Methionine supplementation stimulates mitochondrial respiration

Graphical Abstract (for review) *Highlights (for review) Highlights  Methionine supplementation increases mitochondrial functions  Methionine addition enhances mitochondrial pyruvate uptake and TCA cycle activity  Loss of pyruvate transport in snf1 cells is detrimental in methionine condition *REVISED Manuscript (text UNmarked) Click here to view linked References Methionine Supplementation Stimulates Mitochondrial Respiration 1 2 3 Farida Tripodi1,2*, Andrea Castoldi1,*, Raffaele Nicastro1,, Veronica Reghellin1,, Linda Lombardi1 4 Cristina Airoldi1,2, Ermelinda Falletta3, Elisa Maffioli4, Pasquale Scarcia5, Luigi Palmieri5, Lilia 5 Alberghina1,2, Gennaro Agrimi5**, Gabriella Tedeschi4**, Paola Coccetti1,2** 6 7 1 Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy 8 2 SYSBIO, Centre of Systems Biology, Milan, Italy 9 3 Department of Chemistry, University of Milano, Milan, Italy 10 4 DIMEVET – Department of Veterinary Medicine- University of Milano, Milan, Italy 11 5 Department of Biosciences, Biotechnology and Biopharmaceutics, University of Bari, Italy 12 13 14   Present address: Department of Biology, University of Fribourg, Fribourg, Switzerland Present address: Eurofins BioPharma, Vimodrone, Italy 15 * These authors contributed equally to this work 16 ** To whom correspondence should be addressed: 17 paola.coccetti@unimib.it; gabriella.tedeschi@unimi.it; gennaro.agrimi@uniba.it 18 19 Keywords: Snf1/AMPK, metabolomics, shotgun proteomics, MPC (Mitochondrial Pyruvate 20 Carrier), Saccharomyces cerevisiae, S-adenosyl-methionine. 21 22 23 24 1 25 Abstract 26 Mitochondria play essential metabolic functions in eukaryotes. Although their major role is the 27 generation of energy in the form of ATP, they are also involved in maintenance of cellular redox 28 state, conversion and biosynthesis of metabolites and signal transduction. Most mitochondrial 29 functions are conserved in eukaryotic systems and mitochondrial dysfunctions trigger several 30 human diseases. 31 By using multi-omics approach, we investigate the effect of methionine supplementation on yeast 32 cellular metabolism, considering its role in the regulation of key cellular processes. Methionine 33 supplementation induces an up-regulation of proteins related to mitochondrial functions such as 34 TCA cycle, electron transport chain and respiration, combined with an enhancement of 35 mitochondrial pyruvate uptake and TCA cycle activity. This metabolic signature is more noticeable 36 in cells lacking Snf1/AMPK, the conserved signalling regulator of energy homeostasis. Remarkably, 37 snf1 cells strongly depend on mitochondrial respiration and suppression of pyruvate transport is 38 detrimental for these cells in methionine condition, indicating that respiration mostly relies on 39 pyruvate flux into mitochondrial pathways. 40 These data provide new insights into the regulation of mitochondrial metabolism and extends our 41 understanding on the role of methionine in regulating energy signalling pathways. 42 43 44 2 45 Introduction 46 To tackle the central cell biology issue of how a specific genotype is able to generate a given 47 phenotype in certain environmental conditions, a useful approach is to start by unravelling the 48 complexity of the phenotypic features generated by the interacting genetic and nutritional 49 perturbations. Here, we approach this issue by considering Snf1/AMPK, the key signalling 50 regulator of energy homeostasis in eukaryotes [1] and the amino acid methionine, an essential 51 player of the one-carbon metabolism [2]. In Saccharomyces cerevisiae Snf1 protein complex is a 52 central component of glucose signalling pathway. It promotes respiratory metabolism and 53 gluconeogenesis, being necessary for growth in low glucose or alternative carbon sources [3]. It is 54 made by the catalytic α subunit (Snf1), the γ subunit (Snf4) and one of the three alternative β 55 subunits (Sip1, Sip2, Gal83), which determine the intracellular localization of the kinase [3,4]. 56 Snf1/AMPK is active when the catalytic α subunit is phosphorylated on Thr210 by one of the three 57 constitutive active kinases Sak1, Tos3 or Elm1 [5]. Snf1 activation requires also the association 58 between α and γ subunits, which stabilizes the active conformation of the kinase [5]. In response 59 to high glucose concentrations, Snf1 is inactivated through de-phosphorylation of Thr210 by the 60 phosphatase Glc7/Reg1 [6,7]. Phosphorylation of Ser214, inside the activation loop, has been 61 reported as an additional mechanism for downregulating Snf1/AMPK kinase activity [8]. 62 Upon activation, Snf1 phosphorylates a number of transcription factors, activating some and 63 repressing others [3]. Specifically, active Snf1 causes the translocation to the cytoplasm of Mig1, 64 thus leading to the expression of glucose repressed-genes [9,10]. Besides Mig1, Snf1 activates 65 Cat8 and Sip4, which regulate the expression of gluconeogenic genes [11,12], and Adr1, which 66 activates the expression of the alcohol dehydrogenase gene ADH2 and genes of glycerol 67 metabolism, fatty acid utilization and peroxisome biogenesis [13,14]. Snf1 also phosphorylates and 68 regulates the nuclear localization of Hcm1, a forkhead transcription factor, leading to increased 3 69 transcription of genes involved in respiration during nutrient scarcity [15]. Furthermore, Snf1 70 stimulates the activity of several metabolic enzymes, such as the glycerol-3-phosphate 71 dehydrogenase isoform Gpd2 [16] and the acetyl-CoA carboxylase Acc1 [17,18]. Although 72 Snf1/AMPK function has been mostly studied in respiration-dependent growth [19–21], some 73 reports indicate that it is active even in glucose repression [22–24]. In keeping with these data, we 74 recently reported that Snf1/AMPK phosphorylation on Thr210 is slightly detectable also in high 75 glucose [25] and, in this condition, it regulates G1/S cell-cycle transition, proper spindle 76 orientation and cellular metabolism [25–28]. Moreover, Snf1/AMPK interacts with and 77 phosphorylates the adenylate cyclase Cyr1 in a nutrient-independent manner and negatively 78 regulates intracellular cAMP content as well as PKA-dependent transcription [29]. 