S-Adenosylmethionine Synthetase Is Required for Cell Growth, Maintenance of G0 Phase, and Termination of Quiescence in Fission Yeast.

S-adenosylmethionine is an important compound, because it serves as the methyl donor in most methyl transfer reactions, including methylation of proteins, nucleic acids, and lipids. However, cellular defects in the genetic disruption of S-adenosylmethionine synthesis are not well understood. Here, we report the isolation and characterization of temperature-sensitive mutants of fission yeast S-adenosylmethionine synthetase (Sam1). Levels of S-adenosylmethionine and methylated histone H3 were greatly diminished in sam1 mutants. sam1 mutants stopped proliferating in vegetative culture and arrested specifically in G2 phase without cell elongation. Furthermore, sam1 mutants lost viability during nitrogen starvation-induced G0 phase quiescence. After release from the G0 state, sam1 mutants could neither increase in cell size nor re-initiate DNA replication in the rich medium. Sam1 is thus required for cell growth and proliferation, and maintenance of and exit from quiescence. sam1 mutants lead to broad cellular and drug response defects, as expected, since S. pombe contains more than 90 S-adenosylmethionine-dependent methyltransferases.

Introduction

Methylation, the transfer of a methyl group from one molecule to another, is one of the most important and universal biochemical reactions in cells, where it is involved in numerous cellular processes, such as transcriptional control, lipid metabolism, and signal transduction. The diversity of functions involving methylation is sustained by numerous methyltransferases that are present in all living organisms. Although a broad range of molecules including proteins, nucleic acids, lipids, and cellular metabolites can be substrates for methyltransferases, almost all methyltransferases use S-adenosylmethionine (SAM, also known as SAMe or AdoMet) as the common methyl donor (Chiang et al., 1996). SAM is also required in other important processes, including synthesis of polyamines (Fontecave et al., 2004), which are essential for various cellular functions affecting growth and development (Pegg and Casero, 2011). Thus, SAM is the most versatile biomolecule, second to ATP, in biochemical processes (Loenen, 2006).

SAM is synthesized from methionine and ATP by SAM synthetase (also known as methionine adenosyltransferase [MAT]), and converted to S-adenosylhomocysteine (SAH) via methyl transfer reactions. Then it is regenerated via the methionine cycle (Figure 1A). SAM synthetases are highly conserved among diverse organisms and have been extensively studied in bacteria (Wei and Newman, 2002), fungi (Gerke et al., 2012, Hilti et al., 2000, Thomas and Surdin-Kerjan, 1991), plants (Li et al., 2011, Shen et al., 2002), and animals, including humans (Ding et al., 2015, Obata and Miura, 2015, Ramani and Lu, 2017). However, genetic analyses of SAM synthesis disruption are scarce, because multiple genes encode SAM synthetases in the majority of eukaryotic organisms, including the budding yeast, Saccharomyces cerevisiae.

Figure 1

Isolation of Temperature-Sensitive Mutants of sam1

(A) Schematic illustration of the methionine cycle in the fission yeast, S. pombe. Sam1, SAM synthetase; SPBC8D2.18c, adenosylhomocysteinase (AHCY); Met26, methionine synthase.

(B) Locations of mutations of sam1 mutants determined by nucleotide sequencing in domain architecture based on the three-dimensional structure.

(C) sam1-1–sam1-5 mutants failed to produce colonies at 36°C on both rich YPD and synthetic minimal EMM2 plates, whereas sam1 mutants containing one of two amino acid substitutions in sam1-3–sam1-5 mutants produced colonies at 36°C.

(D) The colony formation defects of sam1-1 and sam1-3 at 36°C were rescued by pREP41 plasmid carrying the sam1 gene. Cells were streaked onto EMM2 plates in the absence of thiamine to induce the expression of sam1.

See also Figures S1 and S2.

The fission yeast, Schizosaccharomyces pombe, is an excellent model organism to study eukaryotic molecular and cellular biology (Hoffman et al., 2015, Yanagida, 2002). S. pombe has more than 90 genes predicted to encode SAM-dependent methyltransferases, according to PomBase (Wood et al., 2012). The physiological roles of methylation have been investigated by inactivating specific methyltransferases involved in a wide range of cellular processes, such as biomolecule synthesis (Hayashi et al., 2014a, Iwaki et al., 2008, Kanipes et al., 1998, Pluskal et al., 2014), ribosome function (Bachand and Silver, 2004, Shirai et al., 2010), transcriptional control (Ekwall and Ruusala, 1994, Morris et al., 2005, Thon et al., 1994), and DNA damage response (Sanders et al., 2004). However, cellular defects in the genetic control of SAM synthesis are not well understood. S. pombe possesses a single gene for SAM synthetase, sam1, which is essential for cell viability, and low expression of sam1 affects growth, mating, and sporulation (Hilti et al., 2000).

