crossing each mutant back to wild type and testing the ability of the resulting diploid to grow on sucrose medium. The assignment into complementation groups was accomplished by intermating mutants of opposite mating types and testing the resulting diploids for growth on BCP-sucrose medium.

The RYE genes were cloned by plasmid complementation of the Suc⁺ growth exhibited by rye mutants. A yeast genomic DNA library constructed in the pSB32 plasmid was introduced into the appropriate rye mutant (SPENCER et al. 1990 ). Transformants were selected at 30° on YM-glucose medium lacking both uracil and leucine; the pSB32 plasmid is marked with the wild-type LEU2 gene. The colonies formed were replica plated to BCP-sucrose medium, incubated at 30°, and those colonies unable to grow on sucrose were identified. The library plasmids present in these Suc^- derivatives were isolated, clonally purified in E. coli, and then reintroduced into the rye mutants. Those plasmids that again corrected the Suc⁺ phenotype of the rye mutant were characterized further. Comparisons between the plasmid sequences and genomic databases were performed with the assistance of analysis programs available at the Saccharomyces Genome Database. Null alleles of the cloned RYE genes were constructed by a PCR-based deletion protocol described previously (BAUDIN et al. 1993 ). The rye strains exhibited a spectrum of phenotypes essentially identical to those of the rye mutants described in this article.

To ensure that the cloned RYE gene corresponded to the locus altered in the original mutant, the appropriate rye null mutant was mated with an original rye isolate. The resulting diploids were then sporulated and the meiotic progeny were characterized for both their growth on sucrose and their propensity to flocculate. At least 30 tetrads were examined for each rye mutant. All of the progeny were found to be Suc⁺ and to flocculate when grown in liquid culture. These data indicated that the cloned genes represented the genomic loci that were altered in the original rye strains.

Invertase assays:
Invertase assays were performed as described previously (JOHNSON et al. 1987 ). One unit of activity is equivalent to the release of 1 nmol of glucose per 30 min per OD₆₀₀ unit of cells. For the environmental stress experiments, PHY1184 cells were grown in YM-glucose medium lacking uracil to midlog phase and then exposed to the following condition for 60 min: 0.3 M NaCl; 7.5% ethanol; 0.4 mM H₂O₂; 0.4 M sorbitol; and incubation at 37°. Following treatment, the cells were processed and assayed for invertase activity. For the ras2-23 and cdc28-4 experiments, the cells were grown in YM-glucose minimal medium to midlog phase at 25°, shifted to 37°, and processed for invertase activity at the indicated intervals.

Assays of stationary phase characteristics:
Stationary phase viability assays were performed on cultures that were grown for 7–10 days in YM-glucose medium. Cells were collected by centrifugation and resuspended in distilled water at a concentration of 1 OD₆₀₀ unit per milliliter. The suspensions were subjected to a series of fivefold dilutions and 200 µl of each suspension was placed into a well of a microtiter plate. These suspensions were plated to YPAD media with a 48-prong replicator and incubated for 3 days at 30°. The relative number of survivors was then determined for each strain analyzed. Similar numbers of survivors were observed from wild-type cultures that had been grown for either 2 or 8 days at 30°.

The heat-shock sensitivity of stationary phase cultures was tested by placing 200-µl aliquots of the cultures into a microcentrifuge tube and incubating at 50° for 20 min. Dilutions of the cultures were plated onto rich medium before and after the heat-shock regimen. These plates were then incubated at 30° for 3 days to allow colony formation. The relative survival rate was determined by comparing the number of colonies formed by the original cultures to the number formed by the same cultures after the heat-shock treatment.

RNA analyses:
Total RNA was prepared from yeast cells by a hot phenol extraction method (AUSUBEL et al. 1995 ). Twenty micrograms of total RNA per lane was loaded onto a formaldehyde-agarose gel and subjected to electrophoretic separation. The gel was blotted to nylon membranes that were then hybridized with the appropriate ³²P-labeled probes (AUSUBEL et al. 1995 ). Typically, these probes were 0.5- to 1.0-kb PCR fragments that were prepared with the Oligolabeling kit (Amersham, Piscataway, NJ). To ensure uniform loading for the stationary phase RNA experiments, rRNA levels were monitored by visual inspection after staining the gel with ethidium bromide.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