79 The sulfur amino acid methionine (Met) is the precursor of S-adenosylmethionine (SAM), the 80 universal cellular methyl donor [30–33]. Methionine metabolism regulates key biological functions 81 in mammalian cells, such as cell proliferation, metabolism, stem cell maintenance and embryonic 82 development [34,35]. It is well-known that methionine-restriction extends lifespan across different 83 species [36] and in human fibroblast this is due to a decrease of mitochondrial oxidative 84 phosphorylation [37]. Importantly, SAM is involved in G1 cell-cycle regulation in yeast [38] and 85 stimulation of SAM synthesis triggers Snf1 activation in yeast [39]. 86 Here, we have uncovered new links between methionine metabolism and the Snf1/AMPK 87 pathway. By using metabolomics profiling, metabolic flux analysis and mitochondrial proteomics, 88 we have discovered a novel function for Snf1/AMPK as a negative regulator of aerobic respiration 89 and mitochondrial pyruvate uptake, in methionine and glucose-repressing conditions. 90 4 91 92 Results 93 Methionine addition affects proliferation and metabolism in the absence of Snf1 94 Stimulation of S-adenosylmethionine (SAM) synthesis leads to Snf1 activation [39]. Accordingly, 95 Snf1-dependent phosphorylation of an acetyl-coenzyme A carboxylase 1 derived reporter [40] 96 increased upon methionine supplementation (Fig. 1A). 97 Then, to better understand the relation between methionine metabolism and Snf1 activity, the 98 prototrophic CEN.PK JT4 snf1Δ strain and its control wild type were grown on synthetic medium 99 containing 2% glucose and increasing concentrations of methionine (from 0.05 g/l up to 1.5 g/l). 100 Methionine and SAM supplementation (Fig. 1B, Supplementary Fig. S1A) affected both 101 proliferation and budding index of cells lacking Snf1 (Fig. S1B). 102 Addition of methionine (0.1 g/l, 0.67 mM), which was uptaken at similar rates by wt and snf1Δ 103 strains, significantly impaired glycerol secretion in cells lacking Snf1, while no alterations were 104 observed on glucose uptake, ethanol, acetate and glycerol secretion rates, which were already 105 lower in the snf1Δ mutant (Fig. 1C). 106 To gain more insight into the relevant intracellular metabolic changes upon SNF1 deletion and 107 methionine addition, we next performed a metabolomic profiling analysis (Fig. 2A, Supplementary 108 Fig. S2A-B, Supplementary Table S1). In the control strain grown in the presence of methionine, 109 most metabolites decreased, mainly amino acids, intermediates of the citric acid cycle and the 110 urea cycle (Fig. 2A, Supplementary Fig. S2B). On the contrary, SNF1 deletion, in combination with 111 methionine addition, promoted an increase in the level of amino acids, tricarboxylic acids, as well 112 as TCA cycle derivatives and urea cycle intermediates, with only few exceptions (Fig. 2A, 113 Supplementary Fig. S2B). One of the most upregulated metabolites was trehalose, which was 114 more abundant in snf1Δ cells compared to wt, and was strongly up-regulated in the presence of 5 115 methionine (Fig. 2A, Supplementary Fig. S2B), possibly due to the increased activity of trehalose-6- 116 phosphate synthase upon methylation [41]. 117 Interestingly, intracellular homocysteine increased in cells lacking Snf1 (Fig. 2A, Supplementary 118 Fig. S2B) and all metabolites of the methionine cycle were more up-regulated in the snf1Δ mutant 119 supplemented with methionine in comparison with the control (Fig. 2A-C). Moreover, in this 120 condition, S-adenosylmethionine and S-adenosylhomocysteine ratio (SAM/SAH) decreased in the 121 wild type, while increased in the snf1Δ mutant, further confirming the unbalance of methionine 122 metabolism due to Snf1 loss (Fig. 2D). 123 Taken together, these data indicate that methionine supplementation induces a general 124 remodelling of metabolism, being more evident in cells lacking Snf1. 125 Mitochondrial proteome shows an increase of proteins involved in aerobic respiration in 126 methionine medium 127 To gain a better insight in the up-regulation of TCA cycle intermediates following SNF1 deletion 128 and methionine treatment, a label-free shotgun proteomics approach was used to investigate the 129 mitochondrial proteome of wild type and snf1Δ cells grown without and with methionine. The 130 corresponding Venn diagram and workflow are shown in Fig. 3A and Supplementary Fig. S3, 131 respectively. Among the 1236 proteins common to all data sets, Supplementary Table S2 reports 132 only the proteins whose differences were statistically significant according to ANOVA test. More 133 than 89% of them were reported as mitochondrial according to Yeast Mine software and [42], 134 indicating a significant enrichment in mitochondrial components. Remarkably, Snf1 was found 135 associated to mitochondria, further confirming its mitochondrial localization [43]. Due to the high 136 sensitivity of the analysis, non-mitochondrial proteins were also identified, as previously reported 137 [42], mainly localized in compartments tightly associated to mitochondria (such as endoplasmic 138 reticulum, Golgi and vacuole). A principal component analysis (PCA), carried out on the four data 6 139 sets (wt, wt + M, snf1Δ, snf1Δ + M) confirmed that, as expected, each condition exerted a specific 140 detectable effect on protein expression. However, the proteome of both wt and snf1∆ moved 141 towards negative values of the Component 1 upon methionine addition (Fig. 3B). 142 We then carried out pairwise analyses focusing on the following comparisons: snf1Δ/wt (Fig. 3C, 143 Supplementary Table S3), wt + M/wt (Fig. 3D, Supplementary Table S4), snf1Δ + M/snf1Δ (Fig. 3E, 144 Supplementary Table S5) and snf1Δ + M/wt + M (Fig. 3F, Supplementary Table S6). SNF1 deletion 145 mostly induced an up-regulation of many proteins related to cellular transport (transmembrane 146 transport, mitochondrial transport, late endosome to vacuole transport, Golgi to endosome 147 transport, heme transport) and amino acids biosynthesis (cellular amino acid biosynthetic process, 148 glutamate biosynthetic process, serine family amino acid biosynthetic process) (Fig. 3C, 149 Supplementary Table S3), in keeping with metabolomics analysis (Fig. 2) and with previously 150 reported transcriptional up-regulation of genes related to transport, amino acids biosynthesis and 151 iron homeostasis of the snf1Δ mutant [26]. 