In this study, we report isolation by PCR random mutagenesis and characterization of temperature-sensitive (ts) mutant strains of fission yeast SAM synthetase and demonstrate that sam1 is a super-housekeeping (SHK) gene, essential for both proliferation and quiescence (Sajiki et al., 2009). Mutations in the sam1 gene block cell growth and cell cycle progression in vegetative culture and also cause failure to exit from nitrogen starvation-induced G0 quiescence. Furthermore, sam1 mutants lose cell viability during G0 quiescence.

Results

Isolation of Temperature-Sensitive Mutants of the sam1 Gene

Because the sam1 gene is essential for cell viability (Hilti et al., 2000, Kim et al., 2010), we examined the effects of SAM limitation on cellular functions by isolating S. pombe ts mutants of SAM synthetase Sam1. To obtain ts mutants of the sam1 gene, we employed a PCR-based random mutagenesis screen (Hayashi et al., 2014b) (Figure S1). The sam1 DNA fragment, in which the hygromycin-resistance-encoding marker gene, hph, was inserted just downstream of the stop codon of the sam1 gene open reading frame, was amplified by PCR under error-prone conditions, containing increased MgCl2 (Eckert and Kunkel, 1990). Mutagenized DNA fragments were introduced into wild-type (WT) cells for replacement of the chromosomal sam1 gene with the mutated gene by homologous recombination. Hygromycin-resistant transformants were selected at 26°C and then tested for colony formation at 36°C on rich YPD medium plates. After confirmation of linkage of the ts phenotype to the hygromycin-resistant phenotype, five ts mutant strains of the sam1 gene were obtained and designated sam1-1 to sam1-5.

We then identified mutation sites in the sam1 gene of the sam1 ts mutants. sam1-1 and sam1-2 contained single amino acid substitutions (F367L and D36N, respectively), whereas sam1-3, sam1-4, and sam1-5 contained two amino acid substitutions (L292S R299H, I24M E115G, and T90A Q370R, respectively) in the sam1 gene (Figure 1B). All mutation sites except for Q370 are conserved among humans, rats, and fission yeast. Based on the three-dimensional structure of the rat ortholog of Sam1 (González et al., 2003), no mutations were found to locate near the binding site of the substrates, ATP and methionine (Figure S2). To identify the mutations responsible for the ts phenotype, we introduced one of the five mutant sequences (sam1-1–sam1-5) or one of two amino acid substitutions in sam1-3–sam1-5 mutants into the WT genome using linearized plasmids carrying the hygromycin resistance marker. The resulting transformants, containing chromosomal gene replacements with the sam1-1–sam1-5 mutant genes, showed the ts phenotype on both rich YPD and synthetic minimal EMM2 plates, whereas the transformants containing one of two amino acid substitutions in sam1-3–sam1-5 mutants did not show the ts phenotype (Figure 1C). In conclusion, sam1 gene mutations in the mutants caused the ts phenotype and both the amino acid substitutions in sam1-3–sam1-5 were necessary for the ts phenotype. Since sam1-3 showed the most severe growth defects and sam1-1 showed a moderate ts phenotype at 36°C on YPD plates (Figure 1C), sam1-1 and sam1-3 were used for further investigation. It was confirmed that the colony formation defects of sam1-1 and sam1-3 at 36°C were rescued by plasmid carrying the sam1 gene (Figure 1D).

Defective SAM Synthetase in sam1 Mutant Cells

To detect Sam1 protein, polyclonal antibodies against a mixture of two peptides (I77GYDDSEKGFDYKTC91 and N320TYGTSSKTSAELV333) of S. pombe Sam1 were raised and used for immunoblots after affinity purification. We validated the specificity of the α-Sam1 antibodies by detecting a single band at the expected molecular weight of ∼42 kD in immunoblots of WT cell extracts (no tag, Figure 2A). The assignment of the band was confirmed using extracts of Sam1-FLAG cells in which the chromosomal sam1 gene was replaced with the sam1-FLAG gene. The band detected by immunoblot using α-Sam1 antibodies was shifted to ∼45 kD in extracts of Sam1-FLAG cells (Figure 2A). Untagged Sam1 was not detected by α-FLAG antibody. Thus, polyclonal antibodies against Sam1 specifically detected Sam1 protein in cell extracts. We then compared protein levels of Sam1 in WT and sam1 mutant cells cultured at 36°C for 0–6 hr in rich YPD medium. Sam1 mutant proteins of sam1-1 and sam1-3 were diminished, compared with WT, even at the permissive temperature of 26°C (0 hr, Figure 2B), and further decreased at 36°C. These results suggest that sam1-1 and sam1-3 mutants are loss-of-function mutants of SAM synthetase Sam1, partly due to the decreased protein level.

