Abstract
Ume6p is a non-essential transcription factor that represses meiotic gene expression during vegetative growth in budding yeast. To relieve this repression, Ume6p is destroyed as cells enter meiosis and is not re-synthesized until spore wall assembly. The present study reveals that spores derived from a ume6 null homozygous diploid fail to germinate. In addition, mutant spores from a UME6/ume6 heterozygote exhibited reduced germination efficiency compared to their wild-type sister spores. Analysis of ume6 spore colonies that did germinate revealed that the majority of cells in microcolonies following the first few cell divisions were inviable. As the colony developed, the viability percentage increased and achieved wild-type levels within approximately six cell divisions indicating that the requirement for Ume6p in cell viability is transient. This function is specific for germinating spores as Ume6p has no or only a modest impact on the return to growth ability of cells arrested at other points in the cell cycle. These results define a new role for Ume6p in spore germination and the first few subsequent mitotic cell divisions.
Keywords: sporulation, transcriptional repressor, cell cycle
Introduction
When deprived of nitrogen and a fermentable carbon source, the budding yeast Saccharomyces cerevisiae will withdraw from the cell cycle and initiate meiosis and spore morphogenesis (Honigberg, et al., 1993). The spores remain quiescent until exposed to the appropriate growth signals at which time they germinate and reenter the mitotic cell cycle. However, spores evaluate growth signals differently than do mitotically dividing cells. Germination and subsequent cell division will occur optimally in the presence of specific growth stimuli such as glucose and ammonia. Other carbon sources that normally promote cell division in vegetative cells (e.g., lactose, pyruvate, ethanol) will not support germination or do so at lower efficiencies (Palleroni, 1961, Sussman & Halvorson, 1966, Rousseau & Halvorson, 1973, Donnini, et al., 1986). These results suggest the existence of a separate (or modified) regulatory system directing the decision of a resting spore to reenter cell division than the one controlling a vegetative cell.
Currently, few details are known concerning the molecular mechanisms governing spore quiescence or germination. Several studies have suggested that germination and return to growth is a complex, multi-step process (Herman & Rine, 1997). For example, the growth arrest in yeast ascospores does not appear to be simply the response to the lack of nutrients that induced sporulation (Miller, 1989). The resting spore contains ample sources of carbon (trehalose, glycogen) and nitrogen (free amino acid pools) that can maintain vegetative cell growth but will not stimulate germination. Initially, germination is marked by the breakdown of the specialized spore wall and storage carbohydrates (reviewed in (Thevelein, 1984). Initially, translation was thought to occur prior to mRNA synthesis (Rousseau & Halvorson, 1973). However, more recent studies have found a robust transcription program that includes both mRNA synthesis (Joseph-Strauss, et al., 2007) and degradation (Brengues, et al., 2002). These studies revealed that spore germination is a dynamic process involving the activity of transcriptional activators, repressors and the mRNA decay machinery.
Ume6p is a transcription factor that represses early meiotic genes (EMG) required for meiotic S phase and recombination (Bowdish & Mitchell, 1993, Strich, et al., 1994). Although Ume6p is not required for mitotic cell division, it is required for meiosis with 95% of null mutants arresting prior to the first meiotic division (Strich, et al., 1994, Steber & Esposito, 1995). UME6 mRNA levels do not vary during meiosis and spore formation (Strich, et al., 1994, Primig, et al., 2000). However, to relieve EMG repression, Ume6p is destroyed as cells enter the meiotic program via ubiquitin-mediated proteolysis (Mallory, et al., 2007). Ume6p levels remain below the limits of detection during meiosis but return to mitotic levels during spore wall assembly. This report identifies a role for Ume6p in spore germination and the first few divisions following germination that is not present in established mitotic cultures.