152 Methionine induced a down-regulation of proteins involved in sterol and ergosterol biosynthesis 153 only in snf1Δ mutant (Fig. 3E, Supplementary Table S5). In addition, proteins involved in ribosome 154 biogenesis and RNA processing were down-regulated in both wt and snf1Δ cells (Fig. 3D, E and 155 Supplementary Tables S4-S5). The identification of this class of proteins is not surprising, in 156 keeping with previous data showing the association of ribosomes with mitochondria [42,44]. 157 Remarkably, in both strains methionine induced an up-regulation of proteins related to 158 mitochondrial functions, such as TCA cycle (i.e. Mdh1, Sdh1,2,4, Cit1, Idh2, Idp1), electron 159 transport chain and aerobic respiration (i.e. Cyt1, Qcr2,7,10, Cir2, Cyb2, Cor1, Cyc1, Rip1, Mam33), 160 as well as proteins related to redox processes (Fig. 3D-E and Supplementary Tables S4-S5), being 161 more up-regulated in snf1Δ mutant (Fig. 3F, Supplementary Table S6). 162 Therefore, proteins related to mitochondrial respiration increase in methionine-medium. 7 163 Methionine addition stimulates mitochondrial respiration in the absence of Snf1 164 To test whether methionine supplementation and SNF1 deletion were involved in the regulation 165 of mitochondrial activity, we measured several parameters associated to active mitochondria. Loss 166 of SNF1 determined a striking increase of mtDNA copy number, mitochondrial membrane 167 potential, as well as oxygen consumption of mitochondria isolated from cells grown in 168 methionine-supplemented media (Fig. 4A-C). In details, although mitochondria isolated from 169 snf1Δ mutant oxidized succinate and NADH to rates comparable to those of the control, they 170 displayed a higher oxidation rate when NADH was used as a substrate in cells grown in methionine 171 medium (Fig. 4C). In keeping with the higher respiration rate, antimycin A, an inhibitor of the 172 mitochondrial electron transport chain complex III [45,46], had a dramatic impact on the growth 173 rate of the snf1Δ mutant in the presence of methionine (Fig. 4D). 174 Despite the more sustained mitochondrial metabolism, intracellular basal ATP levels decreased 175 upon SNF1 deletion in combination with methionine (Fig. 4E), indicating that energy consuming 176 processes, i.e. fatty acids and lipid droplets accumulation (Fig. 2A and Supplementary Fig. S4A, 177 respectively), trehalose (Fig. 2A) and SAM biosynthesis (Fig. 2D) were draining energy in this 178 growth condition. 179 Altogether, these data confirm that methionine has a relevant effect on the metabolism of cells 180 lacking Snf1, highlighting its essential involvement in mitochondrial respiration. 181 Methionine addition stimulates pyruvate transport into mitochondria in the absence of Snf1 182 To gain further insight into mitochondrial substrate utilization, we cultured cells in the presence of 183 [U-13C6] glucose and determined steady-state isotopic labelling from which important intracellular 184 flux partitioning ratios were calculated [47]. Then, a metabolic flux analysis was performed 185 integrating these intracellular flux ratios (Supplementary Fig. S5A), consumption and secretion 186 rates (Fig. 1C) in a yeast model of central carbon metabolism [47,48] (Fig. 5A, Supplementary Fig. 8 187 S5B). snf1Δ mutant displayed a larger flux of carbon towards mitochondria as compared to the 188 control, showing more pyruvate transported into these organelles in both conditions (Fig. 5B, 189 Supplementary Fig. S5B). Strikingly, in methionine supplementation, pyruvate transport towards 190 mitochondria, TCA cycle activity and respiration were more up-regulated in the snf1Δ mutant than 191 wt (Fig. 5A-C, Supplementary Fig. S5B). 192 The fraction of oxaloacetate (OAA) generated from mitochondrial malate by malate 193 dehydrogenase (i.e. oxidative TCA cycle activity) [49] was then calculated. As expected, in the wild 194 type, under glucose repression, TCA cycle oxidative activity was low [49] and increased from 4.6% 195 to 21.5% in methionine growth condition (Fig. 5C). In contrast, a lower glucose-repressed 196 metabolism was detectable in the snf1Δ mutant, being oxidative TCA cycle activity 29.25% without 197 methionine and raising up to 47% in the presence of methionine (Fig. 5C). 198 The downregulation of glycerol secretion suggested a sustained mitochondrial oxidation of 199 cytosolic NADH in the presence of methionine in cells lacking Snf1 (Fig. 1C). Remarkably, the 200 model predicted an increase of the oxygen consumption in the snf1Δ cells (Fig. 5A, Supplementary 201 Fig. S5B), in accordance with the stimulation of mitochondrial respiration in cells lacking Snf1 202 above reported (Fig. 4C). 203 Overall, our results support an inhibitory function of Snf1/AMPK on respiration as well as on TCA 204 cycle in glucose repressed conditions, further enforced in methionine-medium. 205 Mpc1 function has a key role for methionine-dependent respiratory activity in the absence of Snf1 206 Overall our data indicate that methionine stimulates mitochondrial pyruvate transport and 207 respiration. Therefore, we examined the level of the three subunits of the MPC (Mitochondrial 208 Pyruvate Carrier) complex in strains expressing HA-tagged versions of Mpc1,2,3. While Mpc1 209 levels were almost unchanged, the level of Mpc2 and Mpc3 strongly increased in methionine 210 supplementation, both in wt and snf1Δ cells (Fig. 6A), suggesting that the increased mitochondrial 9 211 functionality may depend on the upregulation of MPC subunits. However, although the effect of 212 methionine was similar in both strains, mitochondrial respiration was physiologically more 213 noticeable in cells lacking Snf1 (Fig. 4). Thus, we treated cells with UK5099, which covalently binds 214 to MPC and blocks pyruvate transport [50]. While only a slight decrease of proliferation was 215 observed in both strains, a dramatic slow-down of growth rate occurred in cells lacking Snf1 in the 216 presence of methionine (Fig. 6B). 