YGP1 levels increased upon the loss of Ras pathway activity but not in response to other environmental stresses:
YGP1 expression increases upon nutrient deprivation and entry into stationary phase (DESTRUELLE et al. 1994 ; RIOU et al. 1997 ). Yeast cells starved for either a carbon, nitrogen, or phosphate source exhibit a significant increase in the levels of YGP1 mRNA. These observations suggested that the increase in YGP1 levels could be used as an indicator of the ensuing onset of stationary phase. However, most of the genes that are induced upon nutrient deprivation are not specific to stationary phase. Instead these genes are often associated with a general stress response and can be induced by a variety of conditions, including starvation, heat shock, and exposure to oxidative agents (MAGER and DE KRUIJFF 1995 ; RUIS and SCHULLER 1995 ). Therefore, we tested whether YGP1 levels were induced by a variety of stress conditions that were each capable of eliciting a general stress response.

We found that none of the stress conditions tested, except nutrient deprivation, caused a significant induction in YGP1 levels (Table 2). Each of these conditions triggered the general stress response as the levels of either CTT1 mRNA or a stress-responsive lacZ reporter increased by a factor of 6- to 32-fold in the treated cells (data not shown; see MARCHLER et al. 1993 ). CTT1 encodes a cytoplasmic catalase whose expression is induced by a variety of different stress conditions (SCHULLER et al. 1994 ; MARTINEZ-PASTOR et al. 1996 ). For these experiments, we used a YGP1-SUC2 gene fusion as a reporter of YGP1 expression (DESTRUELLE et al. 1994 ). This reporter is expressed at a very low level during log phase growth and is greatly induced upon entry into stationary phase (Table 2; see DESTRUELLE et al. 1994 ). In our experiments, this induction was >150-fold in response to glucose deprivation. We also found that the induction of YGP1-SUC2 did not require MSN2 or MSN4 gene function (data not shown). The MSN2 and MSN4 genes encode transcription factors that are responsible for a significant portion of the S. cerevisiae transcriptional response to environmental stress (MARCHLER et al. 1993 ; SCHMITT and MCENTEE 1996 ). Thus, the induction of the YGP1-SUC2 reporter was not a general response to environmental stress, but rather was a more specific consequence of stationary phase entry.

The growth rate of yeast cells decreases significantly when an essential nutrient becomes limiting. Therefore, the induction of YGP1 could be a response either to the nutrient deprivation or to the diminished growth rate of the cell. To distinguish between these possibilities, we analyzed YGP1 expression in Ras pathway mutants that arrest growth even in the presence of a nutrient-rich growth medium (reviewed in BROACH 1991 ; THEVELEIN 1994 ). Three mutants that exhibit a temperature-sensitive defect in Ras signaling activity, ras2-23 ras1, cdc25-1, and cyr1-230, were used in this experiment (MATSUMOTO et al. 1985 ; BROEK et al. 1987 ; MITSUZAWA et al. 1989 ). In each case, thermal inactivation of the Ras pathway resulted in a >200-fold increase in the levels of YGP1-SUC2 invertase activity (Fig 1). In contrast, a cdc28-4 mutant that causes a G₁-specific arrest did not lead to a significant increase in YGP1-SUC2 levels (Fig 1). In each of these experiments, the cell cycle block was successfully imposed as >90% of the cells arrested with an unbudded morphology. These data indicated that the stationary phase induction of YGP1 was not caused by the nutrient deprivation per se but was instead a consequence of the accompanying G₀-like growth arrest.

View larger version (23K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Decreased Ras pathway activity resulted in an induction of YGP1-SUC2 expression. Wild-type (PHY2161), ras2-23 (PHY1340), cdc25-1 (PHY1452), and cdc28-4 (PHY1348) cells were grown to midlog phase at 25° in minimal medium and then shifted to 37°. Cells were harvested at the indicated times following the temperature shift and processed for invertase activity as described in MATERIALS AND METHODS. The standard errors were between 10 and 15%. Each value represents the average of three independent experiments.

Isolation of the rye mutants:
The above data suggested that the induction of YGP1 could be used as an indicator for the ensuing entry into stationary phase. Therefore, we set up a genetic selection to identify mutants that exhibited defects in the regulation of YGP1 expression. Specifically, this selection uncovered mutants that expressed the YGP1-SUC2 reporter at elevated levels during the log phase of growth. The underlying rationale was that these defects in YGP1 expression might be harbingers of more global defects in the transcriptional response to nutrient deprivation. Hence, these mutants could identify genes important for stationary phase entry and perhaps more global aspects of growth control, as well.