Materials and methods
Yeast strains, media and plasmids
Yeast strains used in this study are listed in Table 1. Yeast strains were grown and induced to enter meiosis using solid and liquid medium as previously described (Cooper, et al., 2000). The ume6-5 (ume6::LEU2) and ume6Δ (ume6::URA3) mutant alleles were previously described (Strich, et al., 1994, Mallory, et al., 2007). Both ume6Δ and ume6-5 mutant strains exhibited identical phenotypes with respect to EMG repression and sporulation efficiency. To place UME6 under the control of the CLB2 promoter, a one Kbp BglII-PacI fragment containing the CLB2 promoter was inserted into the same sites of pFA6a-TRP1-pGAL (Longtine, et al., 1998) replacing the GAL1 promoter. Oligonucleotides flanking the UME6 promoter were employed to amplify the CLB2 promoter and the resulting fragment used to replace the UME6 promoter using one-step transplacement. The correct insertion was verified by sequencing the amplified genomic sequence.
Table 1.
Yeast Strains.
JX192* | MATa/MATα can1-100 his4-519 leu2-3, 112 lys2-1 trp1-1 ura3-1 | This study |
JX193* | MATa/MATα can1-10 his4-519 leu2-3, 11 lys2-1 trp1-1 ura3-1 UME6/ume6-5 | This study |
RSY194* | MATa/MATα can1-100 his4-519 leu2-3, 112 lys2-1 trp1-1 ura3 ume6-5 | This study |
JX225* | MATa/MATα ade2/ADE2 ADE6/ade6 can1-100/CAN1 his3-11, 15 leu2-3, 112 lys2-1/LYS2 trp1-1 ura3 ume6Δ:URA3/UME6 | This study |
RSY269 | MATαcan1-100 his4-519 leu2-3,11 trp1-1 ura3-1 | This study |
RSY270 | MATa can1-100 his4-519 leu2-3,11 trp1-1 ura3 ume6-5 | This study |
RSY271 | MATa can1-100 his4-519 leu2-3,11 trp1-1 ura3-1 | This study |
RSY280 | MATα can1-100 his4-519 leu2-3,11 trp1-1 ura3 ume6-5 | This study |
JX414* | MATa/MATα ho::LYS2 lys2-1 ura3 ume6Δ::URA3/UME6 | This study |
RSY291 | MATa ade2 ade6 can1-100 his3-11,15 leu2-3,11 trp1-1 ura3-1 ume6::LEU2 | This study |
RSY1295 | MATα ade6 can1-100 his4-519 leu2-3,11 trp1-1 ura3 CLB2pro-UME6::TRP1 | This study |
Alleles are homozygous in diploid strains unless stated otherwise.
Micro-colony viability studies
Tetrads were dissected onto rich medium and incubated for 24 hr at 30°. The number of divisions the spore had accomplished was then determined by counting the number of cells in the microcolony (assuming exponential growth). For microcolonies with >20 cells, the number of generations was estimated. To determine the viability of individual cells within the microcolony, at least eight cells were manipulated away from the colony on the same plate. Following another 24 hr incubation, the ability of the micromanipulated cells to continue cell division was determined microscopically. The genotype of each spore that formed a macrocolony was determined by replica plating onto medium selecting for the presence of the ume6-5 allele.
Return to growth assays
For nutritionally arrested cells, three independent isolates of RSY270 and RSY271 were maintained at stationary phase in liquid culture for three days at 30°. Cells were harvested by centrifugation, washed in water, lightly sonicated to disrupt clumps and counted using a hemocytometer. At least 300 cells from each isolate were plated in triplicate on rich medium and incubated at 30°. Late G1 arrested cells were obtained using α-factor arrest/release protocols (Rita, et al., 1991). Three independent log-phase cultures of RSY270 and RSY271 were treated with 15 μM α-factor (Sigma) for three hours at which time >90% of the cells exhibited the characteristic “shmoo” morphology. Cells were counted and plated on rich agar plates and incubated at 30°. G2 arrest was accomplished by incubating log phase cultures with 10 μM benomyl (DuPont) for three hours at 30° (Guacci, et al., 1997). Growth arrest was monitored both microscopically (>90% of the population containing large buds) and by optical density (650 nm). Cells were treated as described for the G1 arrest above. For nitrogen starvation studies, log phase cultures (in triplicate) were grown to mid-log phase in complete minimal medium. The cells were harvested, washed in sterile water, then resuspended in minimal medium lacking nitrogen or amino acids and timepoints taken every two hrs for 12 hrs. The samples were lightly sonicated as before then serially diluted (1:10) and spotted onto rich medium. The plates were incubated for two days at 30°.