217 Loss of the major structural subunit of the mitochondrial pyruvate carrier, Mpc1, results in 218 defective mitochondrial pyruvate uptake [51,52]. Thus, we tested the effect of MPC1 deletion on 219 the snf1Δ mutant grown with and without methionine in the medium. mpc1Δ cells grew slower 220 than the control, as previously reported [51], also in the presence of methionine (Fig. 6C). 221 Remarkably the snf1Δmpc1Δ double mutant had a major growth defect only in methionine 222 medium (Fig. 6C), in accordance with data obtained with the inhibitor UK5099 (Fig. 6B). The strong 223 reliance on pyruvate transport and respiration of snf1Δ cells was further confirmed by the 224 complete growth arrest of the snf1Δmpc1Δ mutant treated with Antimycin A (Fig. 6D), which 225 however had no effect on cellular viability (data not shown). 226 Taken together, our results indicate that respiration due to Snf1 loss mostly relies on the flux of 227 pyruvate into mitochondria. 228 10 229 Discussion 230 Methionine cycle, being a key metabolic network which integrates biosynthesis, one-carbon 231 metabolism and epigenetics, regulates important biological functions such as cell proliferation, 232 metabolism, stem cell maintenance and embryonic development [34,35]. For these reasons and 233 also because its regulation is mostly unknown, methionine metabolism still needs to be 234 investigated deeper. In the present study, we showed that methionine metabolism has a strong 235 impact on cellular and metabolic features of proliferating yeast cells, collecting evidences that 236 most of them are tightly connected with Snf1/AMPK. 237 Snf1/AMPK, an important cellular energy sensor, is conserved from yeast to humans [1]. In yeast, 238 it is required for the expression of glucose-repressed genes and cells lacking Snf1 are unable to 239 grow on non-fermentable carbon sources, such as glycerol or ethanol. Paradoxically, while in the 240 absence of glucose Snf1 is required to increase respiration, in high glucose condition, oxidative 241 phosphorylation sustains growth and energy production in snf1 cells [26], indicating an 242 unconventional role of Snf1 under glucose repression. Here we showed that although methionine 243 supplemented to the medium was rather low (0.1 g/L) and ineffective to inhibit wild type growth 244 ([53], Fig. 1B), in cells lacking Snf1, it induced a general slow-down of proliferation, combined with 245 enhanced mitochondrial DNA, NADH oxidation, TCA cycle flux, and mitochondrial pyruvate uptake 246 (summarized in Fig. 7). 247 Notably, an imbalance of methionine cycle was evident in cells lacking Snf1 even in methionine- 248 free medium. In fact, intracellular homocysteine, a thiol amino acid, whose dysfunction is 249 associated with a multitude of human diseases [54], increased in snf1 cells and all metabolites of 250 this cycle as well as methylation potential (SAM/SAH) were further up-regulated in the presence of 251 methionine (Fig. 2). In support of this, methionine metabolism has been recently reported to be 252 tightly connected with Snf1/AMPK since SAM accumulation enhances Snf1 activation [39], 11 253 highlighting also a Snf1-dependent function in fine tuning methionine metabolism in a feedback 254 loop (Fig. 7). 255 More interestingly, although in methionine medium many proteins involved in electron transport 256 chain and aerobic respiration were up-regulated both in wild type and in snf1 cells (Fig. 3D-E), 257 inhibition of mitochondrial respiration was detrimental only for cells lacking Snf1 (Fig. 4D), 258 indicating a reliance on mitochondrial function, further supported by the up-regulation of redox 259 processes only in that condition (Fig. 3F). 260 Thus, our results clearly indicate that the addition of a small amount of methionine in the medium 261 has a strong impact only if Snf1/AMPK is inactive and provides novel insights into methionine- 262 dependent regulation of proliferation and mitochondrial metabolism. 263 Several lines of evidences support the connection between methionine metabolism and 264 mitochondrial function: i) metabolism of yeast strains with high intracellular SAM content [55] 265 depends on elevated TCA cycle fluxes and respiration activity [56]; ii) homocysteine metabolism 266 regulates mitochondrial respiration in T cells and mitochondrial membrane potential in yeast 267 [57,58]; iii) in human fibroblast, the activity of oxidative phosphorylation by complex IV decreases 268 in methionine restriction, due to the reduced COX1 level [37]. 269 In support of these data, we confirm the connection between methionine metabolism and 270 mitochondrial respiration showing, in addition, the key role of Snf1/AMPK in such a regulation. 271 It is well known that in glucose containing medium, yeast cells metabolize glucose predominantly 272 through glycolysis, followed by alcoholic fermentation. Since glucose represses functions 273 connected to TCA cycle and respiration, only a relatively small fraction of glycolytically produced 274 pyruvate is translocated into mitochondria and converted to acetyl-CoA. Our metabolic flux 275 analysis indicates that methionine supplementation stimulates pyruvate transport into 276 mitochondria in glucose-repressing conditions (Fig. 5). Remarkably, snf1Δmpc1Δ mutant shows a 12 277 slow growth phenotype in methionine medium (Fig. 6C), highlighting the physiological relevance 278 of Mpc1 function in cells lacking Snf1. 279 The activity of the Mpc1 carrier is crucial to determine the fate of pyruvate, it is involved in the 280 triggering of the Warburg effect and is considered a potential target for cancer therapy [59]. 281 Moreover, the role of protein kinase AMPK in the regulation of MPC1 expression has recently 282 emerged [60]. Decreased MPC1 expression promotes the maintenance of stemness of cancer cells, 283 which become more migratory and resistant to both chemotherapy and radiotherapy [61,62]. 