Figure 2

sam1 Mutants Are Defective in SAM Synthesis and Histone H3K4 Methylation, and Are Rescued by Choline

(A) Immunoblot of extracts of WT (no tag) and Sam1-FLAG cells using affinity-purified anti-Sam1 and anti-FLAG antibodies.

(B) Levels of mutant Sam1 proteins were diminished. Immunoblotting was done using antibodies against Sam1 to detect Sam1 protein in the extracts of WT, sam1-1, and sam1-3 cells cultured at 36°C for 0–6 hr in rich YPD medium. Antibody against α-tubulin (Tub) was used as the loading control.

(C–E) The time course change of the normalized peak areas of SAM and related metabolites. Metabolites were extracted from WT and sam1 mutant cells cultured at 36°C for 0–24 hr in rich YPD medium and analyzed. Normalized peak areas of the following metabolites are shown: (C) S-adenosylmethionine (SAM); (D) S-adenosylhomocysteine (SAH); (E) methionine (Met).

(F) Levels of methylated histone H3 K4 in WT, sam1-1, and sam1-3 cells. WT, sam1-1, and sam1-3 cells were cultured at 36°C for 0–8 hr in rich YPD medium. Antibodies against histone H3 and mono-, di-, and trimethylated H3K4 (H3K4me1, H3K4me2, and H3K4me3, respectively) were used to detect the total H3 and methylated H3K4.

(G) Cells were diluted serially 10-fold, spotted onto EMM2 or EMM2 containing 2 mM choline chloride, and incubated at 30°C, 33°C, or 36°C for 3 days.

If sam1-1 and sam1-3 mutants are indeed defective in SAM synthetic activity, SAM should be decreased in the mutant cells. We employed metabolomic analysis using liquid chromatography-mass spectrometry to assay the levels of SAM and related metabolites in extracts of WT and sam1 mutant cells grown in rich YPD medium. WT and sam1 mutant cells were grown at 26°C and then shifted to 36°C for 0–24 hr. Levels of SAM detected in sam1-1 and sam1-3 cell extracts were rather low, even at the permissive temperature of 26°C (0 hr, Figure 2C), and further decreased at 36°C, whereas the high level of SAM in WT was maintained at 36°C for 0–24 hr (Figure 2C). Similar results were obtained for SAH, a by-product of SAM-dependent methyl transfer reactions (Figure 2D). Levels of methionine, a substrate of Sam1 enzyme, were decreased in WT cells, but not in sam1 mutants at 36°C (Figure 2E). These results strongly support the notion that SAM synthesis was defective in sam1-1 and sam1-3 mutant cells.

Impairment of Methylation Reactions in sam1 Mutants

Because the levels of SAM, a universal methyl group donor, were greatly decreased in sam1 mutant cells, we next investigated histone methylation in sam1 mutant cells. WT and sam1 mutant cells were shifted from 26°C to 36°C for 0–8 hr, and immunoblot analysis was carried out using three antibodies specific to mono-, di-, and trimethylated lysine 4 of histone H3 (H3K4me1, H3K4me2, and H3K4me3, respectively). In sam1-1 and sam1-3 mutant cells, the levels of H3K4me2 and H3K4me3 were greatly decreased after the shift to 36°C, whereas only a slight decrease was observed in H3K4me1 (Figure 2F). These results suggested that a global change of histone methylation was caused by mutations of SAM synthetase and that levels of di- and trimethylated Lys4 of histone H3 were more susceptible to SAM deficiency.

Many eukaryotes, including S. pombe, produce phosphatidylcholine (PC) via two pathways. One is methylation of phosphatidylethanolamine, a major methylation target in mammals and budding yeast (Noga et al., 2003, Stead et al., 2006, Ye et al., 2017). Another PC biosynthetic pathway is the CDP-choline pathway, which is independent of methylation and requires choline (Kanipes et al., 1998, Kent, 1995). We presumed that exogenous choline relieves the ts phenotype of sam1 mutants by rescuing the PC synthesis defect. As expected, colony formation of sam1-1 and sam1-3 at 33–36°C and 33°C, respectively, on EMM2 agar plates was rescued by adding choline (Figure 2G). These results strongly suggested that the sam1 mutations impaired cellular methylation reactions.

