- Open Access
Functional and regulatory profiling of energy metabolism in fission yeast
© The Author(s). 2016
- Received: 15 March 2016
- Accepted: 8 November 2016
- Published: 25 November 2016
The control of energy metabolism is fundamental for cell growth and function and anomalies in it are implicated in complex diseases and ageing. Metabolism in yeast cells can be manipulated by supplying different carbon sources: yeast grown on glucose rapidly proliferates by fermentation, analogous to tumour cells growing by aerobic glycolysis, whereas on non-fermentable carbon sources metabolism shifts towards respiration.
We screened deletion libraries of fission yeast to identify over 200 genes required for respiratory growth. Growth media and auxotrophic mutants strongly influenced respiratory metabolism. Most genes uncovered in the mutant screens have not been implicated in respiration in budding yeast. We applied gene-expression profiling approaches to compare steady-state fermentative and respiratory growth and to analyse the dynamic adaptation to respiratory growth. The transcript levels of most genes functioning in energy metabolism pathways are coherently tuned, reflecting anticipated differences in metabolic flows between fermenting and respiring cells. We show that acetyl-CoA synthase, rather than citrate lyase, is essential for acetyl-CoA synthesis in fission yeast. We also investigated the transcriptional response to mitochondrial damage by genetic or chemical perturbations, defining a retrograde response that involves the concerted regulation of distinct groups of nuclear genes that may avert harm from mitochondrial malfunction.
This study provides a rich framework of the genetic and regulatory basis of energy metabolism in fission yeast and beyond, and it pinpoints weaknesses of commonly used auxotroph mutants for investigating metabolism. As a model for cellular energy regulation, fission yeast provides an attractive and complementary system to budding yeast.
- Electron Transport Chain
- Fission Yeast
- Fermentative Medium
- Respiratory Growth
- Respiratory Medium
Glucose is a common source of energy for cells. Glucose metabolism starts with glycolysis, which produces pyruvate. During fermentation, pyruvate is converted to organic acids, gases or ethanol. Alternatively, pyruvate can be metabolised by respiration via the mitochondrial tricarboxylic acid (TCA) cycle, also called the Krebs or citric acid cycle [1, 2]. In the mitochondrial membrane, electrons are then transferred from NADH and other TCA products to oxygen through the electron transport chain (ETC), which generates a proton gradient across the mitochondrial membrane to produce ATP by oxidative phosphorylation (OXPHOS) [1, 2]. With respect to ATP production, respiration is much more efficient than fermentation, generating a net gain of up to 36 versus only 2 ATP molecules per glucose molecule, respectively. Although respiration and fermentation share the upstream glycolysis pathway, they are to some extent antagonistic and are tuned in response to different nutrient or physiological conditions . Fermentation is preferred in rapidly proliferating cells even in the presence of oxygen, a process also called aerobic glycolysis. Cancer cells, for example, typically grow by aerobic glycolysis (Warburg effect) . Similarly, yeast cells proliferating in nutrient-rich media will induce fermentation and repress respiration (Crabtree effect) . On the other hand, differentiated cells and yeast cells cultured in nutrient-poor media will switch to respiration . Accordingly, the expression of OXPHOS genes in yeast is inversely correlated with the cellular growth rate [6, 7]. Yeast cells exhibit alternating metabolic cycles in which respiration and fermentation are temporally separated and coordinated with the cell cycle [8, 9]. Thus, respiration and fermentation are specifically tuned to environmental or physiological conditions and complement each other to support the cellular energy demands.
Cellular energy metabolism is fundamental for biological processes such as cell proliferation, stress resistance and ageing. In humans, aberrant energy metabolism results in a range of metabolic or degenerative diseases . It is important, therefore, to understand the genetic factors and regulatory mechanisms that affect cellular energy metabolism. Regulation of the balance between respiration and fermentation depends largely on nutrient availability [3, 10], mediated by nutrient-sensing signalling pathways like TOR or PKA which in turn control gene expression [1, 11] as well as by direct metabolic feedback loops . Moreover, it is likely that the cellular metabolic state can control gene expression or protein function via epigenetic mechanisms: the levels of key metabolites such as ATP, acetyl-CoA or NAD/NADH are readouts for energy metabolism; such metabolites can alter global levels of protein phosphorylation, acetylation, or methylation, which in turn will impact genome regulation and protein activities [1, 13, 14].
Yeasts are simple yet powerful model organisms to investigate and manipulate conserved energy metabolism programs under tightly controlled conditions by supplying different carbon sources. The budding yeast, Saccharomyces cerevisiae, has served as a valuable model system to study the genetic and regulatory basis of energy metabolism at a genome-wide scale [4, 5, 7, 8, 15–18]. The fission yeast, Schizosaccharomyces pombe, is only remotely related to budding yeast and shows features that promise valuable complementary insights into energy metabolism. Mitochondria of fission yeast form a dynamic network along microtubules which mediate their inheritance, as is the case in multicellular eukaryotes . The fission yeast mitochondrial genome is compact (~20 kb, 11 protein-coding genes) and mitochondrial RNA processing is similar to in animal cells . Fission yeast can grow using either respiration or fermentation but, in contrast to budding yeast, does not thrive in strictly anaerobic conditions . In the presence of glucose, fission yeast grows mainly by fermentation, but it will switch to respiratory growth with glycerol [21–23] or galactose  as carbon sources. Unlike budding yeast, fission yeast cannot grow on ethanol because it lacks the glyoxylate cycle . Although glucose represses respiration in fission yeast, this effect is weaker than in budding yeast , and low glucose concentrations lead to increased respiration . Unlike budding yeast, fission yeast cannot tolerate the loss of mitochondrial DNA , which may reflect the inability to produce mitochondrial membrane potential in the absence of both the ETC and ATP synthase functions . Thus, fission yeast is much more sensitive than budding yeast to mutations affecting mitochondrial functions even in the presence of glucose and many of its genes involved in respiration are essential.
Here we provide systematic analyses of energy metabolism in fission yeast using both functional and expression profiling to explore the genetic basis and regulatory processes that tune fermentation and respiration to available carbon sources. We also report novel insights into acetyl-CoA production and into the retrograde response which communicates mitochondrial damage to nuclear gene expression. These analyses provide a rich framework to inform future studies on energy metabolism.
Genome-wide screens for genes functioning in respiration
We applied a self-organizing map algorithm to visualise the colony size ratios from all nine screens (Additional file 2: Figure S1). Two distinct clusters contained the mutants whose growth was compromised on respiratory media, either mainly in the prototroph background (cluster P) or in the auxotroph background (cluster A). Both of these clusters were significantly enriched for genes encoding mitochondrial proteins based on Gene Ontology (GO) annotations (Additional file 2: Figure S1). The 88 mutants of cluster P showed the highest growth inhibition in the presence of respiration inhibitors and mostly showed subtle or no growth inhibition in the auxotroph background, whereas the 116 mutants of cluster A showed more growth inhibition in the auxotroph background (Fig. 2a). Only 34 mutants showed growth inhibition in both prototroph and auxotroph backgrounds (Fig. 2b). This core group comprises mostly genes encoding mitochondrial proteins and represents the most conservative hits (Additional file 2: Figure S2a). The distinct screening results from the prototroph and auxotroph libraries point to widespread genetic interactions between auxotroph markers and respiratory functions, highlighting the importance of considering effects from different strain backgrounds in genetic screens (see “Discussion”). Our combined screens using both auxotroph and prototroph backgrounds provide valuable complementary insights into the genetic basis of energy metabolism.