284 Conversely, the inhibition of pyruvate mitochondrial transport by MPC inhibitor UK5099 activates 285 AMPK [63]. Collectively, the above studies, together with our data, support a new link between 286 AMPK pathway and mitochondrial pyruvate transport. Strikingly, the coexistence of glycolysis and 287 functional TCA cycle activity and OXPHOS offers a selective metabolic advantage for cancer cell 288 proliferation and tumorigenesis [64,65]. Moreover, some studies highlight the double-edged 289 function of AMPK in the regulation of tumorigenic potential, showing either an anti-tumorigenic or 290 a pro-tumorigenic function [66,67]. Therefore, in future studies it will be interesting to dissect the 291 molecular mechanism of pyruvate metabolism by Snf1/AMPK along with its conservation in 292 eukaryotic systems. 293 Besides respiratory function, in snf1Δ cells grown in the presence of methionine, there was also a 294 significant decrease of several proteins involved in ergosterol biosynthesis, among which Erg1, 295 Erg4, Erg6 and Scs7. Remarkably, mutants with deficiency in ergosterol biosynthesis accumulate S- 296 adenosylmethionine [55], in accordance with our data showing a significant increase of this 297 metabolite in snf1Δ cells (Fig. 2C). Moreover, the down-regulation of SAH1 expression impairs 298 sterol synthesis, leading to a 4-fold elevated squalene levels [54]. Egr1, involved in squalene 299 biosynthesis, is downregulated in cells lacking Snf1, which also show S-adenosylhomocysteine 300 accumulation (Fig. 2C). 13 301 Interestingly, the proteomics analysis also highlights the down-regulation of glycosylation and 302 vacuolar transport processes in snf1Δ cells grown in methionine condition (Fig. 5F, Supplementary 303 Table S6). Kar2, the ATPase involved in protein import/export into the ER [68], was down- 304 regulated too, in accordance with studies reporting the involvement of Snf1 in ER stress response 305 [69–71]. 306 Finally, histone deacetylation proteins were down-regulated in the absence of Snf1 (Fig. 3C, 307 Supplementary Table S3). It was previously reported that inactivation of SNF1 globally decreases 308 intracellular pool of acetyl-CoA as well as histone acetylation [72]. Therefore, we hypothesize that 309 the downregulation of histone deacetylation functions here presented could be a consequence of 310 the reduced level of acetylation in the snf1∆ strain [72]. 311 Taken together, our results shed significant light on the interplay among Snf1/AMPK activity, 312 methionine and mitochondrial metabolism in glucose-repressing conditions and extend our 313 mechanistic understanding of how methionine can influence cell fate. 314 315 14 316 317 Material and methods 318 Yeast strains and growth conditions 319 S. cerevisiae strains used in this study are reported in Table 1. Synthetic medium (SD) contained 320 2% glucose, 6.7 g/L of Yeast Nitrogen Base without amino acids (Difco). Methionine was added to 321 the concentrations indicated in figure legends. In these conditions, cells exhibit exponential 322 growth between OD600nm = 0.1 (approximately equivalent to 2*106 cells/ml) and OD600nm = 2.5 323 (5*107 cells/ml); all experiments were performed in exponential phase of growth (OD600nm0.5-1). 324 Antimycin A was added to a final concentration of 1 µg/ml from 2 mg/ml stock in 100% ethanol, 325 UK5099 was added to a final concentration of 50 µM from 10 mM stock in 100% DMSO; the same 326 volume of solvent was added in the control cultures. To evaluate Snf1 activity, phosphorylation of 327 the reporter ACC1-HA was assayed in a strain transformed with the plasmid pYX242-ACC1-GFP-HA 328 (or pYX242-ACC1-S79A-GFP-HA as negative control) [40]. 329 Protein extraction and immunoblotting 330 Cells were harvested by filtration and lysed in 250 µl ice-cold lysis buffer (50 mM Tris pH 7.5, 150 331 mM NaCl, 0.1, Nonidet p-40, 10% glycerol) with 1 mM PMSF, protease inhibitor mix (Complete 332 EDTA free protease inhibitor mixture tablets; Roche) and phosphatase inhibitor mix (Cocktail II, 333 Sigma-Aldrich). An equal volume of acid-washed glass beads (Sigma-Aldrich) was added before 334 disruption. Cells were broken by 20 cycles of vortex and ice of 1 min each. Protein concentration 335 was determined with Bio-Rad protein assay. After addition of SDS-sample buffer, crude extract 336 was boiled at 98 °C for 5 min. 337 Anti-phospho-Acetyl-CoA Carboxylase (Ser79) antibody (Cell Signaling Technology®) and anti-HA 338 antibody (Roche) were used to perform immunoblotting following the manufacturer’s instruction. 339 mtDNA quantification 15 340 Relative mtDNA was quantified by real-time PCR. Primers based on the cDNA sequences of 341 nuclear-encoded ACT1 and mitochondrial-encoded COX1 genes were designed with Primer 342 Express 3.0 (Applied Biosystems, Life Technologies) and purchased from Invitrogen (Life 343 Technologies, sequences available upon request). For each strain analyzed the difference of the 344 threshold cycle number (CT) between COX1 and ACT1 (ΔCt) was used to calculate the mtDNA copy 345 number per cell, which was equal to 2-ΔCT [73]. 346 Metabolites analysis 347 Intracellular metabolites were extracted and analysed by GC-MS. Briefly, around 5 mg of cells in 348 exponential phase of growth (0.7 OD600nm) were harvested by filtration and quenched with 1.5 ml 349 50% MeOH at T<-40 °C, then samples were centrifuged at 13000 rpm for 1 min and supernatant 350 was discarded. To extract metabolites, 400 µl of ice-cold chloroform, 800 µl of 50% MeOH and 20 351 µl of 2 mM norvaline (as internal standard) were added and samples were stirred by vortex for 30 352 min at 4 °C. After 5 min centrifugation at 4 °C, the obtained supernatant was concentrated 353 through evaporation. Samples were subsequently derivatized with MSTFA (N-Methyl-N- 354 (trimethylsilyl) trifluoroacetamide) in an automated WorkBench (Agilent Technologies) and 355 analysed with 7200 accurate-mass Q-TOF GC/MS (Agilent Technologies). Data processing and 356 analysis were performed with Mass Hunter and Mass Profiler Professional software (Agilent 357 Technologies). Raw data were normalized on the norvaline (internal standard) signal and on the 358 collected cell dry weight and reported in Supplementary Table S1. 