Diminished Cell Growth and Cell-Cycle Arrest in G2 Phase Occur in sam1 Mutants

We examined the cellular phenotypes of sam1-1 and sam1-3 mutant cells at the restrictive temperature (36°C). WT and sam1 mutant cells were grown at 26°C and then shifted to 36°C in rich YPD medium. The cell number increase of sam1 mutants stopped after one or two cell divisions at 36°C (Figure 3A), although the high viability of mutant cells was still maintained after 8 hr (Figure 3B). Since fluorescence microscopic observation of DNA-specific probe DAPI -stained cells demonstrated that most sam1-1 and sam1-3 cells were mononucleate 8 hr after the shift to 36°C (Figure 3C), the sam1 mutants probably arrest the cell cycle at the higher temperature. The length of sam1 mutant cells did not increase significantly compared with WT after the shift to 36°C. Average cell lengths of WT, sam1-1, and sam1-3 were 11.6 ± 2.3, 11.5 ± 2.4, and 11.8 ± 2.4 μm, respectively, after 8 hr at 36°C (Figure 3D), suggesting that cell growth (i.e., the extension of cell length) halted at 36°C in sam1 mutant cells, unlike cdc mutants that develop extremely elongate cells because of growth without cell division (Nurse et al., 1976).

Figure 3

Diminished Cell Growth and Cell-Cycle Arrest in G2 Phase Occur in sam1 Mutants

(A–F) WT, sam1-1, and sam1-3 cells were grown at 26°C in rich YPD medium and then shifted to 36°C. The cell number increase (A), the viability change (B), fluorescence microscopic images of DAPI-stained cells (C), the average cell length with standard deviations (D), the proportion of cells with a septum (septation index) (E), and the distributions of cellular DNA content (F) are shown. Cell viability was measured by plating cells onto YPD agar and counting the number of colonies formed after incubation at 26°C.

(G and H) Time courses of changes in spindle index (G) and fluorescence microscopic images (H) of WT, sam1-1, and sam1-3 cells expressing GFP-fused α-tubulin and mCherry-fused Cut11 after the shift from 26°C to 36°C in YPD medium. GFP and mCherry fluorescence is shown in green and magenta, respectively.

To monitor cell cycle progression in WT and sam1 mutant cells at 36°C, we measured the septation index (percentage of cells with a septum), the cellular DNA content, and the spindle index (percentage of cells with mitotic spindle). The septation index was maintained at ∼12% in WT cells, whereas it dropped to 0.8% 4 hr after the shift to 36°C in sam1-3 mutant cells and to 1.2% at 8 hr in sam1-1 mutant cells (Figure 3E). These results indicate that sam1 mutant cells were arrested before the onset of cytokinesis. In cellular DNA content analysis by fluorescence-activated cell sorting (FACS), a single peak appeared before the shift to 36°C (0 hr) in WT and sam1 mutants, meaning 2C DNA content, because most vegetatively growing S. pombe cells are mononucleate in post-replicative (G2) phase and pre-replicative nuclei exist in binucleate cells (Figure 3F). After the shift to 36°C (2–8 hr), only one peak at the 2C DNA content was still present in sam1-1 and sam1-3 mutants, like WT cells, indicating that cell-cycle arrest in sam1 mutants at the restrictive temperature occurred after completion of DNA replication. The spindle index was measured using WT and sam1 mutant strains expressing α-tubulin-GFP and Cut11-mCherry, which visualized spindle microtubules and the nuclear envelope, respectively (Figures 3G and 3H). The spindle index was maintained around 8%–10% in WT cells at 36°C for 0–6 hr, whereas it dropped to 0% at 4 hr after the shift to 36°C in sam1-3 mutant cells and to 0.4% at 6 hr in sam1-1 mutant cells (Figure 3G), indicating that sam1 mutant cells arrested before the onset of M phase. Consequently, sam1 mutant cells arrest specifically in G2 phase at the restrictive temperature in rich YPD medium.

sam1 Mutant Cells Lose Cell Viability in the Maintenance of G0 Phase Induced by Nitrogen Starvation