Among the 204 genes of clusters A and P, 18 were in common with the 47 genes annotated as affecting growth on glycerol in fission yeast and present in our experimental data  (Fig. 2c). These 18 genes were strongly enriched for those encoding mitochondrial proteins (Additional file 2: Figure S3). The limited overlap likely reflects differences in the mutant screens and assays used; also, most annotated genes have been identified in experiments using minimal media , which affect respiratory metabolism (Fig. 1). The respiratory-deficient phenotype shows high variability as noticed previously in budding yeast . The results from our screens therefore provide complementary information and fresh insights.
Among the 204 genes of clusters A and P, 171 have orthologs in budding yeast. Only 58 genes of that group were in common with the 350 conserved budding yeast genes implicated in respiratory functions (Fig. 2c). These 58 genes involved in respiration in both yeasts showed overall stronger growth defects in respiratory media than the 146 genes associated with respiration only in fission yeast (Fig. 2d). The 58 common genes were highly enriched for genes encoding mitochondrial proteins (65%). We conclude that as much as ~70% of the genes identified here affect respiration in fission yeast but not in budding yeast. On the other hand, over 80% of the respiration genes identified in budding yeast were not identified in our screens. Although some of the genes associated with respiration in only one yeast may reflect experimental noise or features of the particular screens, this comparison highlights substantial differences in respiratory metabolism between the two species.
Transcriptomes of cells growing in respiratory versus fermentative conditions
Most of the induced genes appeared randomly distributed along the three chromosomes, but several genes near chromosome ends featured large changes in differential expression (Fig. 3e). De-repression of normally silenced regions at chromosome ends also occurs during meiotic differentiation . Neighbouring genes can impact each other’s expression ; we therefore searched for instances where several neighbouring genes are differentially expressed. To this end, we determined the differential expression in sliding windows of ten neighbouring genes (coding or non-coding) (Fig. 3e). Besides the chromosome ends, this analysis uncovered several additional regions which contained at least seven of ten neighbouring genes that were induced or repressed (Fig. 3e). For example, the cluster at the left arm of chromosome 3 contains seven neighbouring genes that were coordinately induced on glycerol (Additional file 2: Figure S7); this cluster includes the genes encoding the high-affinity glucose transporter Ght1 and the Zwf2 enzyme for the pentose-phosphate pathway. We also noticed that at least 70% of the differentially expressed non-coding RNAs were positioned adjacent to a differentially expressed coding gene (Additional file 2: Figure S4; Additional file 1: Table S2), likely reflecting local changes in chromatin or cis-regulatory effects.
The co-regulated regions typically contained genes showing large changes in expression (Fig. 3e), consistent with the idea that highly expressed genes can impact their neighbourhood via chromatin changes . In some cases, the neighbouring genes had related functions. The gene cluster at the right end of chromosome 2 contains three genes encoding galactose metabolism enzymes (Gal1, Gal7 and Gal10) that were strongly induced on glycerol (Additional file 2: Figure S8). Notably, growth on galactose was specifically affected in our genetic screen by deletion of the gal1 or gal7 genes, and in the prototroph background also by deletion of the chromatin silencing genes cid12 and set3 (Additional file 2: Figure S9). These results raise the possibility that gal genes are regulated via changes in chromatin.
Tuning of energy metabolism
In the absence of glucose, genes for the main glycolytic pathway were repressed, with genes responsible for the last steps of fermentation, conversion of pyruvate to acetaldehyde and ethanol (adh4, pdc101), showing the strongest repression (Fig. 4). On the other hand, genes encoding enzymes of the TCA cycle and OXPHOS complex components were induced, reflecting re-direction of pyruvate to mitochondria.
Glycerol enters the glycolytic pathway through the action of three enzymes, Gld1, Dak1 and Dak2 ; gld1 and dak1 were induced on glycerol, as was fbp1, which encodes the main gluconeogenesis enzyme which produces glucose-6-P from glycerol. Glucose-6-P could fuel trehalose metabolism, the genes for which were also induced in glycerol (Fig. 4). Trehalose serves to store glucose but is also an antioxidant; the production of reactive oxygen species and thus the risk of oxidative damage are increased on respiratory media (which was also reflected by the induction of oxidative stress genes; Fig. 3c). Glucose-6-P can also be metabolised to pyruvate via the pentose phosphate pathway, the genes for which were induced on glycerol. Respiring cells could additionally benefit from flow through the pentose phosphate pathway because the resulting NADPH fuels the reduction of glutathione, which in turn can support antioxidant protection.
Sugars are directed through glycolysis to the TCA cycle, and genes for cytoplasmic and mitochondrial enzymes (e.g., prs5, ser2, ilv5, lys4) that use intermediate metabolites of this pathway for anabolic processes were down-regulated (Additional file 1: Table S2). This reduction of anabolic pathways likely reflects the slower growth rate on glycerol (Additional file 2: Figure S6) and thus decreased demand for biomolecule synthesis. Conversely, genes for enzymes that direct metabolites into the TCA cycle were induced, like those encoding glutamate dehydrogenases (Gdh1, Gdh2), which convert glutamate into the TCA cycle intermediate 2-oxoglutarate, or SPAC4H3.08, which is probably involved in beta oxidation to restore acetyl-CoA from fatty acids (Fig. 4). Taken together, our data reveal coherent transcriptome changes that mirror the metabolic rewiring under steady-state respiratory and fermentative conditions.
Acetyl-coenzyme A metabolism
The acetyl-CoA synthase Acs1 provides an alternative pathway for acetyl-CoA production from glucose (Fig. 5a) . The acs1 gene has been reported to be non-essential based on large-scale deletion analyses . We independently deleted acs1 and found that the deletion cells were not viable (Fig. 5e). We therefore conclude that Acs1 is actually essential and propose that this protein functions as the main enzyme for acetyl-CoA production in fission yeast. Consistent with this view, the substrate of Acs1, acetate, was sufficient to re-establish normal levels of histone acetylation under glucose depletion (Fig. 5f). It is unlikely that acetate was converted into glucose given that fission yeast lacks the glyoxylate cycle and thus cannot use acetate as a carbon source. We conclude that Acs1, but not the citrate lyase, is critical for acetyl-CoA synthesis under both respiratory and fermentative conditions.
Dynamic gene regulation during adaptation to respiratory medium
In budding yeast, the volume and protein content of mitochondria increase on respiratory media . We also observed more punctate mitochondrial patterns, suggesting extensive fission of mitochondria, in cells grown on glycerol compared to cells grown on glucose (Additional file 2: Figure S10). Given this adaptation to respiration, it seems surprising that only 63 transcripts for mitochondrial proteins were significantly induced on glycerol medium (Fig. 3d). This analysis included cells grown for several generations under steady-state respiratory or fermentative conditions; the main effects on gene regulation, however, may be transient and more pronounced shortly after the medium shift, when cells adapt to the new carbon source. Transient transcript changes in response to respiratory conditions such as stress or quiescence can lead to longer term changes in protein levels [47, 48]. To capture this dynamic transition, we used microarrays to profile gene expression before and at six time points after the shift from fermentative to respiratory medium.