Results
Ume6p is required for efficient spore colony formation
Ume6p negative regulates genes (e.g., IME2, SPO13) involved in early meiotic processes (Bowdish & Mitchell, 1993, Strich, et al., 1994). Ume6p-dependent repression is removed when it is destroyed as cells enter meiosis (Mallory, et al., 2007). Although Ume6p levels remained below the limits of detection during the meiotic nuclear divisions, they returned to vegetative concentrations as the cells formed spores. These observations suggested a possible role for Ume6p late in the sporulation process. To address this question, a homozygous diploid strain (JX194) harboring an insertion allele (ume6-5) for both copies of UME6 was sporulated on solid medium. As previously described (Strich, et al., 1994), the sporulation efficiency of the mutant diploid was significantly reduced compared to wild type (Table 2). Of the few tetrads obtained, the outer spore wall and DAPI staining nuclei appeared normal compared to wild type (data not shown). However, dissection analysis revealed that none of the spores derived from twenty ume6-5 tetrads were able to form colonies (Table 2). Microscopic examination revealed that the spores remained as single cells (data not shown). As Ume6p is not required for normal mitotic cell division (Strich, et al., 1994), these results suggest that either Ume6p is required for spore germination or that the spores fail to germinate due to aberrant meioses.
Table 2.
Requirement of Ume6p for spore colony formation.
Strain | % Ascia | Tetrads Dissected | % CFSb Total | % CFSc UME6 | % CFSc ume6 |
---|---|---|---|---|---|
JX192 UME6/UME6 |
79 | 10 | 100 | 100 | NA |
JX193 UME6/ume6-5 |
83 | 43 | 67 | 97 | 38 |
JX194 ume6-5/ume6-5 |
4.5 | 20 | 0 | NA | 0 |
JX414 (SK1) UME6/ume6Δ |
92 | 27 | 39 | 83 | 17 |
JX225 (W303) UME6/ume6Δ |
47 | 20 | 60 | 75 | 45 |
% asci was calculated as the sum of two-, three- and four-spored cells in the sporulated culture divided by the total cells counted (n = >200).
% colony forming spores (CFS) was calculated by dividing the number of spore colonies observed after a five day incubation by the number of spores dissected.
% colony forming spores for UME6 and ume6 mutant spores as determined by the presence of the ume6 disruption marker LEU2 or URA3. For spores that did not form colonies, genotypic designations were assigned assuming 2:2 segregation of the UME6 locus. Numbers are normalized to 100% of each genotype (UME6 or ume6) expected from each tetrad. NA = not applicable.
To address this question, the experiments were repeated with a UME6/ume6-5 heterozygote. The rationale behind this experiment is that if Ume6p levels return only at the onset of spore wall assembly, spores harboring the ume6-5 allele may possess reduced amounts of Ume6p. Tetrad analysis of the UME6/ume6-5 diploid (JX193) revealed a 33% reduction in colony formation ability of the resulting spores compared to the wild-type control (Table 2, Figure 1). Further examination revealed that less than half of the spores carrying the ume6-5 allele were able to form colonies. In contrast, the colony forming ability of the UME6 spores derived from the heterozygote were similar to that observed for spores from the homozygous wild type strain (97% vs. 100%). To control for possible strain variations, these experiments were repeated in the W303 (JX225) and SK1 (JX414) backgrounds. These studies revealed variations in ume6Δ spore viability with respect to these strains. Mutant ume6Δ spores derived from the W303 background were more likely to form colonies (45%) than from the SK1 strain (17%). However, mutant spores were still less likely to form colonies than their wild-type sister spores. These results indicate that there are strain differences with respect to the viability of ume6Δ spores. Finally, to test whether the spore colony forming ability of ume6 mutant spores was due to the presence of an extragenic suppressor, three ume6-5 spore colonies were mated to a wild-type strain and the resulting diploids sporulated. Tetrad dissection analysis revealed a similar loss in colony forming ability in the ume6-5 spores as observed previously (data not shown) indicating that ume6-5 spore colony forming ability is not due to a second site suppressor. These results indicate that Ume6p is required for normal colony forming ability of yeast spores. Furthermore, these results indicate that the viability defect associated with the ume6 allele is spore autonomous i.e., the viability of the wild-type spores in the same ascus was not affected.