359 Glucose uptake and glycerol, acetate, pyruvate and ethanol productions were determined by HPLC 360 analysis using a Waters Allianc 2695 separation module (Waters, Milford, MA, USA) equipped with 361 a Rezex ROA-Organic Acid H+ (8%) 300 mm × 7.8 mm column (Phenomenex Inc., USA), coupled to 362 a Waters 2410 refractive index detector and a Waters 2996 UV detector. Separation was carried 363 out at 65 °C with 0.005 M H2SO4 as the mobile phase at a flow rate of 0.6 ml/min. The 16 364 physiological parameters: maximum specific growth rate, biomass yield on glucose and specific 365 glucose consumption rate were calculated during the exponential growth phase, as described [74]. 366 Methionine uptake was measured by H-NMR on the medium of exponentially growing cells. 367 Briefly, cells grown in the presence of 0.1 g/L methionine were collected by filtration and 368 resuspended in fresh medium containing 0.1 g/L methionine at a final concentration of 0.1 369 OD600nm. Media were then collected every hour until cells reached the concentration of 1 OD 600nm. 370 S-Adenosyl methionine and S-adenosyl homocysteine were measured using the SAM-SAH ELISA kit 371 assay (Cells Biolabs©) following the manufacturer’s instructions. 372 13 373 All labelling experiments were performed in batch cultures assuming pseudo-steady-state 374 conditions during the exponential growth phase in respiro-fermentative conditions [47,75]. 375 labelling of proteinogenic amino acids was achieved by growth on 20 g/L glucose as a mixture of 376 80% (w/w) unlabelled and 20% (w/w) uniformly labeled [U-13C]glucose (13C, 99 %; Cambridge 377 Isotope Laboratories, Inc). Cells from an overnight minimal medium culture were washed and used 378 for inoculation below an OD600 of 0.03. 379 centrifugation during the mid-exponential growth phase at an OD600 of ≤1. Cells (about 0.3 mg of 380 dry biomass) were washed once with sterile water and hydrolysed in 150 µL 6 M HCl at 105 °C for 381 6 h. The hydrolysate was dried in a heating block at 80 °C under a constant airflow. Before the 382 GC/MS analyses all samples were subjected to a derivatization step as follows. Each sample was 383 resuspended in 30 µL of acetonitrile, followed by 30 µL of MBDSTFA (N-methyl-N-ter- 384 butyldimethylsilyl-trifluoroacetamide). The resulting mixture, contained in a closed vial, was 385 stirred for 10 min and centrifuged for 15 sec. Then, the vial was incubated at 85 °C. After 1 h, the 386 sample slowly reached room temperature and was analysed. All the derivatized samples were 387 processed by using a ISQ™ QD Single Quadrupole GC-MS (Thermo Fisher) equipped with a VF-5ms C-labelling experiments 13 13 C- C-labelled biomass aliquots were harvested by 17 388 (30 m x 0.25 mm i.d. x 0.25 µm; Agilent Technology). Injection volume: 1 µL. Oven program: 140 °C 389 for 1 min; then 10 °C/min to 310 °C for 1 min; Run Time 15 min. Helium was used as the gas 390 carrier. SS Inlet: Mode Split. Split flow: 15 mL/min. Split ratio: 1/15. Inlet temperature: 270 °C. 391 Flow 1.0 mL/min. MS transfer line: 280 °C. Ion source: 280 °C. Ionization mode: electron impact: 392 70 eV. Acquisition mode: full scan. 393 A METAFOR (metabolic flux ratio) analysis [47] was perfomed on the generated GC-MS data: the 394 mass isotopomer distribution of proteinogenic amino acids was used to calculate the split ratios of 395 key branching points of yeast central metabolism using the software FIAT FLUX [48]. 396 13 397 Intracellular flux ratios , consumption and secretion rates were integrated in a model of yeast 398 central metabolism using the NETTO subprogram of the Fiat Flux software, to obtain network- 399 wide absolute fluxes [47,48]. 400 The stoichiometric model for 401 pathways of yeast central carbon metabolism [76]. The model used contains 30 fluxes and 28 402 metabolites. To calculate intracellular fluxes, the stoichiometric model was constrained with five 403 extracellular fluxes (growth rate, glucose uptake rate and production rates of ethanol, glycerol and 404 acetate) and five intracellular flux ratios (fraction of cytosolic oxaloacetate originating from 405 cytosolic pyruvate, fraction of mitochondrial oxaloacetate derived through anaplerosis, fraction of 406 phosphoenol-pyruvate originating from cytosolic oxaloacetate, upper and lower bounds of 407 mitochondrial pyruvate derived through malic enzyme). Only NADH-dependent isocitrate 408 dehydrogenase activity (Idh1 and Idh2) was considered in the model; NADP-specific isocitrate 409 dehydrogenase activity was neglected. Mass balances of O2, CO2 and ATP production and 410 consumption were excluded from the analyses. The overly constrained system was solved by a 411 least square optimization as described in [77]. C-constrained metabolic flux analysis 13 C-constrained metabolic flux analysis comprises the major 18 412 ATP assay 413 ATP content was quantified using the BacTiter-Glo™ Luminescent Assay (Promega®), following the 414 manufacturer’s instructions. Briefly, cells in exponential growth phase were collected and diluted 415 at 0.3 OD600nm and 100 µl of each sample was assayed in triplicate in a 96-well plate. An equal 416 volume of BacTiter-GLO™ was then added to each well and the measurement was carried out 417 after a brief incubation at room temperature (7 min). The measurement was performed at a 418 wavelength of 560 nm with a Cary Eclipse© Luminometer. 419 Flow cytofluorimetric (FACS) analysis 420 FACS analysis were performed with the FACSCalibur© Cytofluorimeter and the CellQuest Pro© 421 software as described [27]. Cells were collected at the indicated time points and independently 422 stained with 175 nM 3,3’-dihexyloxacarbocyanine Iodide (DiOC6) to measure mitochondrial 423 potential, with 20 nM Mitotracker green (MTG) to measure the total mitochondrial content. FACS 424 data were analysed with Flowing Software© 2 or with the WinMDI© software which provided 425 quantifications and statistical analysis. 426 Mitochondrial purification 427 The isolation of mitochondria was carried out as previously reported [78]. In details, cell walls 428 were enzymatically degraded with Zymolyase. The resulting spheroplasts were disrupted by 13 429 strokes in a cooled Potter-Elvehjem homogenizer in hypotonic medium. Cytosolic and 430 mitochondrial fractions were separated by differential centrifugation. Mitochondria were spun 431 down gently for 10 min at 10,000 x g and were resuspended in a buffer containing 0.6 M mannitol, 432 20 mM Hepes/KOH pH 7.4, 1 mM EGTA, 0.2% bovine serum albumin (BSA) at approximately 10 mg 433 of protein/ml. For the proteomic analysis, mitochondria were further purified using a 434 discontinuous sucrose gradient as described [78]. 