We analysed the genes that were differentially induced or repressed at each time point, including the genes encoding mitochondrial proteins. Many more mitochondrial genes became induced than repressed after the glycerol shift; this induction was somewhat delayed compared to the other induced genes, with as many as 119 mitochondrial genes peaking in expression at 1 h after the medium shift (Fig. 6c). Thus, many mitochondrial genes were up-regulated in response to glycerol, peaking in expression ~15–30 minutes after the bulk of the other induced genes. Many mitochondrial genes were then induced again at 24 h when cells approached stationary phase (Fig. 6c).
We separated all genes detected by microarrays into 12 clusters based on their expression profiles across the time course (Additional file 2: Figure S11). Each cluster was analysed for enrichments of GO categories. The clusters showing substantial expression changes were strongly enriched in categories that represent distinct metabolic functions (Fig. 6d). Clusters containing genes important for respiration (TCA cycle and OXPHOS) were induced together, peaking in expression at 1 h after the medium shift. Most notably, all of the genes encoding the ETC and the ATP synthase complex were coordinately up-regulated upon the shift to glycerol (Additional file 2: Figure S12). Moreover, genes encoding enzymes involved in carbon metabolism, which were induced or repressed under steady-state growth on glycerol (marked with coloured squares in Fig. 4), were also differentially regulated during the adaptation to glycerol (Additional file 2: Figure S13). Again, most of these genes showed the highest induction or repression at 1 h after the medium shift, and this time point therefore shows the most pronounced changes with respect to gene regulation relevant for metabolism.
Among the down-regulated clusters, one was enriched in genes functioning in iron and pyruvate metabolism. Immediately after the shift to glycerol, genes involved in pyruvate production and its transformation to ethanol were strongly repressed (Fig. 6e). Intriguingly, genes for different isoforms of enolase, pyruvate decarboxylase and alcohol dehydrogenase were induced at corresponding time points (Fig. 6e). This finding raises the possibility that the enzymes used for the last steps of glycolysis and for fermentation are replaced by these isoforms, which may have specialised functions in shifting the metabolism towards respiration. Moreover, the mpc1 and mpc2 genes, encoding mitochondrial pyruvate importers, were transiently induced at 1 h (Fig. 6e).
It is noteworthy that almost all the gene expression changes detected after 1 h in glycerol were largely repeated when cells approached stationary phase at 24 h. This finding suggests that the cells undergo similar metabolic changes under conditions of nutrient shortage and diminished growth. This finding suggests that the gene expression and metabolic changes during the transition to respiratory growth are similar to those during entry into stationary phase when nutrients become limiting and growth diminishes.
We conclude that during the transition from fermentative to respiratory growth the expression of many genes functioning in key metabolic pathways is strongly regulated, with maximal changes around 1 h after the shift to glycerol. Many of these genes, however, remain differentially expressed during steady-state conditions as reflected by the strong overall agreement between our RNA-seq and microarray data. Genes encoding isoforms of metabolic enzymes are often antagonistically regulated during the adaptation from fermentative to respiratory growth, raising the possibility that they have specialised roles in either growth condition.
Communication between mitochondria and nucleus: defining a retrograde response
Mitochondrial damage can impact nuclear transcription through the retrograde signalling response . In budding yeast, loss of mitochondrial DNA or chemical inhibition of the ETC lead to nuclear gene regulation in response to the mitochondrial dysfunction . In fission yeast, different deletion mutants of respiratory metabolism genes show altered expression of a similar group of nuclear transcripts, suggesting the existence of a retrograde response [51, 52].
Most of the genes that were repressed in the retrograde response encode mitochondrial proteins, most notably components of the ETC and ATP synthase complex (Fig. 7c, d). Block or dysfunction of the ETC leads to increased production of reactive oxygen species, and the down-regulation of OXPHOS genes can protect cells by reducing oxidative damage. Surprisingly, genes encoding the respiratory complex 2 were not repressed in the three data sets (Fig. 7d). The succinate dehydrogenase of complex 2 is part of the TCA cycle; it may therefore be necessary to maintain complex 2 activity because metabolites produced by the TCA cycle are important for anabolic reactions.
The 28 genes induced during the retrograde response (Fig. 7c) were enriched for CESR and oxidative stress (P <9.0E-10 and <1.7E-8, respectively). The induced retrograde response also included nine genes which encode oxidoreductases, some of which have poorly understood functions. Increased fermentation might lead to an accumulation of NADH in the cytoplasm , and up-regulation of cytoplasmic oxidoreductases could help to stabilise the cellular redox balance. The high-affinity glucose transporter gene ght5  was also induced (Fig. 7c). These results are consistent with the higher glucose consumption observed in antimycin A-treated cells (Fig. 7a). When the carbon flux is restricted to cytoplasmic glycolysis, cells need to utilise more glucose to provide similar amounts of ATP for supporting growth rates similar to untreated cells. Taken together, we define here a retrograde response in fission yeast. This response involves both the repression and induction of distinct, functionally coherent groups of genes that together may ameliorate the effects of mitochondrial damage.
We investigated energy metabolism of fission yeast using complementary functional and expression profiling approaches. We screened a non-essential deletion library for mutants with deficient respiratory growth, compared the transcriptomes of cells proliferating under steady-state fermentative and respiratory conditions, analysed the dynamic changes in gene expression during adaptation to respiratory conditions and identified critical enzymes for acetyl-CoA production and genes regulated in response to mitochondrial dysfunction.
Only few genes were both differentially expressed on respiratory media and also required for respiratory growth (Fig. 3a). This finding is consistent with results in budding yeast showing that genes that are differentially expressed under a given condition overlap only little with the corresponding mutants that show phenotypes under this condition . While genetic screens tend to uncover response regulators, expression profiling typically identifies metabolic pathways and responses. Accordingly, the changes in transcript levels as a function of different carbon sources reflect predicted changes in energy metabolism (Fig. 4). These coherent changes fit the expected metabolic differences between fermenting and respiring cells very well [2, 5], and expression profiling can thus serve to probe cellular metabolic states. The higher uptake of intermediary metabolites for catabolic processes in fermenting cells, together with more rapid proliferation, resembles the metabolic changes in cancer cells for which it can serve as a basic model system .
The manipulation of energy metabolism using different carbon sources has been used to assay mitochondrial function and to screen for respiratory mutants in budding yeast . Different types of growth media, even with identical carbon source, can also affect energy metabolism and need to be carefully considered for such experiments (Fig. 1). Our results, supported by literature evidence, provide a general overview of the genetic and regulatory basis of energy metabolism in fission yeast. The genetic basis for respiratory growth appears to be remarkably distinct between fission and budding yeast: we uncovered 154 genes that are important for respiratory growth in S. pombe but whose orthologs have not been identified in corresponding S. cerevisiae screens or which do not have orthologs in S. cerevisiae. Out of these 154 genes, 92 are conserved in metazoa, with at least 15 reported to be associated with human diseases . Fission yeast thus provides a valuable complementary model system to associate energy metabolism with basic cellular function. On the other hand, the genes being differentially expressed as a function of energy metabolism showed much higher overall concordance between the two yeasts: for example, ~50% of the S. pombe genes regulated in glycerol are also regulated during the S. cerevisiae post-diauxic shift  (Additional file 2: Figure S14). This finding is in accordance with other processes, like the cellular stress response , where regulatory mechanisms evolve more rapidly than the genes being regulated.