Fig. 1.
Ume6p is required for spore colony formation. (a) Tetrad dissection of the JX193 diploid (UME6/ume6-5) following three day incubation at 30°. The panel on the right indicates wild type (+) and ume6-5 (−) mutants. Solid boxes indicate no colony formation. The tetrads (numbers) and spores (letters) are indicated. (b) Growth rates of cultures derived from UME6 spore clones 6C (JX269), 6D (JX271) and ume6-5 spore clones 6A (RSY270), 6B (RSY280).
Ume6p is required for normal spore colony development
To further investigate the role of UME6 in spore colony formation, the growth characteristics of wild type and mutant spores were compared. The UME6 spores derived from a UME6/ume6-5 heterozygote produced colonies of consistent size and shape following a two day incubation (Fig. 1A). However, ume6-5 spore colonies were of variable size. Some asci produced wild type and mutant colonies of similar size (e.g., tetrad 14). However, most of the ume6-5 spore colonies were smaller than their wild-type sisters (e.g., tetrads 6 and 20). This reduced colony size may be explained if the individual ume6-5 mutants exhibited a slower growth rate. A previous study found that ume6 mutants in a similar strain background grew approximately 10% slower compared to wild type (Strich, et al., 1994). To test this possibility, the spore colonies from tetrad #6 were picked, colony purified and inoculated into liquid rich medium. The analysis of the growth rates for these cultures revealed generation times for the ume6-5 strains of 2.4 and 3.2 hrs for 6A and 6B, respectively (Fig. 1B). The wild-type control cultures (6C and 6D) doubled every 2.4 and 2.7 hrs. These results suggest that the differences observed in spore colony size are probably not due to a variation in the growth rates among individual spore colonies.
Ume6p does not regulate bud emergence kinetics in spores
The analysis described above indicated that growth rates were not responsible for the disparity in ume6 spore colony size. An alternative possibility is that ume6-5 spores germinate and reinitiate mitotic cell division slower than wild-type spores. This model would predict that colony size would be a function of the appearance of the first bud. To examine the return to growth kinetics of ume6-5 and UME6 spores, eleven tetrads derived from JX193 were dissected on rich medium and the spores followed microscopically to detect the appearance of the first and second bud. No buds were observed for the first three hrs. By four hrs, buds were observed and scored. The percentage of the spores that formed their first bud at each timepoint was calculated from the total number of spores monitored. The genotypes (wild type or ume6-5) of the resulting colonies were determined by replica plating to leucine drop out medium. For spores that did not grow into colonies, their genotype was designated assuming a 2:2 segregation pattern. No significant difference was observed in the appearance of the first bud (Fig. 2A) or the second bud (data not shown) between wild type and mutant. The significance of the oscillation observed in bud emergence for both spore genotypes is not known. Next, the relationship between bud emergence and colony size were directly examined. Eight tetrads derived from JX193 were dissected and the germination status of the spore was examined microscopically 8 hrs later. As expected, no distinction between the germination kinetics of wild type and ume6-5 spores was noted (Fig. 2B, bottom panel). The plate was incubated an additional two days then photographed and the ume6-5 spores were identified by the presence of the LEU2 marker. Although no difference was observed in germination kinetics, ume6-5 spores still produced smaller colonies compared to wild type (Fig. 2B, top panel). Taken together, these results indicate that the small colony phenotype observed with the ume6-5 mutation is not due to delayed germination.
Fig. 2.
Bud emergence kinetics for UME6 and ume6-5 spores. (a) Wild type and ume6-5 mutant spores were monitored microscopically following dissection to determine when the first bud appeared for each spore. The percentage of the population that produced their initial bud for a given timepoint is plotted. For UME6, n=22, ume6-5, n=19. (b) Germination kinetics and colony size. The germination status of spores dissected from eight tetrads was examined microscopically 14 hrs following dissection. The number in each box indicates the number of cells found at this timepoint. "B" indicates the presence of a bud. Open boxes indicate UME6 spores, shaded indicate a ume6-5 spores, solid boxes indicate spores that did not form colonies.