To determine the capacity of sam1 mutants to maintain viability in the nitrogen starvation-induced quiescent G0 phase, we monitored cell viability under nitrogen starvation (Sajiki et al., 2009, Shimanuki et al., 2007). WT and sam1 mutant cells first grown in EMM2 medium were transferred to the nitrogen-deficient EMM2−N at 26°C for 24 hr to enter a quiescent G0 state. After 24 hr, the cell number increase in the sam1-1 mutant was only 2.4-fold, whereas that in WT and sam1-3 mutants was around 3.6-fold (Figure 4A). FACS analysis revealed that ∼80% of WT and sam1-3 cells contained 1C DNA, whereas 1C DNA-containing sam1-1 cells accounted for only ∼40% (Figure 4B). These results indicated that the sam1-1 mutant was somewhat defective in pre-quiescence division under nitrogen starvation at 26°C. Nitrogen starvation-induced quiescent G0 cells were further cultivated at either 26°C or 36°C for 12 days. G0-arrested WT and sam1-3 cells maintained high viability at 26°C, whereas G0-arrested sam1-1 cells gradually lost viability even at 26°C (Figure 4C). At 36°C, the viability of sam1-1 and sam1-3 decreased to <25% after 4 days and to <0.3% after 8 days, whereas the viability of WT was ∼40% after 12 days at 36°C (Figure 4D). Together, functional Sam1 is required for the maintenance of G0 phase under nitrogen starvation.

Figure 4

sam1 Mutant Cells Lose Cell Viability in the Maintenance of Nitrogen Starvation-Induced G0 Phase

(A and B) WT, sam1-1, and sam1-3 cells were brought into the G0 quiescent state at 26°C under nitrogen-deficient medium, EMM2−N for 24 hr. Increase in cell number as the fold increase (A) and the distribution of cellular DNA content (B) after 24 hr in EMM2−N medium at 26°C are shown.

(C and D) Resulting G0 quiescent cultures were kept at 26°C (C) or shifted to 36°C (D) for 12 days. The cell viability percentages were scored at different time points (days). sam1-1 mutant cells lost cell viability both at 26°C and 36°C, whereas sam1-3 mutant cells lost cell viability only at 36°C.

(E) Levels of Sam1 and methylated histone H3 K4 in WT, sam1-1, and sam1-3 cells in nitrogen starvation-induced G0 phase at 26°C. WT, sam1-1, and sam1-3 cells were brought into the G0 quiescent state under nitrogen-deficient medium, EMM2−N for 24 hr (day 0), and further incubated at 26°C for 8 days. Immunoblotting was done using antibodies against Sam1, α-tubulin (Tub), histone H3, and mono-, di- and trimethylated H3K4 (H3K4me1, H3K4me2, and H3K4me3, respectively).

Since sam1-1 lost viability at 26°C during nitrogen starvation, whereas sam1-3 did not, we compared the mutant Sam1 protein abundance in sam1-1 with that in sam1-3 mutants in G0 phase 0–8 days after culture for 24-hr quiescence entry in the EMM2−N medium. Although the Sam1 proteins of both sam1-1 and sam1-3 were diminished compared with that of WT under nitrogen starvation at 26°C, the mutant Sam1 protein level was lower in sam1-1 than in sam1-3 (Figure 4E). In addition, levels of H3K4me2 and H3K4me3 were already greatly decreased in sam1-1 at day 0 in G0 (24 hr after nitrogen removal), whereas only a slight decrease of H3K4me3 was observed in sam1-3 (Figure 4E). These results are consistent with the data in Figure 4C.

sam1 Mutant Cells Fail to Exit from Nitrogen-Starved Arrest

We next addressed the question of whether sam1 mutant cells could exit from a nitrogen starvation-induced G0 state in which cells became small and contained pre-replicative 1C DNA (Su et al., 1996) (see also Figures 5C and 5D). WT and sam1 mutants were first cultured in nitrogen-deficient medium, EMM2−N, at 26°C for 24 hr to induce G0 arrest, and then shifted to rich YPD medium for release from G0 arrest at 36°C. The cell number increase was completely blocked in sam1-1 and sam1-3 mutant cells after transfer to rich YPD medium at 36°C, whereas the cell number of WT started increasing after 6 hr (Figure 5A). However, the high viability of sam1-1 and sam1-3 mutant cells was maintained for 8 and 24 hr, respectively, in rich YPD medium at 36°C (Figure 5B). sam1-1 and sam1-3 mutant cells remained small and round even after 24 hr in rich YPD medium, whereas WT cells were a normal rod shape after 8 hr (Figure 5C), demonstrating that sam1 mutants were unable to initiate cell growth at the restrictive temperature. FACS analysis showed that sam1-1 and sam1-3 mutant cells did not initiate DNA replication even after 24 hr, whereas WT cells completed DNA replication after 4 hr (Figure 5D). These results imply that Sam1 is necessary to initiate cell growth and DNA replication after the release from nitrogen starvation-induced G0 state.