The key feature of this YGP1-SUC2 reporter was its low expression during the log phase of growth (Table 2). SUC2 encodes an enzyme, invertase, that is required for S. cerevisiae growth on sucrose (JOHNSTON and CARLSON 1992 ). As a result, wild-type cells that contained only this fusion version of SUC2 were unable to grow on sucrose media (see Fig 2). The rye mutants were isolated by plating such suc2 strains, carrying the YGP1-SUC2 reporter, onto a sucrose medium and selecting for mutants that could form colonies. For all rye mutants, the growth on sucrose was dependent upon the presence of the YGP1-SUC2 fusion. Of 48 independent rye mutants isolated, 41 contained recessive mutations (Table 3). By intermating the recessive mutants, we found that they defined eight different complementation groups and that almost half of all the mutants were members of the rye1 group (Table 3). One of the dominant mutants defined a separate gene that was designated RYE9. The remainder of this article will concentrate upon the five complementation groups, rye1 to rye5, that had multiple members.

View larger version (40K): [in this window] [in a new window] [Download PPT slide]   Figure 2. The RYE2, RYE3, and RYE5 genes encode Ssn/Srb components of the RNA polymerase II holoenzyme. (A) The rye mutations were complemented by the presence of the appropriate SSN/SRB gene. Yeast strains were grown with the indicated plasmids for 2–3 days at 30° on either YPAD (Glc) or BCP-sucrose (Suc) media. The strains used were wild type (PHY1220), rye2 (PHY1378), rye3 (PHY1382), and rye5 (PHY1397). (B) Null alleles of the SSN/SRB genes resulted in a Rye- phenotype. The following strains were grown for 3 days at 30° on either YPAD (Glc) or BCP-sucrose (Suc) media: wild type (PHY1220), srb11 (PHY1616), srb9 (PHY1606), and srb10 (PHY1615).

Three RYE genes encode Ssn/Srb components of the RNA pol II holoenzyme:
Mutants within the rye2, rye3, and rye5 complementation groups shared an interesting set of phenotypes that distinguished these mutants from those in the other groups (see below). These data suggested that the products of the RYE2, RYE3, and RYE5 genes might act at a similar step in the regulation of stationary phase biology. These three RYE genes were cloned and each was found to encode a protein associated with a subcomplex of the RNA polymerase II holoenzyme. RYE2 was identical to SSN8/SRB11, RYE3 to SSN2/SRB9, and RYE5 to SSN3/SRB10 (Fig 2A). The three Rye proteins, Rye2p/Srb11p, Rye3p/Srb9p, and Rye5p/Srb10p, together with a fourth protein, Srb8p, constitute the functional Ssn/Srb complex (CARLSON 1997 ; MYER and YOUNG 1998 ). This Ssn/Srb complex appears to function primarily as a negative regulator of RNA pol II transcription at a subset of yeast promoters (CARLSON 1997 ; HENGARTNER et al. 1998 ; HOLSTEGE et al. 1998 ). We constructed null alleles of these three RYE loci and found that each null mutant was viable and exhibited a Rye^- phenotype (Fig 2B). These data were consistent with previous reports indicating that these genes were not essential for yeast cell viability (SUROSKY et al. 1994 ; HENGARTNER et al. 1995 ; KUCHIN et al. 1995 ; LIAO et al. 1995 ; SONG et al. 1996 ). For the remainder of this article, we will refer to these genes by both their RYE and SRB designations but future communications will use only the more-established SRB names.

The rye and srb mutants exhibited defects in the regulation of YGP1:
The ability of the rye mutants to grow on sucrose-containing media suggested that these mutants expressed the YGP1-SUC2 reporter at elevated levels during log phase growth. Indeed, the rye mutants had 13- to 57-fold higher levels of invertase activity than wild-type cells during this growth phase (Table 4). In general, the relative rate of growth on sucrose media was directly proportional to the level of invertase produced from the fusion construct. Previous studies have indicated that the expression of YGP1 is controlled at the level of mRNA production (DESTRUELLE et al. 1994 ). Our data were consistent with this observation as log phase cultures of the rye mutants possessed elevated levels of the YGP1-SUC2 mRNA (Fig 3A). The rye mutations also affected the endogenous YGP1 gene as expression from this promoter was also elevated in log phase cultures of the rye mutants (data not shown). Therefore, the RYE genes were required for the proper regulation of YGP1 gene expression.