Ume6p is required to maintain mitotic cell division following spore germination
The analysis of ume6-5 spores that could form colonies did not provide an explanation for the variation in mutant spore viability or the variation in colony size. Therefore, the studies were then focused on the ume6-5 spores that did not form macroscopic colonies. Over half of ume6-5 spores from JX193 failed to form a colony (Table 2). Microscopic examination revealed that 76% of these mutant spores had executed approximately one to four rounds of cell division (as inferred by the estimated number of cells in the microcolony) but failed to continue dividing. This observation was paradoxical to the previous finding that UME6 is a non-essential gene (Strich, et al., 1994). Specifically, it was not clear why cells that were able to undergo one round of cell division could not continue to form a colony. One possibility was that Ume6p performed a function during the re-entry into mitotic cell division that was not required for established logarithmic growth. To test this possibility, the heterozygote JX193 was again dissected onto rich medium. Following 24 hrs, eight cells from UME6 and ume6-5 microcolonies that had undergone approximately 2, 3, 6 and 9 generations (as calculated by the number of cells in a microcolony assuming exponential growth) were micromanipulated away from their siblings and re-incubated for 24 hrs. The genotype of each colony was determined later by the presence of the ume6-5 allele. Individual cells from ume6-5 microcolonies executing two or fewer cell divisions after 24 hr were not able to continue growth in the six examples followed. These cells predominantly arrested as a single large cell or as equal sized mothers and daughters. In addition, these mothers and daughters were easily separated suggesting that they had undergone septation. In S. cerevisiae, the relative position in the cell cycle for an individual cell can be approximated by the presence and size of the bud (Pringle & Hartwell, 1981). Therefore, these cells may have ceased cell division in the G1 (or G0) stage of the cell cycle.
In ume6-5 microcolonies able to execute three cell divisions within 24 hrs, approximately 90% of the cells were unable to continue growth (closed arrows, Fig. 3A). The one cell in this example that was able to continue dividing (open arrow) eventually formed a macroscopic colony. Microcolonies that had concluded approximately six (Fig. 3B) or nine (Fig. 3C) divisions contained an increasing percentage of viable cells. As shown in Fig. 3D, a linear relationship was observed between the number of cells in a colony and the percentage of viable cells. As expected, cells derived from wild-type microcolonies were >95% viable regardless of the colony age (data not shown). These results indicate that UME6 is required for the efficient transition between resting spore to mitotic division. Moreover, these findings provide an explanation for the small colony phenotype. Colonies initially producing a high percentage of inviable cells would exhibit slower development. However, this requirement appears transient as the ume6-5 microcolonies able to execute approximately 10 cell divisions exhibit nearly wild-type growth rates.
Fig. 3.
Ume6p is transiently required for cell division following germination. Eight cells were micromanipulated from a ume6-5 microcolony executing approximately three (a), six (b) or nine (c) generations at 24 hrs after dissection. The images were taken after another 24 hr incubation when the ability of the individual cells to continue growth was determined. Closed arrowheads indicate cells that failed to continue growth, open arrowheads indicate cells that continued growth. The mother colony in Panel C expanded into the manipulated single cell colonies allowing the analysis of five manipulated cells in this example. Bar = 30 μM. (d) The percentages cells continuing growth was plotted versus the number of cells in the microcolony 24 hrs after dissection. At least three microcolonies were assayed for each point. The bars indicate the range at each point, the data points indicate the average.