Figure 5

sam1 Mutant Cells Fail to Exit from Nitrogen-Starved Arrest

(A–D) WT, sam1-1, and sam1-3 were first cultured in nitrogen-deficient medium, EMM2−N, at 26°C for 24 hr to induce the G0 arrest. G0-arrested, small, round cells were then released to rich YPD medium at 36°C. The cell number increase (A), the viability change (B), fluorescence microscopic images of DAPI-stained cells (C), and the distributions of cellular DNA content (D) are shown.

Rapamycin Rescues sam1 Mutants

The target of rapamycin (TOR) complex is the main regulator of cell growth and controls nutrient signaling pathways in eukaryotes (Kennedy and Lamming, 2016). We investigated the effect of rapamycin (an inhibitor of TOR kinase) on the ts phenotype of sam1 mutants on rich YPD medium, as sam1 mutants were defective in cell growth, as described above (Figures 3C and 5C). Rapamycin (0.1 μg/mL) rescued the growth defect of sam1-1 mutant cells at 36°C and slightly suppressed the ts phenotype of sam1-3 mutants at 33°C (Figure 6A), suggesting that reducing TOR activity relieved the growth defects of sam1 mutants. Rapamycin may rescue sam1 mutants in a way different from protein level restoration because the mutant Sam1 protein amount was not affected in sam1-1 and sam1-3 mutants by the addition of rapamycin at 26°C and 36°C (Figures 6B and 6C).

Figure 6

sam1 Mutants Are Rescued by Rapamycin

(A) sam1-1 and sam1-3 were spotted on YPD plates in the presence or absence of rapamycin and then incubated at the indicated temperatures for 2–3 days. Wild-type and tor2-287 (hypersensitive to rapamycin) (Hayashi et al., 2007) were used as controls.

(B) Levels of mutant Sam1 proteins in sam1-1 and sam1-3 cells in the presence of rapamycin at 26°C. sam1-1 and sam1-3 cells were cultured in YPD with 0.2 μg/mL rapamycin at 26°C. Immunoblotting was done using antibodies against Sam1 and α-tubulin (Tub).

(C) Levels of mutant Sam1 proteins in sam1-1 and sam1-3 cells in the presence or absence of rapamycin at 36°C. Rapamycin (0.2 μg/mL) or DMSO was added to YPD medium 2 hr before the shift up from 26°C to 36°C. Immunoblotting was done using antibodies against Sam1 and α-tubulin (Tub).

(D and E) Viability of WT, sam1-1, and sam1-3 in nitrogen starvation-induced G0 phase with or without rapamycin. WT, sam1-1, and sam1-3 cells were brought into the G0 quiescent state at 26°C under nitrogen-deficient medium with or without 0.2 μg/mL rapamycin (Rap) for 24 hr. Resulting G0 quiescent cultures were kept at 26°C (D) or shifted to 36°C (E) for 12 days. The cell viability percentages were scored at different time points (days).

sam1 mutants were also defective in the maintenance of G0 phase induced by nitrogen starvation (Figures 4C and 4D), so we next tested whether rapamycin could rescue the viability loss of sam1 mutants in G0 phase. Viability loss of sam1-1 in G0 at 26°C was significantly suppressed by the addition of rapamycin, and the viability was >40% after 12 days, whereas the viability in the absence of rapamycin was ∼3% after 8 days (Figure 6D). At 36°C, viability loss of sam1-1 and sam1-3 in G0 was delayed in the presence of rapamycin (Figure 6E). Thus, rapamycin rescued sam1 mutants in both proliferative and quiescent phases.

sam1 Mutants Show Pleiotropic Drug Sensitivities

To search further the physiological roles of Sam1, we examined the drug sensitivity of sam1 mutants. The response of sam1-1 and sam1-3 mutants to various stress agents is summarized in Table 1 (data are shown in Figure S3). Both mutants were sensitive to 25 mM nicotinamide, which inhibits class III histone deacetylases (HDACs) (Sauve et al., 2006), but not to 12.5 μg/mL trichostatin A (TSA), an inhibitor of class I and class II HDACs (Yoshida et al., 1995), at 30°C. These mutants were also sensitive to 4 μg/mL actinomycin D, an inhibitor of transcription (Bensaude, 2011), at 30°C. At 33°C, sam1-1 was moderately sensitive to 4 mM hydroxyurea (HU, an inhibitor of DNA synthesis) (Singh and Xu, 2016), 8 μg/mL phleomycin (inducing double-strand DNA breaks) (Moore, 1988), and 4 μM camptothecin (CPT, an inhibitor of topoisomerase I that induces single-strand DNA breaks) (Pommier, 2006) and showed similar sensitivity to 100 J/m2 UV irradiation (inducing the formation of thymine dimers) compared with the WT strain. No significant difference in the sensitivity of sam1-3 to HU, phleomycin, CPT, and UV irradiation was observed, partly due to no growth at 33°C even without the treatments. Thus, sam1 mutants showed hypersensitivity to a variety of drugs related to DNA damage and chromatin remodeling.