Our genetic screens using both auxotroph and prototroph mutant libraries uncovered strong genetic interactions between the auxotroph mutants (ade6, leu1 or ura4) and deletion mutants affecting respiratory function. A large number of mutants were required for respiratory growth specifically in either the auxotroph or prototroph backgrounds (Fig. 2b). For example, the mutants only identified in the auxotroph background require the presence of auxotroph mutants for the respiratory phenotype to manifest, pointing to negative genetic interactions with the auxotroph markers. The ura4 deletion mutant, defective in uracil synthesis, is likely the main cause of this effect for the following reasons: 1) this deletion results in decreased growth on glycerol ; 2) the pyrimidine synthesis pathway is linked to reduction of coenzyme Q which may directly impact the ETC and antioxidant defence [49, 60]; and 3) this deletion affects cell wall integrity , which could indirectly compromise respiratory metabolism. Such genetic interactions may complicate functional analyses of the corresponding respiratory genes. The distinct results obtained from mutant libraries differing in their genetic background highlight the importance of considering effects from auxotrophies, especially when studying metabolic processes. This point is also highlighted by a recent report of extensive gene expression epistasis as a function of the metabolic-genetic background in S. cerevisiae strains . Here we obtained valuable complementary insights into the genetics of energy metabolism by using both auxotroph and prototroph libraries.
The shift from fermentation to respiration is controlled by multiple pathways. The glucose-sensing Pka1 pathway is repressed during respiration . Our RNA-seq data, however, showed that transcripts functioning in the Pka1 pathway are slightly higher expressed in respiratory than in fermentative conditions (Additional file 2: Figure S15). This finding could reflect a sensitization, in that cells prepare to rapidly return to fermentation when conditions allow. We also found that deletion of tor1 (functioning in the TORC2 complex) inhibits respiratory growth, adding to recent evidence that TORC2 is involved in the regulation of carbon metabolism [39, 62].
Genes encoding the two transcription factors Rsv1 and Rsv2 were strongly induced in response to respiratory conditions; their orthologs in budding yeast (Mig1–3) are implicated in glucose repression . The rsv1 deletion was missing from our deletion library, and the rsv2 deletion did not affect respiratory growth. Rsv1 is required to maintain viability under glucose depletion during stationary phase . Rsv2 has been shown to induce stress-related genes during spore formation, while Rsv1 represses glucose metabolism genes . The scr1 gene, encoding a transcription factor related to Rsv1/2, was also induced in response to respiratory conditions in our experiments, consistent with data showing that scr1 is induced in response to glucose starvation ; Scr1 is regulated by the Ssp2 kinase and involved in glucose derepression . Php3 is another transcriptional regulator involved in energy metabolism based on our data, as it was required for respiratory growth. Php3 is a component of the CCAAT-binding complex, which regulates the glucose-repressible fbp1 gene in S. pombe . Accordingly, the orthologous complex in budding yeast (Hap2–5) acts as the main activator of respiratory genes . The Reb1 transcription factor  was also required for respiratory growth, consistent with findings that it functions as an activator of nuclear-encoded respiratory genes (M. R.-L., unpublished data). The transcripts for several other transcription factors were induced during respiratory conditions, which may include additional regulators of energy metabolism (Additional file 1: Tables S2 and S3).
Our genetic screens identified several chromatin proteins that are involved in respiratory growth, including the argonaute silencing factor Ago1  and the chromatin remodelling complex subunits Rsc1 (RSC complex)  and Ies2 (Ino80 complex). Moreover, we detected several clusters of co-regulated genes in respiratory medium (Fig. 3e), and the chromatin silencing factors Cid12 and Set3 were required for respiratory growth on galactose (Additional file 2: Figure S9). These findings suggest that changes in chromatin states are important in regulating the metabolic shift between fermentation and respiration.
We also uncovered several RNA-binding proteins in our genetic screens as being important for respiratory growth. Examples are Mlo3 , the polyA-binding protein Nab2  and Mcp2, an ortholog of budding yeast Puf3 that regulates the translation and stability of mRNAs encoding mitochondrial proteins . These results indicate that post-transcriptional levels of regulation play important roles in the control of energy metabolism.
Respiration is essential for meiotic differentiation . Our data indicate that the link between these two processes could be hardwired, in that respiration may even be sufficient to trigger meiotic differentiation. Under respiratory conditions, the meiotic gene expression program was induced and cells managed to efficiently undergo meiosis and sporulation on yeast extract media containing glycerol (Additional file 2: Figure S5). This is a surprising finding as yeast extract is normally a strong repressor of meiotic differentiation in fission yeast.
In multicellular eukaryotes, the ATP citrate lyase activity generates acetyl-CoA used for histone acetylation and thus epigenetically links gene expression with glucose metabolism [42, 78]. The citrate lyase is absent in budding yeast. It has not been known how carbon metabolism is linked to acetyl-CoA synthesis in fission yeast. We show here that the acetyl-CoA synthase Acs1, rather than the citrate lyase, is an essential player in cellular acetyl-CoA synthesis, as is the case in budding yeast . The acetyl-CoA synthase is instrumental for the growth of cancer cells [80, 81], further illustrating the similarities between yeast and cancer metabolism. Further work will be required to test whether the fission yeast citrate lyase has any specialised role in acetyl-CoA metabolism.
We provide a systematic survey of genes that are required for respiration and analyse gene regulation during the switch from fermentation to respiration. These two sets of genes show remarkably little overlap and provide complementary insights into the functional richness and intricate regulation of energy metabolism. We also study aspects of acetyl-CoA metabolism and define the retrograde response to prevent damage from dysfunctional mitochondria in fission yeast. Our analyses provide rich information on metabolic processes and also serve as a framework for future research on energy metabolism and crosstalk between respiration and other cellular processes.
Yeast strains and growth media
Strains used in this study are listed in Additional file 1: Table S5. For the genetic screening, the auxotroph Bioneer library v2.0  or its prototroph derivative  was used. Cells were grown in rich yeast extract (YE) medium with 3% glucose (fermentative medium) or 2% glucose (Fig. 1) or in minimal (EMM) medium with 2% glucose. For respiratory media, YE medium was supplemented with both 3% glycerol and 0.1% glucose or with 2% galactose and 0.1% glucose. For screening of respiratory deficient mutants, solid YE medium was additionally supplemented with adenine, uracil, leucine, histidine and lysine (YES). Where indicated, media were supplemented with antimycin A (0.1 ng/ml for genetic screening or 0.15 μg/ml for inhibition of respiration) or with 2,4-dinitrophenol (1 μg/ml) . For cell mating, malt extract agar (MEA) medium was used.
Genetic screens for respiratory mutants
The Bioneer haploid deletion mutant library v2.0 (3005 mutants) or prototroph library (2847 strains) was arrayed using a RoToR HDA robot (Singer Instruments) onto solid YES media in 1536 format, with each mutant spotted in quadruplicate. Subsequently, arrays were copied onto fermentative and respiratory media. Plates were incubated at 32 °C for 2 days, and images were acquired using a Canon camera and multidoc imagining system (UVP). Quantification of colony sizes was performed with the gitter R package . Colony sizes were normalised to the median colony size of the plate, and colony sizes for each mutant strain were calculated as a median of four replicate colonies analysed per mutant. Subsequently colony size ratios of strains grown on respiratory relative to fermentative media were calculated.