Restricting UME6 expression in ascospores reduces viability
Our data suggest that Ume6p is required for spore germination. However, the sporulated UME6/ume6-5 diploid reduced, but did not eliminate, the ability of the ume6-5 spore to form colonies. These results suggest two possibilities. The loss of Ume6p function in a spore may result in partial loss in germination ability. Alternatively, the presence of the wild-type allele may allow Ume6p to be present in a genetically null spore. The amount of Ume6p packaged in the ume6Δ spore would then influence the germination ability of the spore. To test these models, we took advantage of the finding that the B-type cyclin CLB2 is transcribed poorly during meiosis (Primig, et al., 2000) but is induced shortly after the exposure of spores to glucose (Joseph-Strauss, et al., 2007). Therefore, we put genomic UME6 under the control of the CLB2 promoter (see Materials and methods). This construct was functional as determined by its ability to repress a spo13-lacZ reporter gene during vegetative growth (Fig. 4A). This strain was mated to a ume6-5 strain and the subsequent diploid sporulated in either liquid or on solid medium. After 24 hrs, the liquid sporulation cultures were examined and found to contain 54% asci, comparable to the W303 background (66%, Table 2) from which it was derived. Tetrads were dissected (N=33) and viable spore colonies were assayed for the presence of the CLB2pro-UME6 (Trp+) or ume6-5 (Leu+) alleles by replica plating. We obtained 77% of the expected number of Trp+ spore colonies (see Fig. 4B for representative plate) similar to the percentage observed for UME6 spores in the W303 heterozygote (Table 2). However, only 16% of the ume6-5 spores derived from the CLB2pro-UME6/ume6-5 heterozygote formed colonies. This value is three-fold lower than the ume6Δ spores from the W303 heterozygote (45%, Table 2). These findings indicate that the reduction of Ume6p levels in sporulating cells has a negative effect on ume6Δ mutant spore colony formation.
Fig. 4.
CLB2pro-UME6 promotes germination. (a) Haploid UME6 (RSY269), ume6-5 (RSY291) and CLB2pro-UME6 (RSY1295) strains transformed with pBW2 harboring a spo13-lacZ reporter gene were assayed for β-galactosidase expression using X-gal containing top agar (see Materials and methods). X-gal cleavage (dark color) indicates β-galactosidase expression. (b) Tetrads resulting from sporulated CLB2pro-UME6/ume6-5 diploid (RSY1295 × RSY291) were dissected onto rich medium and incubated for three days at 30°. The left panel is a image of a representative plate with tetrads (numbers) and spores (letters) indicated. Subsequent genotyping of spore colonies is indicated on the right. + = CLB2pro-UME6, = ume6-5. Closed squares indicate spores that did not form colonies.
Ume6p is required for efficient return to growth from stationary phase arrest
The findings presented above suggest that Ume6p is required for the successful transition from quiescent spore to mitotic cell division. To determine if this finding represents a general requirement for Ume6p following arrest at other points in the cell cycle, wild type (RSY271) and ume6-5 (RSY270) haploid strains were subjected to growth arrest in early G1, late G1 or G2 stages of the cell cycle (see Materials and methods for details). Early G1 (or G0) arrest was accomplished through nutrient deprivation by growing triplicate cultures to saturation density in liquid rich medium and maintaining these cultures at 30° for three days. Haploids were used to prohibit the cultures from entering meiosis under these starvation conditions. The cells were harvested, counted and their viability assayed by plating onto rich medium. These experiments revealed that ume6-5 mutants exhibited a twofold reduction in their plating efficiency (Table 3). To determine if this loss in plating efficiency was dependent on the cells being growth arrested or whether ume6-5 cultures always contain a significant subpopulation of inviable cells, the plating efficiency of log phase RSY271 and RSY270 cultures were determined. No difference was observed in the colony forming ability between these strains (Table 3) indicating that cell cycle arrest is required for the observed return to growth defect. These results suggest that Ume6p is required for the efficient return to growth of starvation induced G0 arrested cells.
Table 3.
Requirement of Ume6p for return to growth following cell cycle arrest.
Strain | Log phasea % cfu | Stationary phaseb % cfu |
α-factor arrest % cfu | benomyl arrest % cfu | |
---|---|---|---|---|---|
3 d | 100 d | ||||
RSY270 ume6-5 |
92 ± 1.1 | 53 ± 3 | 31 ± 9 | 100 ±7 | 33 ± 16 |
RSY271 UME6 |
92 ± 3.1 | 81 ± 1 | 65 ± 14 | 98 ± 5 | 21 ± 2 |
Log phase represents 5.6–6.5 × 106 cells per ml in rich liquid medium. The % cfu was calculated as the number of colonies observed divided by the number of cells plated as determined by direct counting.