Table 1

Summary of Colony Formation Abilities of WT and sam1 Mutants in the Presence of Various Stress Agents

	30°C			33°C
	WT	sam1-1	sam1-3	WT	sam1-1	sam1-3
Control	++++	++++	++++	++++	+++	–
25 mM nicotinamide	+++	–	–	+++	–	–
12.5 μg/mL TSA	+++	+++	+++	+++	++	–
4 μg/mL actinomycin D	++	–	–	+++	–	–
4 mM HU	++	++	++	+++	+	–
8 μg/mL phleomycin	+++	+++	+++	+++	±	–
4 μM CPT	+++	+++	+++	+++	+	–
100 J/m2 UV	++++	++++	++++	++++	+++	–

++++, normal growth; +, slow growth; ±, diminished colony formation; −, no colony formation.

See also Figure S3.

Extensive Changes Occur in the Metabolic Profiles of sam1 Mutants

We performed metabolomic analysis of WT and sam1 mutant cells grown at 36°C for 8 hr in rich YPD medium to understand the impact of SAM limitation on metabolic homeostasis beyond the methionine cycle (Table S1). A comparison of WT with sam1-1 is shown in a scatterplot (Figure 7A). The normalized peak area showed more than a 2-fold difference for 54 compounds (61%) out of 88 identified metabolites in comparison of WT with sam1-1, whereas only ∼10% of the identified metabolites usually change more than 2-fold in comparisons of two independent experiments under identical conditions (Pluskal et al., 2010), indicating that the profile of metabolites was significantly altered in sam1-1. Similar results were obtained from comparisons of sam1-3 with WT: the normalized peak areas of approximately 64% of identified metabolites had greater than a 2-fold change (Figure 7B). Beside the remarkable decrease of SAM and SAH described above (Figures 2C and 2D), eight compounds (phosphoribosyl pyrophosphate [PRPP], hercynylcysteine sulfoxide, CDP, UMP, adenine, UDP, sedoheptulose-7-phosphate, and pentose-phosphate) decreased over 10-fold in sam1-1 and/or sam1-3 mutant cells (Figure 7C). Hercynylcysteine sulfoxide is a precursor of ergothioneine, an antioxidant compound (Pluskal et al., 2014), and the others are involved in nucleotide metabolism. These extensive changes in the metabolic profiles of sam1 mutants may reflect the importance of SAM in diverse cellular functions implicated in anti-oxidation, nucleotide metabolism, and pentose pathways.

Figure 7

Metabolomic Analysis of sam1 Mutants

(A and B) A scatterplot comparing the normalized peak areas of all identified metabolites in extracts of cells cultured at 36°C for 8 hr in rich YPD medium. WT versus sam1-1 (A) and WT versus sam1-3 (B) are shown. Red diagonal lines indicate a 2-fold difference. Most changed compounds are annotated according to the list in (C).

(C) Normalized peak areas of compounds that decreased more than 10-fold in sam1-1 or sam1-3 compared with WT.

See also Table S1.

Discussion

SAM-dependent methylation occurs in a wide variety of cellular compounds, and in the fission yeast, S. pombe, one enzyme, the product of the sam1+ gene, is solely responsible for the synthesis of SAM from methionine and ATP. There are ∼90 methyltransferase genes in the genome of S. pombe, so these enzymes are the sources of diversity of methylated substrates. In this study, we examined the cellular defects produced by the genetic impairment of SAM synthesis in the fission yeast, S. pombe, using five sam1 mutants that produced the ts phenotype. Two of them, moderate sam1-1 and severe sam1-3, were further investigated. Both mutants showed Sam1 protein instability, and a great decline of SAM and SAH, whereas increased methionine was observed at the restrictive temperature. As expected, the degree of histone H3 K4 methylation was diminished at the restrictive temperature. The ts phenotype was suppressed by the presence of choline at the semi-restrictive and restrictive temperatures. Exogenous choline, containing three methyl groups, may relieve the ts phenotype by suppressing the defect of PC synthesis and/or reducing the required amount of SAM for cell proliferation. The findings, indeed, indicated that sam1 mutations cause deterioration of a wide spectrum of methylation reactions.