View larger version (70K): [in this window] [in a new window] [Download PPT slide]   Figure 3. mRNA levels in the rye and srb mutants. (A) YGP1-SUC2, CTT1, HSP12, and HSP26 mRNA levels are elevated during log phase in the rye and srb mutants. The steady-state levels of YGP1-SUC2, CTT1, HSP12, HSP26, and ACT1 mRNA in midlog cultures of the indicated strains were measured by Northern RNA blot analysis. Twenty micrograms of total RNA were loaded for each sample. (B) The rye and srb mutants exhibited defects in the stationary phase repression of ACT1. The steady-state level of ACT1 mRNA in stationary phase cultures of the indicated strains was measured by Northern blot analysis. Total RNA was prepared from cultures grown for 6 days at 30° in minimal medium. Twenty micrograms of total RNA were loaded for each sample. As a control for loading, the rRNA levels were assessed by visual inspection after staining the gel with ethidium bromide. The strains analyzed were as listed in the legend to Table 4; the relevant genotype is shown.

The above observations suggested that the rye mutants might be defective in a transcriptional response to nutrient deprivation and/or growth arrest. Therefore, we examined the expression of three additional genes, HSP12, HSP26, and CTT1, that are controlled, in part, by the growth status of the cell. In wild-type cells, these genes are expressed at very low levels during exponential growth but are induced severalfold during the diauxic shift (PETKO and LINDQUIST 1986 ; PRAEKELT and MEACOCK 1990 ; DERISI et al. 1997 ). The mRNA level of each of these genes was elevated in log phase cultures of most of the rye mutants tested (Fig 3A). The rye2/srb11 and rye5/srb10 mutants exhibited the highest level of derepression with levels four- to sevenfold higher than those seen in wild-type cells. The rye1 mutations generally resulted in a three- to fivefold increase in CTT1 and HSP12 levels but had only a very modest effect upon HSP26. Thus, the rye mutant defects were not specific to YGP1 and instead represented a more general defect in growth control.

The growth characteristics of the rye mutants were further analyzed under a variety of conditions. The rye mutants exhibited relatively normal growth rates at 25° and 30° on media containing glucose, galactose, or a nonfermentable carbon source. However, several of the rye strains exhibited a slow growth rate at 38° (Table 3). In addition, rye1 and RYE9-1 mutants and, to a lesser degree, rye4 grew poorly on media lacking inositol (Table 3). Many of the rye mutants also displayed a propensity to flocculate in liquid culture (Table 3). Finally, many of the rye mutants exhibited abnormal cellular morphologies during exponential growth. This was most severe in the rye1 strains where the cells were often highly elongated and sometimes contained multiple buds. These mitotic defects could either indicate a mitotic role for the RYE gene products or be a secondary consequence of the mitotic expression of genes normally required during periods of growth arrest.

The rye and srb mutants failed to enter into a normal stationary phase in response to nutrient deprivation:
The entry into stationary phase appears to involve a tightly regulated program of changes in gene expression (CHODER 1991 ; WERNER-WASHBURNE et al. 1993 , WERNER-WASHBURNE et al. 1996 ). Disruption of this program therefore could result in a failure to enter into a normal stationary phase. The above data indicated that the rye mutants did indeed exhibit defects in the regulation of several genes that are induced during the diauxic shift or upon entry into stationary phase. Therefore, we asked whether the rye mutants would show defects in other responses normally associated with stationary phase entry.

Upon stationary phase entry, the level of most mRNAs decreases dramatically (CHODER 1991 ). This repression phenomenon has been best characterized for the ACT1 locus and thus we measured the levels of ACT1 mRNA in the rye mutants at different times following nutrient deprivation. In wild-type cells, the level of ACT1 mRNA dropped precipitously after 2 days of growth in minimal medium (data not shown). This observation was similar to that made in other studies of stationary phase gene expression (CHODER 1991 ; CHODER and YOUNG 1993 ). In contrast, the levels of ACT1 mRNA remained significantly elevated after 6 days of growth in all of the rye mutants tested except rye4 (Fig 3B). Therefore, the rye mutants did not exhibit a normal transcriptional response to this nutrient limitation.