435 Proteomic analysis by shotgun mass spectrometry and label free quantification 19 436 After reduction and derivatisation [79], the mitochondrial proteins were digested with trypsin 437 sequence grade (Roche) for 16 h at 37 °C using a protein:trypsin ratio of 20:1 [80]. LC-ESI-MS/MS 438 analysis was performed on a Dionex UltiMate 3000 HPLC System with a PicoFrit ProteoPrep C18 439 column (200 mm, internal diameter of 75 μm) (New Objective, USA). Gradient: 1% ACN in 0.1 % 440 formic acid for 10 min, 1-4 % ACN in 0.1% formic acid for 6 min, 4-30% ACN in 0.1% formic acid for 441 147 min and 30-50 % ACN in 0.1% formic for 3 min at a flow rate of 0.3 μl/min. The eluate was 442 electrosprayed into an LTQ Orbitrap Velos (Thermo Fisher Scientific, Bremen, Germany) through a 443 Proxeon nanoelectrospray ion source (Thermo Fisher Scientific) as previously described [81]. Data 444 acquisition was controlled by Xcalibur 2.0 and Tune 2.4 software (Thermo Fisher Scientific). Mass 445 spectra were analysed using MaxQuant software (version 1.3.0.5). The spectra were searched by 446 the Andromeda search engine against the Uniprot sequence database Saccharomyces cerevisiae 447 CEN.PK113-7D (release 15.12.2016). Protein identification required at least one unique or razor 448 peptide per protein group (FDR 0.01). Quantification in MaxQuant was performed using the built 449 in XIC-based label free quantification (LFQ) algorithm using fast LFQ 450 Statistical 451 www.biochem.mpg.de/mann/tools/). An Anova test (FDR 0.05) was carried out to identify 452 proteins differentially expressed among the different conditions. PCA analysis was performed by 453 Perseus software. Focusing on specific comparison, proteins were considered differentially 454 expressed if they were present only in one condition or showed significant t-test (p value = 0.05). 455 Bioinformatic 456 (http://yeastmine.yeastgenome.org) to cluster enriched annotation groups of Biological Processes 457 and Kegg Pathways within the set of identified proteins. Functional grouping was based on p-value 458 ≤0.05 and at least two counts. The mass spectrometry proteomics data have been deposited to analyses were analyses performed were using carried the Perseus out by software (version YeastMine 1.5.5.3, software 20 459 the ProteomeXchange Consortium via the PRIDE [82] partner repository with the dataset identifier 460 PXD007644. 461 Oxygen Consumption 462 Oxygen consumption by isolated mitochondria was determined at 30 °C using an Oxygraph-2 k 463 system (Oroboros, Innsbruck, Austria) equipped with two chambers in a buffer containing 0.6 M 464 mannitol, 20 mM Hepes/KOH pH 6.8, 10 mM potassium phosphate pH 6.8, 2 mM MgCl 2, 1 mM 465 EGTA, and 0.1% BSA. Data was analysed using DatLab software. Measurements were started by 466 adding 1.25 mM NADH or 5 mM succinate, followed by the addition of 0.25 mM ADP. 467 Statistical analysis 468 Data are reported as means ± SDs from at least three independent experiments. Statistical 469 significance of the measured differences was assessed by two-tailed Student’s t-test (* p < 0.05). 470 471 472 473 474 21 475 476 Acknowledgments 477 This work has been supported by grants to P. Coccetti from the Italian Government (FAR) and to L. 478 Alberghina and P. Coccetti from the SYSBIO, Centre of Systems Biology, a MIUR initiative of the 479 Italian Roadmap of European Strategy Forum on Research Infrastructures (ESFRI). A.C., R.N. and 480 V.R. were supported by fellowships from SYSBIO, while F.T. has been supported by fellowship from 481 MIUR. 482 We gratefully acknowledge helpful discussion and comments from Dr. Evelina Gatti and Prof. 483 Marco Vanoni. 484 We kindly thank Prof. Filip Rolland for pYX242-ACC1 and pYX242-ACC1-S79A plasmids. 485 We thank Prof. Uwe Sauer for FiatFlux software permission and Prof. Lars M. Blank and Dr. Birgitta 486 Ebert for their help with the metabolic flux analysis. 487 488 Author contribution 489 F.T., A.C., V.R., L.L. performed cell biology experiments; F.T., A.C., R.N., C.A, E.F. performed 490 metabolomics experiments; E.M., G.T. performed proteomics experiments and data analysis; G.A., 491 P.S. performed metabolic flux analysis experiments and mitochondria purification; G.T., G.A., L.P., 492 L.A. edited the manuscript; P.C., F.T., G.A. wrote the manuscript and conceived the experiments; 493 P.C. coordinated the project; all the authors read and approved the entire paper. 494 495 Competing interest 496 The authors declare no competing interest. 22 497 498 Table 1. Yeast strains used in this study. Strain wt snf1Δ mpc1Δ mpc1Δ snf1Δ W303 [ACC1-HA reporter] W303 [ACC1-S79A-HA reporter] Mpc1-4HA Mpc1-4HA snf1Δ Mpc2-4HA Mpc2-4HA snf1Δ Mpc3-4HA Mpc3-4HA snf1Δ Genotype CEN.PK JT4 MATa URA3 LEU2 TRP1 HIS3 LCR1 CEN.PK JT4 MATa URA3 LEU2 TRP1 HIS3 LCR1 snf1::HPH CEN.PK JT4 MATa URA3 LEU2 TRP1 HIS3 LCR1 mpc1::NAT1 CEN.PK JT4 MATa URA3 LEU2 TRP1 HIS3 LCR1 mpc1::NAT1 snf1::HPH W303-1A leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 [pYX242-ACC1-GFP-HA] W303-1A leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 [pYX242-ACC1-S79A-GFP-HA] CEN.PK JT4 MATa URA3 LEU2 TRP1 HIS3 LCR1 MPC14HA:KANMX4 CEN.PK JT4 MATa URA3 LEU2 TRP1 HIS3 LCR1 snf1::HPH MPC1-4HA:KANMX4 CEN.PK JT4 MATa URA3 LEU2 TRP1 HIS3 LCR1 MPC24HA:KANMX4 CEN.PK JT4 MATa URA3 LEU2 TRP1 HIS3 LCR1 snf1::HPH MPC2-4HA:KANMX4 CEN.PK JT4 MATa URA3 LEU2 TRP1 HIS3 LCR1 MPC34HA:KANMX4 CEN.PK JT4 MATa URA3 LEU2 TRP1 HIS3 LCR1 snf1::HPH MPC3-4HA:KANMX4 Origin [49] This study This study This study This study This study This study This study This study This study This study 499 500 501 Figure legends 502 Figure 1. Methionine addition impairs the growth rate of the snf1Δ mutant. 