Stationary phase indicates cells grown to a density of 2–3 × 108 cells/ml in rich liquid medium followed by an additional two day incubation at 30°. Percentages presented are averages from three independent cultures plated in triplicate. The 100 day arrest was conducted by placing the three day stationary culture at 4° for an additional 97 days.
Reduced plating efficiency following nutritional deprivation can be due to either the failure to properly arrest, the inability to reenter cell division, or both. For example, cells carrying constitutively activated RAS2val19 alleles do not arrest correctly and exhibit reduced viability upon starvation (Tatchell, et al., 1985). Two phenotypes are commonly associated with an aberrant growth arrest phenotype. First, these cells will stop dividing randomly throughout the cell cycle (Toda, et al., 1985). Normally, cells arrest growth in G1 in response to nutrient deprivation. Second, prolonged maintenance in a growth arrested state increasingly reduces viability compare to wild-type cells. However, growth arrested ume6-5 cells are predominantly large and unbudded indicative of G1 (data not shown). Moreover, the plating efficiency of ume6-5 cells held for 100 days at 4° showed only modest reduction in viability (31%) compared to the wild type (65%, Table 3). In addition, starving ume6Δ mutant cultures for nitrogen alone for 12 hrs did not adversely affect cell viability compared to the wild-type control (Fig. 5). These results suggest that ume6 mutants are most likely defective in their ability to return to growth, not growth arrest.
Fig. 5.
Ume6p is not required for viability following nitrogen starvation. Haploid UME6 (RSY269) and ume6-5 (RSY270) strains were grown to mid-log phase in minimal complete medium. The cultures were harvested, washed and transferred to minimal medium lacking a nitrogen source. At the times indicated after the shift to nitrogen depleted medium, cells were taken, serially diluted (1:10) and plated on rich solid medium. The plates were incubated two days at 30° then the image collected.
Ume6p is not required to reinitiate the cell cycle following late G1 or G2 arrest
To examine the requirement of Ume6p for cells to continue cell division following blocks at other points in the cell cycle, triplicate wild type (RSY271) and ume6-5 (RSY270) log phase cultures were arrested in late G1 with the mating pheromone α-factor or in G2 with benomyl, an inhibitor of microtubule assembly. Growth arrest was monitored microscopically for the expected cell morphology (i.e., shmoo formation for α-factor arrest, large budded cells for the benomyl block, see Materials and methods for details). Arrested cultures were plated on rich solid medium lacking either compound. No significant differences in plating efficiency were observed between the ume6-5 or wild-type strains (Table 3). Both strains exhibited a significant but equal reduction in plating efficiency following benomyl arrest due to the toxic effects of this drug. These results indicate that Ume6p is not generally required for cells to resume mitotic cell division following cell cycle arrest but instead is specific for cell returning to the cell cycle upon nutrient stimulation.
Discussion
The requirement for Ume6p in spore colony formation appears different than those generally observed in other studies. Three factors have been described that influence spore viability. First, mutations that disrupt mitotic cell division prevent spore colony formation. However, loss of Ume6p function produces only a modest growth defect in established cultures. Second, mutations causing spore wall defects reduce viability when the spore is challenged with enzymatic digestion or other cellular stresses (Briza, et al., 1990, Krisak, et al., 1994). The finding that ume6-5 mutant spores can undergo 2–3 rounds of division before arresting suggests that the spores are intact. In addition, spore colony formation did not occur even when intact tetrads from ume6/ume6 diploids were examined. Finally, spore viability is reduced due to aberrant chromosome segregation during meiosis (Shonn, et al., 2000). A previous study found that recombination/gene conversion at one locus was reduced about fourfold in a ume6 mutant (Steber & Esposito, 1995). Therefore, the loss of viability in spores derived from the ume6/ume6 diploid could be attributed (at least in part) to the recombination defect. However, this explanation does not account for the observation that ume6 spores derived from the heterozygote also display a 2–5 fold reduction in colony forming ability. Since the ume6-5 allele is recessive, the presence of wild-type UME6 should promote normal development. This idea is consistent with the finding that no reduction in sporulation levels (Table 2) or kinetics (data not shown) is observed in heterozygous diploids compared to homozygous wild-type controls. Second, if problems did occur during meiosis due to haplo-insufficiency in the heterozygote (e.g., aberrant chromosome segregation), the wild-type spores should also exhibit a reduced viability since a defect in meiosis would not be spore autonomous. However, no differences in viability were observed in UME6 spores derived from either UME6/ume6-5 or UME6/UME6 diploids. These results argue that Ume6p functions during the transition between spore quiescence and the establishment of normal logarithmic growth.