One of the hallmarks of stationary phase cells is the ability to survive prolonged periods of starvation (WERNER-WASHBURNE et al. 1993 ). Mutants that cannot enter into this resting state rapidly lose viability upon nutrient limitation (CANNON and TATCHELL 1987 ; TODA et al. 1987 ; WERNER-WASHBURNE et al. 1993 ). Therefore, we assessed the relative number of survivors in stationary phase cultures of wild-type and rye mutant strains. As expected, we found that wild-type cells remained viable after 8 days of growth in minimal medium (Fig 4). In contrast, the rye mutants rapidly lost viability following nutrient deprivation. After 8 days of growth, the rye1, srb9, srb10, and srb11 strains all exhibited a survival rate at least 200-fold lower than that seen with the isogenic wild type (Fig 4). The viability defect associated with rye4 mutants was less severe. In general, the rye4 cultures had 5- to 7-fold fewer survivors than the wild-type control. Therefore, the rye mutants rapidly lost viability upon nutrient limitation and hence failed to assume one of the most basic properties normally associated with stationary phase cells.

View larger version (86K): [in this window] [in a new window] [Download PPT slide]   Figure 4. The rye and srb mutants exhibited decreased viability following an extended period of nutrient deprivation. Wild-type and the indicated mutant strains were grown at 30° for 8 days in YM-glucose minimal medium. Cells from these cultures were collected by centrifugation, washed, and resuspended in distilled water at a concentration of 1 OD600 unit per milliliter. Fivefold serial dilutions of these suspensions were then plated to YPAD medium and incubated for 3 days at 30°. The number of colonies formed was a measure of the number of survivors in the original cultures. The strains analyzed were as listed in the legend to Table 4; the relevant genotype is shown.

Wild-type stationary phase cells also exhibit an elevated resistance to heat-shock regimens that would kill log phase cells (WERNER-WASHBURNE et al. 1993 ). Therefore, the heat-shock sensitivity of rye cultures grown for 4 days at 30° in minimal medium was tested. At this time, the stationary phase viability defect associated with the rye cultures was not as severe as that observed after 8 days. Nonetheless, we found that all of the rye mutants were much more sensitive than wild-type cells to a 20-min incubation at 50° (Fig 5). The increase in sensitivity varied from 7-fold with rye1 mutants to 50-fold with rye4. Therefore, these data provided independent evidence that the rye mutants were unable to enter into a normal stationary phase.

View larger version (27K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Stationary phase cultures of rye and srb mutants were sensitive to a brief heat shock. The indicated strains were grown at 30° for 4 days in YM-glucose minimal medium and then subjected to a 50° heat shock for 20 min. The percentage of cells surviving this treatment is shown. The strains analyzed were as listed in the legend to Table 4; the relevant genotype is shown. The RAS2val19 control is the wild-type strain (PHY1220) carrying a plasmid with the dominant RAS2val19 allele. The standard errors were typically <10%. Each value represents the average of three independent experiments.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The RYE genes are required for stationary phase entry:
We are interested in understanding the mechanisms regulating cell growth and stationary phase biology in S. cerevisiae. In this study, we identified a collection of mutants that were unable to enter into a normal stationary phase. These stationary phase defects were likely due to a defective transcriptional response to nutrient limitation. In wild-type cells, nutrient deprivation elicits an orderly series of changes in gene expression that culminates in the proper entry into stationary phase (CHODER 1991 ; DERISI et al. 1997 ). The rye mutations identified in this study disrupted this transcriptional program; genes that were normally expressed only upon nutrient limitation were instead expressed constitutively during log phase growth. As a result, the cells were unable to undergo a normal growth arrest and hence failed to assume many of the characteristics normally associated with stationary phase cells. Altogether, these data indicated that the RYE genes were required for the proper entry into the stationary phase of growth.