sam1 mutants showed diverse phenotypes. Two sam1 mutants (sam1-1, sam1-3) are hypersensitive to drugs such as nicotinamide, actinomycin D, HU, phleomycin, and CPT. Considering these drug sensitivities, broad cellular functions implicating methylation seem to necessitate Sam1 enzyme. In addition, rapamycin relieved the defects of sam1 mutants in both proliferative and quiescence phases. A possible explanation is that the TOR signaling regulates Sam1 via direct or indirect phosphorylation as rapamycin attenuates TOR kinase. Another possibility is that rapamycin, perhaps through TOR kinase, alters methyltransferase activity to give priority to methylation reactions essential to growth. We also identified 12 other groups of ts mutants that were rescued by rapamycin (Sajiki et al., 2018); thus, perhaps Sam1 with some methyltransferases, composes an additional group. According to the report proposing that rapamycin re-balances TOR activity with the activities of identified gene products (Sajiki et al., 2018), the balance between TOR kinase and Sam1 appears important for cell proliferation, and severe impairment of the balance blocks cell growth. The detailed mechanism of this rapamycin-induced rescue should be the subject of further investigation.

Mutations in the S. pombe sam1 gene affected cell growth (cell size increase) and cell cycle progression at two transition points: the G2/M transition in vegetative culture and the G1/S transition after release from nitrogen-starvation-induced G0 phase. Since fission yeast cells need to increase their size to a critical threshold to pass these two regulatory points (Nurse and Thuriaux, 1977, Nurse, 1975, Shiozaki, 2009), we interpreted our results to mean that a primary defect of mutants resided in cell growth, rather than in the transition of specific cell cycle stages. Our metabolomic analysis of sam1 mutants suggested that the sam1 mutation disturbed many metabolic pathways, especially attenuating nucleotide biosynthesis. These metabolic changes appear to cause defects of both cell growth and cell cycle progression, although the precise roles of Sam1 in cell growth remain to be determined.

Sam1 is also required in G0 phase quiescence induced by nitrogen starvation, so Sam1 belongs to a family of SHK gene products, involved in the control of G0 phase, as well as cell growth (Sajiki et al., 2009). sam1-1 lost viability at 26°C in G0 phase, whereas sam1-3 did not. Why is sam1-1 more sensitive to this condition than sam1-3 although the ts phenotype of sam1-1 was less severe in vegetative culture? One reason is that sam1-1 seems more defective at 26°C as the SAM content of sam1-1 is about half of sam1-3 at 26°C (Figure 2C, 0 hr) and the doubling time of sam1-1 (3.4 hr) is longer than that of sam1-3 (3.2 hr) at 26°C. It is also possible that Sam1 protein is differently regulated in G0 phase, as the mutant Sam1 protein level in G0 phase was lower in sam1-1 than in sam1-3 (Figure 4E), contrary to the case in vegetative culture. In G0 phase, the level of SAM increases (Shimanuki et al., 2013), and nitrogen sources are recycled for the degradation and synthesis of nucleic acids, proteins, and other nitrogen-containing substrates. Hence, methylated compounds may be central to recycling of nitrogen-containing metabolites.

We isolated and characterized fission yeast ts mutants for the SAM synthetase gene. As the yeast is an excellent model organism for genetic analyses, further study by using these sam1 mutant strains is expected. However, this single-cell eukaryote does not reveal higher order phenotypes at the level of tissues or organs. In humans, mutations in MAT I/III have been identified as clinical blood variations with hypermethionine and various brain symptoms (Chamberlin et al., 2000, Nagao et al., 2013). Accumulation of methionine seems to be characteristic to all SAM synthetase mutants isolated in different species. In C. elegans, S-adenosylmethionine synthetase 1 (SAMS-1) mutants produced enlarged lipid droplets throughout their life cycle and impaired PC synthesis (Ehmke et al., 2014). In both C. elegans and S. pombe, methyl group-enriched PC is a key metabolite, perhaps because choline is the basic compound for phospholipid metabolism. In Arabidopsis thaliana, a mutation in the S-adenosylmethionine synthetase 3 gene caused high free methionine and decreased lignin (Goto et al., 2002, Shen et al., 2002). In Neurospora crassa, mutations of the S-adenosylmethionine synthetase-encoding gene has been identified as an allele resistant to the toxic methionine analog, ethionine (Barra et al., 1996). Taken together, although the phenotypes of different organisms are not universally understood, except for the abundance of methionine, further study of SAM synthetase mutants may open a new avenue to understand the dynamic metabolic regulation of methylation-related biological events, which may be quite complex.

Methods

All methods can be found in the accompanying Transparent Methods supplemental file.