Since Ume6p is not required for vegetative growth, it is not clear why newly germinated ume6 mutants were unable to continue growth even following an initial round of cell division (see Fig. 4). A previous study identified genes that were repressed during spore germination (Joseph-Strauss, et al., 2007). Many of these genes were involved in oxidative phosphorylation that were repressed as spores germinated on dextrose medium. In addition, components of the 26S proteasome were also down regulated. These genes could be potential targets of Ume6p-dependent repression. Comparing the list of down regulated genes with those loci repressed by Ume6p (Williams, et al., 2002) revealed only three overlaps in these data sets. One is Pre10p, a component of the 26S proteasome. It could be envisioned that aberrant up regulation of proteasome activity could have a detrimental impact on cell division. However, the remaining proteasome subunits are not regulated by Ume6p and are therefore still repressed upon germination. Therefore, it seems unlikely that aberrant expression of one subunit of this complex is important when the remaining subunits are still repressed. The other two genes are Sol4p, a 6-phosphogluconolactonase that is part of the pentose-phosphate shunt and Cox5A, a component of cytochrome c oxidase. The derepression of these genes would not be expected to induce growth arrest on rich glucose medium. In addition, other genes repressed by Ume6p (e.g., CAR1, CAR2, INO1) were not down regulated as spores germinated. Therefore, no potential targets of Ume6p repression that would regulate spore germination were identified.
A similar defect in spore germination, but not mitotic cell division, was observed with mutants lacking the ubiquitin conjugating enzyme Ubc1p (Seufert, et al., 1990). Ubc1p is involved in ER-associated protein degradation in response to stress (for review, see (Kostova, et al., 2007). There are several similarities between Ume6p and Ubc1p. First, UBC1 mRNA is induced late in meiosis (Cho, et al., 1998) coincident with the return of Ume6p levels. In addition, both Ubc1p and Ume6p exert negative regulation through protein destruction and transcriptional repression, respectively. As discussed earlier, yeast spores decide to re-enter the cell cycle based on the quality of the environmental nutrients. To control this decision, a regulatory system may be in place to prevent germination in response to sub-optimal conditions. Therefore, one possible role for Ume6p and Ubc1p would be to down regulate this pathway(s) in response to rich growth signals. Failure to do so may divert the cell back into a spore-like quiescence even in the presence of optimal growth conditions. This model is consistent with the role of Ume6p in repressing genes involved in arginine catabolism (CAR1 and CAR2) in the presence of a rich nitrogen source (Park, et al., 1992, Strich, et al., 1994). Barkai and co-workers proposed a two-step process for spore germination (Joseph-Strauss, et al., 2007). The first step, triggered by dextrose, initiates changes in the spore wall and subcellular localization of septins. The second step requires the presence of nitrogen and allows transition into the mitotic cell cycle. This model would be consistent with Ume6p functioning in the second step to promote late germination events and reentry into the mitotic cell cycle.
Conclusion
Ume6p is a non-essential protein that represses the transcription of diverse loci including early meiotic genes. In this report, we find that Ume6p promotes colony formation of germinating yeast ascospores. However, this requirement is transient as no significant effect on cell viability is observed in established (>10 generations) ume6 mutant cultures. Finally, this role is specific for spores reentering mitotic cell division as Ume6p is not required for efficient return to growth of cells arrested in G1 or G2 although a small reduction in viability is observed in cells arrested for a extended time in G0. Our findings reveal a new role for Ume6p in the transition of a resting spore to mitotic cell division.
Acknowledgments
I thank K. F. Cooper and M. Law for critical reading and helpful discussions. This work was supported in part by grants from the National Institutes of Health General Medicine (GM086788) and the National Cancer Institute (CA099003).
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