Ssn/Srb proteins of the RNA pol II holoenzyme are required for stationary phase entry:
This study established that at least three components of a particular complex within the RNA pol II holoenzyme play a role in stationary phase entry. This complex is made up of four proteins, Rye2p/Srb11p, Rye3p/Srb9p, Rye5p/Srb10p, and Srb8p, that appear to function primarily as negative regulators of RNA pol II transcription (CARLSON 1997 ; MYER and YOUNG 1998 ). Moreover, a recent study of gene expression in a srb10 null mutant has suggested a role for this Ssn/Srb complex in the transcriptional response to nutrient deprivation (HOLSTEGE et al. 1998 ). The primary defect in the srb10 strain was the elevated log phase expression of many genes, including YGP1, that are normally expressed during nutrient deprivation. Our data were consistent with these observations and extended them by showing that Srb10p was required for the proper entry into stationary phase. When faced with nutrient deprivation, our rye5/srb10 mutants failed to attain many of the properties normally associated with stationary phase cells. In addition, our results showed that other members of the Srb10p subcomplex, in particular Srb9p and Srb11p, were also required for the normal transcriptional response to nutrient limitation and for stationary phase entry.

One interesting possibility is that this Ssn/Srb complex serves as a target for those signal transduction pathways that control cell growth in response to environmental stimuli (CARLSON 1997 ; HOLSTEGE et al. 1998 ; COOPER et al. 1999 ). In such a model, nutrient deprivation would generate a signal that would ultimately lead to the inactivation of this Ssn/Srb complex, resulting in the increased expression of those genes required during this period of nutrient limitation. A possible mechanism for this inactivation has already been suggested by previous studies of this regulatory complex. These studies have shown that Srb10p and Srb11p are rapidly degraded in response to glucose deprivation and, in the case of Srb11p, several other stress conditions (COOPER et al. 1997 , COOPER et al. 1999 ; HOLSTEGE et al. 1998 ). However, it is not yet clear what signals are being generated by nutrient deprivation and what signal transduction pathway is subsequently responsible for triggering the degradative response. Perhaps the additional rye mutants will provide some insights into the mechanisms regulating this Ssn/Srb complex.

The RYE genes and the control of yeast cell growth:
Although we have been specifically discussing the Ssn/Srb complex as an effector of environmental signals on gene expression, it is interesting to consider whether such a regulatory mechanism might be applied more generally in eukaryotic cells. Many of the accessory proteins within the RNA pol II holoenzyme have been shown to interact with specific subsets of DNA-bound transcriptional regulators (BARBERIS et al. 1995 ; PTASHNE and GANN 1997 ; KEAVENEY and STRUHL 1998 ; KOH et al. 1998 ; HAN et al. 1999 ). Therefore, individual holoenzyme proteins may be responsible for the transcription from distinct sets of promoters (HAMPSEY and REINBERG 1999 ; HAN et al. 1999 ). By targeting these holoenzyme proteins directly, the cell would be able to control transcription from multiple promoters in a single step. Such a regulatory mechanism could allow the cell to bring about rather global changes in the patterns of transcription in a very rapid and precise manner. This type of a regulatory approach would be very useful in those instances when cells undergo dramatic changes in their overall physiology, such as during the entry into a G₀-like resting state.

Despite the significant advances made in recent years, many of the events that lead to stationary phase entry remain poorly understood. For example, although the Ras pathway is clearly an important regulator of stationary phase, the precise targets of this pathway relevant for growth control have remained elusive (BROACH 1991 ; WERNER-WASHBURNE et al. 1993 ; THEVELEIN 1994 ; HERMAN and RINE 1997 ; REINDERS et al. 1998 ). These difficulties could be due to factors such as the essential nature of the Ras proteins and overlapping functions among the targets of this pathway. The rye selection described here might be able to overcome such complications by allowing for the identification of mutants with only modest defects in Ras signaling (our unpublished data). Therefore, the other RYE genes identified in this study are of great interest as they may shed additional light on the signaling pathways regulating stationary phase biology.

	ACKNOWLEDGMENTS

We thank Drs. D. Balciunas, S. Emr, F. Estruch, D. Klionsky, H. Mitsuzawa, and J. Thorner for providing strains and plasmids used in this study. This study was supported by grants from the American Cancer Society, the Ohio Cancer Research Associates, and The Ohio State University Seed Grant program (to P.K.H.), and the National Institutes of Health (GM 35827 to J.R.). During the early stages of this work, P.K.H. was supported by a Special Fellow Award from the Leukemia Society of America.

Manuscript received June 26, 2000; Accepted for publication September 12, 2000.

	LITERATURE CITED

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