The colony size ratios calculated for the different screens (Additional file 1: Table S1) were imported into GeneSpring GX13 software (Agilent Technologies). Lower values of colony size ratios were set at a threshold of 0.2. Mutants with data missing for any of the conditions were removed. Using the Self-Organizing Map clustering method with default settings, the mutants were grouped into 12 clusters. GO category enrichments in each cluster were calculated using the AnGeLi web tool . S. cerevisiae genes associated with phenotype categories “respiratory growth: decreased rate” and “respiratory growth: absent” were checked for S. pombe orthologues using the manually curated orthologue list available in PomBase . This list was then restricted to the strains included in the Bioneer collection v2.0 and compared to genes identified in the screen and to the S. pombe phenotype category FYPO:0001934 (“abolished cell growth on glycerol carbon source”) .
Wild-type yeast cells (972 h-) were grown on YES or YE media with 3% glycerol and 0.1% glucose and harvested at early exponential growth phase (OD 0.5), and total RNA was isolated by hot-phenol extraction . RNA quality was assessed on a Bioanalyzer instrument (Agilent), treated with DNase (Turbo DNA-free, Ambion) and subsequently 4 μg of RNA was treated with a beta version of Ribo-Zero Magnetic Gold Kit Yeast (Epicentre) to deplete rRNAs. RNA-seq libraries were prepared from rRNA-free RNA using a strand-specific library preparation protocol  and sequenced on an Illumina HiSeq instrument. Sequence data analysis was carried out as described , with the exception of using only annotated regions (7022 annotated genes) and 51-bp reads. The significance of overlapping gene lists was calculated with the hypergeometric probability formula using the phyper R function.
Generation of deletion mutants and tetrad dissection
Diploid strains were selected on EMM media from a cross of ade6-210 h + and ade6-216 h- strains and afterwards grown on YES media. Diploids were transformed with a deletion cassette for acs1 containing the hygromycin marker [86, 87]. Positive clones were selected and the deletion junctions were checked by PCR. The diploid strain was then sporulated on MEA medium, and tetrads were dissected on YES medium using a MSM 400 dissection microscope (Singer Instruments). The grown spore colonies were then replicated onto YES medium with hygromycin (0.1 mg/ml).
Total protein extracts were prepared using the FastPrep-24 equipment (MP) in PBS buffer with protease inhibitors. Protein concentrations of the soluble fractions were adjusted using the BCA Protein Assay (Thermo Scientific). About 10 μg of proteins from the soluble fractions were separated on the NuPAGE 4–12% acrylamide gels (Novex) and transferred to nitrocellulose membranes (mini Trans-Blot Cell BioRad). Antibodies against actin, histone H3 and histone H3K9 (Ambion), and appropriate secondary antibodies, were used according to the manufacturer’s instructions.
Time course and antimycin A experiments using microarrays
For the time course analyses, cells were grown to early exponential phase (OD 0.5) in fermentative medium, washed once in sterile water and re-suspended in the same volume of respiratory medium. Transcriptomes were analysed before (time point 0) and at six time points after the change of carbon source, up to 24 h. RNA from cell pellets was isolated using hot phenol extraction, followed by labelling of the single samples and a pool of all the samples which served as reference . Agilent 8 × 15 K custom-made S. pombe expression microarrays were used, and hybridizations and subsequent washes were performed according to the manufacturer’s protocols. Microarrays were scanned using a GenePix 4000 B laser scanner, and fluorescence signals were analysed using GenePix Pro software (Axon Instruments). The resulting data were processed using customized R scripts for quality control and normalization and analysed using GeneSpring GX13 [84, 88]. Two independent biological repeats with a dye swap were performed. The K-means algorithm was used for clustering (Additional file 2: Figure S11).
For investigating the retrograde response, wild-type cells with or without antimycin A treatment were grown in YES medium to OD ~0.5. The RNA of these cells was then isolated and processed for microarray analysis as described above. Differentially labelled RNA from treated versus untreated cells was analysed from three independent biological repeats including a dye swap.
Determining glucose and ethanol concentration in media
Cells were grown in YES medium with or without antimycin A (0.15 μg/ml). At the indicated time points, 1 ml of cell culture was precipitated, and the supernatant was assayed for ethanol and glucose concentrations using the Ethanol Assay Kit (Abcam) or Glucose (HK) Assay (Sigma), respectively.
We thank Ivan Gout, Antonia Lock, Markus Ralser and Peter Rich for comments on the manuscript, Christopher Herbert for advice and Mimoza Hoti for help with the ArrayExpress submission.
This work was funded by a Wellcome Trust Senior Investigator Award and a Royal Society Wolfson Research Merit Award to J.B. (grant number 095598/Z/11/Z) and a Royal Society Newton International Fellowship to M.M. (ref. NF130840).
Availability of data and material
RNA-sequencing data have been submitted to the European Nucleotide Archive under accession number PRJEB12865. Microarray data have been submitted to ArrayExpress under accession numbers E-MTAB-4518 and E-MTAB-4520.
MM and JB designed the experiments and drafted the manuscript; MM performed most of the experiments and analyses; DAB performed the initial analysis of RNA-seq data; MRL helped with microarray experiments; CR helped with analysing the genetic screen data; NGC performed growth analyses of selected respiratory mutants; GCS and DAB helped and advised on aspects of data analysis. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Ethics approval and consent to participate
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- Wallace DC, Fan W. Energetics, epigenetics, mitochondrial genetics. Mitochondrion. 2010;10:12–31.View ArticlePubMedGoogle Scholar
- Lunt SY, Vander Heiden MG. Aerobic glycolysis: meeting the metabolic requirements of cell proliferation. Annu Rev Cell Dev Biol. 2011;27:441–64.View ArticlePubMedGoogle Scholar
- Molenaar D, van Berlo R, de Ridder D, Teusink B. Shifts in growth strategies reflect tradeoffs in cellular economics. Mol Syst Biol. 2009;5:323.View ArticlePubMedPubMed CentralGoogle Scholar
- Turcotte B, Liang XB, Robert F, Soontorngun N. Transcriptional regulation of nonfermentable carbon utilization in budding yeast. FEMS Yeast Res. 2010;10:2–13.View ArticlePubMedGoogle Scholar
- DeRisi JL, Iyer VR, Brown PO. Exploring the metabolic and genetic control of gene expression on a genomic scale. Science. 1997;278:680–6.View ArticlePubMedGoogle Scholar
- Pancaldi V, Schubert F, Bähler J. Meta-analysis of genome regulation and expression variability across hundreds of environmental and genetic perturbations in fission yeast. Mol Biosyst. 2010;6:543–52.View ArticlePubMedGoogle Scholar
- Brauer MJ, Huttenhower C, Airoldi EM, Rosenstein R, Matese JC, Gresham D, Boer VM, Troyanskaya OG, Botstein D. Coordination of growth rate, cell cycle, stress response, and metabolic activity in yeast. Mol Biol Cell. 2008;19:352–67.View ArticlePubMedPubMed CentralGoogle Scholar
- Chen Z, Odstrcil EA, Tu BP, McKnight SL. Restriction of DNA replication to the reductive phase of the metabolic cycle protects genome integrity. Science. 2007;316:1916–9.View ArticlePubMedGoogle Scholar
- Silverman SJ, Petti AA, Slavov N, Parsons L, Briehof R, Thiberge SY, Zenklusen D, Gandhi SJ, Larson DR, Singer RH, Botstein D. Metabolic cycling in single yeast cells from unsynchronized steady-state populations limited on glucose or phosphate. Proc Natl Acad Sci U S A. 2010;107:6946–51.View ArticlePubMedPubMed CentralGoogle Scholar
- Takeda K, Starzynski C, Mori A, Yanagida M. The critical glucose concentration for respiration-independent proliferation of fission yeast. Schizosaccharomyces pombe. Mitochondrion. 2015;22:91–5.View ArticlePubMedGoogle Scholar
- Roux AE, Leroux A, Alaamery MA, Hoffman CS, Chartrand P, Ferbeyre G, Rokeach LA. Pro-aging effects of glucose signaling through a G protein-coupled glucose receptor in fission yeast. PLoS Genet. 2009;5, e1000408.View ArticlePubMedPubMed CentralGoogle Scholar
- Gruning NM, Rinnerthaler M, Bluemlein K, Mulleder M, Wamelink MM, Lehrach H, Jakobs C, Breitenbach M, Ralser M. Pyruvate kinase triggers a metabolic feedback loop that controls redox metabolism in respiring cells. Cell Metab. 2011;14:415–27.View ArticlePubMedPubMed CentralGoogle Scholar
- Huang Z, Cai L, Tu BP. Dietary control of chromatin. Curr Opin Cell Biol. 2015;34:69–74.View ArticlePubMedPubMed CentralGoogle Scholar
- Chandel NS. Evolution of mitochondria as signaling organelles. Cell Metab. 2015;22:204–6.View ArticlePubMedGoogle Scholar
- Steinmetz LM, Scharfe C, Deutschbauer AM, Mokranjac D, Herman ZS, Jones T, Chu AM, Giaever G, Prokisch H, Oefner PJ, Davis RW. Systematic screen for human disease genes in yeast. Nat Genet. 2002;31:400–4.PubMedGoogle Scholar
- Jambhekar A, Amon A. Control of meiosis by respiration. Curr Biol. 2008;18:969–75.View ArticlePubMedPubMed CentralGoogle Scholar
- Ocampo A, Liu J, Schroeder EA, Shadel GS, Barrientos A. Mitochondrial respiratory thresholds regulate yeast chronological life span and its extension by caloric restriction. Cell Metab. 2012;16:55–67.View ArticlePubMedPubMed CentralGoogle Scholar
- Zampar GG, Kummel A, Ewald J, Jol S, Niebel B, Picotti P, Aebersold R, Sauer U, Zamboni N, Heinemann M. Temporal system-level organization of the switch from glycolytic to gluconeogenic operation in yeast. Mol Syst Biol. 2013;9:651.View ArticlePubMedPubMed CentralGoogle Scholar
- Yaffe MP, Stuurman N, Vale RD. Mitochondrial positioning in fission yeast is driven by association with dynamic microtubules and mitotic spindle poles. Proc Natl Acad Sci U S A. 2003;100:11424–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Schafer B, Hansen M, Lang BF. Transcription and RNA-processing in fission yeast mitochondria. RNA. 2005;11:785–95.View ArticlePubMedPubMed CentralGoogle Scholar
- Heslot H, Goffeau A, Louis C. Respiratory metabolism of a "petite negative" yeast Schizosaccharomyces pombe 972 h. J Bacteriol. 1970;104:473–81.PubMedPubMed CentralGoogle Scholar
- Klement T, Dankmeyer L, Hommes R, van Solingen P, Buchs J. Acetate-glycerol cometabolism: cultivating Schizosaccharomyces pombe on a non-fermentable carbon source in a defined minimal medium. J Biosci Bioeng. 2011;112:20–5.View ArticlePubMedGoogle Scholar
- Klein T, Heinzle E, Schneider K. Metabolic fluxes in Schizosaccharomyces pombe grown on glucose and mixtures of glycerol and acetate. Appl Microbiol Biotechnol. 2013;97:5013–26.View ArticlePubMedGoogle Scholar
- Chiron S, Gaisne M, Guillou E, Belenguer P, Clark-Walker GD, Bonnefoy N. Studying mitochondria in an attractive model: Schizosaccharomyces pombe. Methods Mol Biol. 2007;372:91–105.View ArticlePubMedGoogle Scholar
- Rhind N, Chen Z, Yassour M, Thompson DA, Haas BJ, Habib N, Wapinski I, Roy S, Lin MF, Heiman DI, et al. Comparative functional genomics of the fission yeasts. Science. 2011;332:930–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Zuin A, Gabrielli N, Calvo IA, Garcia-Santamarina S, Hoe KL, Kim DU, Park HO, Hayles J, Ayte J, Hidalgo E. Mitochondrial dysfunction increases oxidative stress and decreases chronological life span in fission yeast. PLoS One. 2008;3, e2842.View ArticlePubMedPubMed CentralGoogle Scholar
- Moreno S, Klar A, Nurse P. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 1991;194:795–823.View ArticlePubMedGoogle Scholar
- Zuin A, Carmona M, Morales-Ivorra I, Gabrielli N, Vivancos AP, Ayte J, Hidalgo E. Lifespan extension by calorie restriction relies on the Sty1 MAP kinase stress pathway. EMBO J. 2010;29:981–91.View ArticlePubMedPubMed CentralGoogle Scholar
- Egli T. Microbial growth and physiology: a call for better craftsmanship. Front Microbiol. 2015;6:287.View ArticlePubMedPubMed CentralGoogle Scholar
- Kim DU, Hayles J, Kim D, Wood V, Park HO, Won M, Yoo HS, Duhig T, Nam M, Palmer G, et al. Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe. Nat Biotechnol. 2010;28:617–23.View ArticlePubMedPubMed CentralGoogle Scholar
- Alam MT, Zelezniak A, Mülleder M, Shliaha P, Schwarz R, Capuano F, Vowinckel J, Radmaneshfar E, Krüger A, Calvani E, et al. The metabolic background is a global player in Saccharomyces gene expression epistasis. Nat Microbiol. 2016;1:15030.Google Scholar
- Sideri T, Rallis C, Bitton DA, Lages BM, Suo F, Rodriguez-Lopez M, Du LL, Bähler J. Parallel profiling of fission yeast deletion mutants for proliferation and for lifespan during long-term quiescence. G3 (Bethesda). 2014;5:145–55.Google Scholar
- Harris MA, Lock A, Bähler J, Oliver SG, Wood V. FYPO: the fission yeast phenotype ontology. Bioinformatics. 2013;29:1671–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Merz S, Westermann B. Genome-wide deletion mutant analysis reveals genes required for respiratory growth, mitochondrial genome maintenance and mitochondrial protein synthesis in Saccharomyces cerevisiae. Genome Biol. 2009;10:R95.View ArticlePubMedPubMed CentralGoogle Scholar
- Chen D, Toone WM, Mata J, Lyne R, Burns G, Kivinen K, Brazma A, Jones N, Bähler J. Global transcriptional responses of fission yeast to environmental stress. Mol Biol Cell. 2003;14:214–29.View ArticlePubMedPubMed CentralGoogle Scholar
- Chen D, Wilkinson CR, Watt S, Penkett CJ, Toone WM, Jones N, Bähler J. Multiple pathways differentially regulate global oxidative stress responses in fission yeast. Mol Biol Cell. 2008;19:308–17.View ArticlePubMedPubMed CentralGoogle Scholar
- Mata J, Lyne R, Burns G, Bähler J. The transcriptional program of meiosis and sporulation in fission yeast. Nat Genet. 2002;32:143–7.View ArticlePubMedGoogle Scholar
- Batada NN, Urrutia AO, Hurst LD. Chromatin remodelling is a major source of coexpression of linked genes in yeast. Trends Genet. 2007;23:480–4.View ArticlePubMedGoogle Scholar
- Saitoh S, Mori A, Uehara L, Masuda F, Soejima S, Yanagida M. Mechanisms of expression and translocation of major fission yeast glucose transporters regulated by CaMKK/phosphatases, nuclear shuttling, and TOR. Mol Biol Cell. 2015;26:373–86.View ArticlePubMedPubMed CentralGoogle Scholar
- Matsuzawa T, Ohashi T, Hosomi A, Tanaka N, Tohda H, Takegawa K. The gld1+ gene encoding glycerol dehydrogenase is required for glycerol metabolism in Schizosaccharomyces pombe. Appl Microbiol Biotechnol. 2010;87:715–27.View ArticlePubMedGoogle Scholar
- Hynes MJ, Murray SL. ATP-citrate lyase is required for production of cytosolic acetyl coenzyme A and development in Aspergillus nidulans. Eukaryot Cell. 2010;9:1039–48.View ArticlePubMedPubMed CentralGoogle Scholar
- Wellen KE, Hatzivassiliou G, Sachdeva UM, Bui TV, Cross JR, Thompson CB. ATP-citrate lyase links cellular metabolism to histone acetylation. Science. 2009;324:1076–80.View ArticlePubMedPubMed CentralGoogle Scholar
- Choudhary C, Weinert BT, Nishida Y, Verdin E, Mann M. The growing landscape of lysine acetylation links metabolism and cell signalling. Nat Rev Mol Cell Biol. 2014;15:536–50.View ArticlePubMedGoogle Scholar
- Kwon ES, Jeong JH, Roe JH. Inactivation of homocitrate synthase causes lysine auxotrophy in copper/zinc-containing superoxide dismutase-deficient yeast Schizosaccharomyces pombe. J Biol Chem. 2006;281:1345–51.View ArticlePubMedGoogle Scholar
- Nakamura T, Pluskal T, Nakaseko Y, Yanagida M. Impaired coenzyme A synthesis in fission yeast causes defective mitosis, quiescence-exit failure, histone hypoacetylation and fragile DNA. Open Biol. 2012;2:120117.View ArticlePubMedPubMed CentralGoogle Scholar
- Egner A, Jakobs S, Hell SW. Fast 100-nm resolution three-dimensional microscope reveals structural plasticity of mitochondria in live yeast. Proc Natl Acad Sci U S A. 2002;99:3370–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Lackner DH, Schmidt MW, Wu S, Wolf DA, Bähler J. Regulation of transcriptome, translation, and proteome in response to environmental stress in fission yeast. Genome Biol. 2012;13:R25.View ArticlePubMedPubMed CentralGoogle Scholar
- Marguerat S, Schmidt A, Codlin S, Chen W, Aebersold R, Bähler J. Quantitative analysis of fission yeast transcriptomes and proteomes in proliferating and quiescent cells. Cell. 2012;151:671–83.View ArticlePubMedPubMed CentralGoogle Scholar
- Kotiadis VN, Duchen MR, Osellame LD. Mitochondrial quality control and communications with the nucleus are important in maintaining mitochondrial function and cell health. Biochim Biophys Acta. 1840;2014:1254–65.Google Scholar
- Epstein CB, Waddle JA, Hale W, Dave V, Thornton J, Macatee TL, Garner HR, Butow RA. Genome-wide responses to mitochondrial dysfunction. Mol Biol Cell. 2001;12:297–308.View ArticlePubMedPubMed CentralGoogle Scholar
- Guha S, Lopez-Maury L, Shaw M, Bähler J, Norbury CJ, Agashe VR. Transcriptional and cellular responses to defective mitochondrial proteolysis in fission yeast. J Mol Biol. 2011;408:222–37.View ArticlePubMedGoogle Scholar
- Hasan A, Cotobal C, Duncan CD, Mata J. Systematic analysis of the role of RNA-binding proteins in the regulation of RNA stability. PLoS Genet. 2014;10, e1004684.View ArticlePubMedPubMed CentralGoogle Scholar
- Hoffmann B, Nickel J, Speer F, Schafer B. The 3' ends of mature transcripts are generated by a processosome complex in fission yeast mitochondria. J Mol Biol. 2008;377:1024–37.View ArticlePubMedGoogle Scholar
- Rodriguez-Sanchez L, Rodriguez-Lopez M, Garcia Z, Tenorio-Gomez M, Schvartzman JB, Krimer DB, Hernandez P. The fission yeast rDNA-binding protein Reb1 regulates G1 phase under nutritional stress. J Cell Sci. 2011;124:25–34.View ArticlePubMedGoogle Scholar
- Vemuri GN, Eiteman MA, McEwen JE, Olsson L, Nielsen J. Increasing NADH oxidation reduces overflow metabolism in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 2007;104:2402–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Heiland S, Radovanovic N, Hofer M, Winderickx J, Lichtenberg H. Multiple hexose transporters of Schizosaccharomyces pombe. J Bacteriol. 2000;182:2153–62.View ArticlePubMedPubMed CentralGoogle Scholar
- Yeger-Lotem E, Riva L, Su LJ, Gitler AD, Cashikar AG, King OD, Auluck PK, Geddie ML, Valastyan JS, Karger DR, et al. Bridging high-throughput genetic and transcriptional data reveals cellular responses to alpha-synuclein toxicity. Nat Genet. 2009;41:316–23.View ArticlePubMedPubMed CentralGoogle Scholar
- Diaz-Ruiz R, Rigoulet M, Devin A. The Warburg and Crabtree effects: on the origin of cancer cell energy metabolism and of yeast glucose repression. Biochim Biophys Acta. 1807;2011:568–76.Google Scholar
- Wood V, Harris MA, McDowall MD, Rutherford K, Vaughan BW, Staines DM, Aslett M, Lock A, Bähler J, Kersey PJ, Oliver SG. PomBase: a comprehensive online resource for fission yeast. Nucleic Acids Res. 2012;40:D695–9.View ArticlePubMedGoogle Scholar
- Fang J, Uchiumi T, Yagi M, Matsumoto S, Amamoto R, Takazaki S, Yamaza H, Nonaka K, Kang D. Dihydro-orotate dehydrogenase is physically associated with the respiratory complex and its loss leads to mitochondrial dysfunction. Biosci Rep. 2013;33, e00021.PubMedPubMed CentralGoogle Scholar
- Matsuo Y, Nishino K, Mizuno K, Akihiro T, Toda T, Matsuo Y, Kaino T, Kawamukai M. Polypeptone induces dramatic cell lysis in ura4 deletion mutants of fission yeast. PLoS One. 2013;8, e59887.View ArticlePubMedPubMed CentralGoogle Scholar
- Hatano T, Morigasaki S, Tatebe H, Ikeda K, Shiozaki K. Fission yeast Ryh1 GTPase activates TOR Complex 2 in response to glucose. Cell Cycle. 2015;14:848–56.View ArticlePubMedPubMed CentralGoogle Scholar
- Hao Z, Furunobu A, Nagata A, Okayama H. A zinc finger protein required for stationary phase viability in fission yeast. J Cell Sci. 1997;110(Pt 20):2557–66.PubMedGoogle Scholar
- Mata J, Wilbrey A, Bähler J. Transcriptional regulatory network for sexual differentiation in fission yeast. Genome Biol. 2007;8:R217.View ArticlePubMedPubMed CentralGoogle Scholar
- Matsuzawa T, Fujita Y, Tohda H, Takegawa K. Snf1-like protein kinase Ssp2 regulates glucose derepression in Schizosaccharomyces pombe. Eukaryot Cell. 2012;11:159–67.View ArticlePubMedPubMed CentralGoogle Scholar
- Janoo RT, Neely LA, Braun BR, Whitehall SK, Hoffman CS. Transcriptional regulators of the Schizosaccharomyces pombe fbp1 gene include two redundant Tup1p-like corepressors and the CCAAT binding factor activation complex. Genetics. 2001;157:1205–15.PubMedPubMed CentralGoogle Scholar
- Liu Z, Butow RA. Mitochondrial retrograde signaling. Annu Rev Genet. 2006;40:159–85.View ArticlePubMedGoogle Scholar
- Sullivan LB, Gui DY, Hosios AM, Bush LN, Freinkman E, Vander Heiden MG. Supporting aspartate biosynthesis is an essential function of respiration in proliferating cells. Cell. 2015;162:552–63.View ArticlePubMedPubMed CentralGoogle Scholar
- Birsoy K, Wang T, Chen WW, Freinkman E, Abu-Remaileh M, Sabatini DM. An essential role of the mitochondrial electron transport chain in cell proliferation is to enable aspartate synthesis. Cell. 2015;162:540–51.View ArticlePubMedPubMed CentralGoogle Scholar
- Mercier A, Watt S, Bähler J, Labbe S. Key function for the CCAAT-binding factor Php4 to regulate gene expression in response to iron deficiency in fission yeast. Eukaryot Cell. 2008;7:493–508.View ArticlePubMedPubMed CentralGoogle Scholar
- Encinar Del Dedo J, Gabrielli N, Carmona M, Ayte J, Hidalgo E. A cascade of iron-containing proteins governs the genetic iron starvation response to promote iron uptake and inhibit iron storage in fission yeast. PLoS Genet. 2015;11, e1005106.View ArticlePubMedPubMed CentralGoogle Scholar
- Todd BL, Stewart EV, Burg JS, Hughes AL, Espenshade PJ. Sterol regulatory element binding protein is a principal regulator of anaerobic gene expression in fission yeast. Mol Cell Biol. 2006;26:2817–31.View ArticlePubMedPubMed CentralGoogle Scholar
- Kowalik KM, Shimada Y, Flury V, Stadler MB, Batki J, Bühler M. The Paf1 complex represses small-RNA-mediated epigenetic gene silencing. Nature. 2015;520:248–52.View ArticlePubMedPubMed CentralGoogle Scholar
- Monahan BJ, Villen J, Marguerat S, Bähler J, Gygi SP, Winston F. Fission yeast SWI/SNF and RSC complexes show compositional and functional differences from budding yeast. Nat Struct Mol Biol. 2008;15:873–80.View ArticlePubMedPubMed CentralGoogle Scholar
- Thakurta AG, Gopal G, Yoon JH, Kozak L, Dhar R. Homolog of BRCA2-interacting Dss1p and Uap56p link Mlo3p and Rae1p for mRNA export in fission yeast. EMBO J. 2005;24:2512–23.View ArticlePubMedPubMed CentralGoogle Scholar
- Grenier St-Sauveur V, Soucek S, Corbett AH, Bachand F. Poly(A) tail-mediated gene regulation by opposing roles of Nab2 and Pab2 nuclear poly(A)-binding proteins in pre-mRNA decay. Mol Cell Biol. 2013;33:4718–31.View ArticlePubMedPubMed CentralGoogle Scholar
- Lee CD, Tu BP. Glucose-regulated phosphorylation of the PUF protein Puf3 regulates the translational fate of its bound mRNAs and association with RNA granules. Cell Rep. 2015;11:1638–50.View ArticlePubMedPubMed CentralGoogle Scholar
- Morciano P, Carrisi C, Capobianco L, Mannini L, Burgio G, Cestra G, De Benedetto GE, Corona DF, Musio A, Cenci G. A conserved role for the mitochondrial citrate transporter Sea/SLC25A1 in the maintenance of chromosome integrity. Hum Mol Genet. 2009;18:4180–8.View ArticlePubMedGoogle Scholar
- Takahashi H, McCaffery JM, Irizarry RA, Boeke JD. Nucleocytosolic acetyl-coenzyme a synthetase is required for histone acetylation and global transcription. Mol Cell. 2006;23:207–17.View ArticlePubMedGoogle Scholar
- Comerford SA, Huang Z, Du X, Wang Y, Cai L, Witkiewicz AK, Walters H, Tantawy MN, Fu A, Manning HC, et al. Acetate dependence of tumors. Cell. 2014;159:1591–602.View ArticlePubMedPubMed CentralGoogle Scholar
- Mashimo T, Pichumani K, Vemireddy V, Hatanpaa KJ, Singh DK, Sirasanagandla S, Nannepaga S, Piccirillo SG, Kovacs Z, Foong C, et al. Acetate is a bioenergetic substrate for human glioblastoma and brain metastases. Cell. 2014;159:1603–14.View ArticlePubMedPubMed CentralGoogle Scholar
- Wagih O, Parts L. gitter: a robust and accurate method for quantification of colony sizes from plate images. G3 (Bethesda). 2014;4:547–52.Google Scholar
- Bitton DA, Schubert F, Dey S, Okoniewski M, Smith GC, Khadayate S, Pancaldi V, Wood V, Bähler J. AnGeLi: a tool for the analysis of gene lists from fission yeast. Front Genet. 2015;6:330.View ArticlePubMedPubMed CentralGoogle Scholar
- Lyne R, Burns G, Mata J, Penkett CJ, Rustici G, Chen D, Langford C, Vetrie D, Bähler J. Whole-genome microarrays of fission yeast: characteristics, accuracy, reproducibility, and processing of array data. BMC Genomics. 2003;4:27.View ArticlePubMedPubMed CentralGoogle Scholar
- Bitton DA, Rallis C, Jeffares DC, Smith GC, Chen YY, Codlin S, Marguerat S, Bähler J. LaSSO, a strategy for genome-wide mapping of intronic lariats and branch points using RNA-seq. Genome Res. 2014;24:1169–79.View ArticlePubMedPubMed CentralGoogle Scholar
- Bähler J, Wu JQ, Longtine MS, Shah NG, McKenzie 3rd A, Steever AB, Wach A, Philippsen P, Pringle JR. Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast. 1998;14:943–51.View ArticlePubMedGoogle Scholar
- Hentges P, Van Driessche B, Tafforeau L, Vandenhaute J, Carr AM. Three novel antibiotic marker cassettes for gene disruption and marker switching in Schizosaccharomyces pombe. Yeast. 2005;22:1013–9.View ArticlePubMedGoogle Scholar
- Rallis C, Codlin S, Bähler J. TORC1 signaling inhibition by rapamycin and caffeine affect lifespan, global gene expression, and cell proliferation of fission yeast. Aging Cell. 2013;12:563–73.View ArticlePubMedPubMed CentralGoogle Scholar