503 (A) Snf1 activity was evaluated in wt cells grown with or without 0.1 g/l methionine and expressing 504 the Acc1-pS79 reporter or the non-phosphorylatable Acc1-S79A version. The asterisk indicates a 505 non-specific protein band recognized by the anti-HA antibody, used as loading control. (B) Mass 506 duplication time (MDT) of wt and snf1Δ cells grown in the presence of the indicated 507 concentrations of methionine. *p<0.05 (C) Glucose and methionine consumption rate, ethanol, 508 acetate and glycerol secretion rates of wt and snf1Δ cells grown in the presence or absence of 0.1 509 g/l methionine. *p<0.05. 23 510 Figure 2. Metabolomic analysis of wt and snf1Δ cells grown in the presence or absence of 511 methionine (0.1 g/l). 512 (A) wt and snf1Δ cells were collected in exponential phase and cell lysates were analyzed by 513 GC/MS spectrometry as reported in material and methods. Heat map diagram shows the log2 514 differential levels of the indicated metabolites. (B) Schematic representation of the methionine 515 cycle in yeast. Solid arrows indicate direct reactions, while dashed arrows indicate reactions with 516 multiple steps. (C) Relative levels of S-adenosylmethionine (SAM) and S-adenosylhomocysteine 517 (SAH) in wt and snf1Δ cells. *p<0.05. (D) Ratio between SAM and SAH in wt and snf1Δ cells. 518 *p<0.05. 519 Figure 3. Mitochondrial proteome analysis of wt and snf1Δ cells grown in the presence or 520 absence of methionine (0.1 g/l). 521 (A) Venn diagram of the shotgun proteomic analysis on mitochondria purified from wt and snf1Δ 522 cells grown with or without methionine. (B) Principal component analysis of the proteins common 523 among the data sets of wt and snf1Δ cells in the presence or absence of 0.1 g/L methionine, whose 524 differences were statistically significant according to ANOVA test (FDR 0.05). (C-F) Biological 525 process enrichment analysis of the proteins differentially expressed between the following pairs of 526 conditions: snf1Δ vs wt (C), wt+M vs wt (D), snf1Δ+M vs snf1Δ (E) and snf1Δ+M vs wt+M (F). For 527 each comparison, BP-enrichment classes with the lowest p-value are shown. For the full lists see 528 Supplementary Tables S3-S6. Bars represent the number of annotated genes in the input list. 529 Figure 4. Methionine addition stimulates mitochondrial respiration in cells lacking Snf1. 530 (A) Relative mtDNA copy numbers of wt and snf1Δ cells grown in the presence or absence of 0.1 531 g/l methionine. (B) Ratio between mitochondrial membrane potential and total mitochondrial 532 content of wt and snf1Δ cells grown in the presence or absence of 0.1 g/l methionine. Data were 533 obtained by flow cytometric analysis with DiOC6 staining to evaluate the mitochondrial potential 24 534 and with Mitotracker Green staining to measure the total mitochondrial content. *p<0.01. (C) 535 Respiration rate of isolated mitochondria from wt and snf1Δ cells grown in the presence or 536 absence of 0.1 g/l methionine, in the presence of the indicated oxidizable substrates. *p<0.02. (D) 537 Effect of 1 µg/ml antimycin A on mass duplication time (MDT) of wt and snf1Δ cells grown in the 538 presence or absence of 0.1 g/l methionine. * p<0.001. (E) ATP relative level of wt and snf1Δ cells 539 grown in the presence or absence of 0.1 g/l methionine. *p<0.005. 540 Figure 5. Methionine addition enhances mitochondrial pyruvate transport in cells lacking Snf1. 541 (A) Schematic representation of changes in flux through metabolic pathways in snf1Δ+M relative 542 to wt+M. (B) Pyruvate transport into mitochondria in wt and snf1Δ cells determined by metabolic 543 flux analysis as mmoles of pyruvate per 100 mmoles of glucose taken up by yeast cells. (C) 544 Oxidative TCA cycle activity in wt and snf1Δ cells, reported as percentage of mitochondrial 545 oxaloacetate produced from the TCA cycle activity and calculated from 546 proteinogenic aminoacids, as described [49]. 547 Figure 6. Cells lacking Snf1 depend on Mpc1 activity. (A) Mpc1,2,3 levels in wt and snf1Δ cells 548 expressing HA-tagged versions of the three proteins, grown with or without 0.1 g/l methionine. 549 Anti-Cdc34 antibody was used as loading control. (B) Mass duplication time (MDT) of wt, snf1Δ 550 treated with 50 µM UK5099, in the presence or absence of 0.1 g/L methionine. *p<0.05. (C) (B) 551 Mass duplication time (MDT) of wt, snf1Δ, mpc1Δ and mpc1Δsnf1Δ cells grown in the presence or 552 absence of 0.1 g/L methionine. *p<0.05. (D) Effect of 1 µg/ml antimycin A on mass duplication 553 time (MDT) of mpc1Δ and mpc1Δsnf1Δ cells grown in the presence or absence of 0.1 g/l 554 methionine. 555 Figure 7. Relevant connections between Snf1 and methionine metabolism. Schematization of the 556 role of Snf1 and methionine cycle on cellular metabolism. Methionine cycle activates 557 mitochondrial functions and stimulates Snf1/AMPK activity. Snf1/AMPK inhibits glucose uptake 13 C-labeling patterns of 25 558 from the medium, pyruvate flux into the mitochondria and respiration. Snf1-dependent function 559 in fine tuning methionine metabolism in a feedback loop is also shown [39]. 560 References 561 [1] Nat. Rev. Mol. Cell Biol. 8 (2007) 774–85. doi:10.1038/nrm2249. 562 563 [2] [3] [4] M.C. Schmidt, R.R. McCartney, beta-subunits of Snf1 kinase are required for kinase function and substrate definition., EMBO J. 19 (2000) 4936–43. doi:10.1093/emboj/19.18.4936. 568 569 K. Hedbacker, M. 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(A) Mass duplication time (MDT) of wt and snf1Δ cells in the presence or absence of 0.67 mM (0.1 g/l) methionine or 0.2 mM S-adenosylmethionine (SAM). * p<0.05. (B) Budding index of wt and snf1Δ cells exponentially growing in the presence or absence of 0.1 g/l methionine. Figure S2. (A) PCA scores plot of GC/MS analysis showing the distinct clustering for the biological replicates of the four conditions, i.e. wt (brown squares), wt+M (brown triangles), snf1Δ (red squares), snf1Δ+M (red triangles). (B) Table of the fold changes (expressed as log2) of the heat map diagrams shown in Figure 2A. Figure S3. Workflow of the proteomic analysis of Figure 3. Figure S4. Lipid droplet content of wt and snf1Δ cells grown in the presence or absence of 0.1 g/l methionine. Data were obtained by Flow Cytometric Analysis with BODIPY staining. *p<0.05. Figure S5. (A) Intracellular flux ratios (fraction of cytosolic oxaloacetate originating from cytosolic pyruvate, fraction of mitochondrial oxaloacetate derived through anaplerosis, fraction of phosphoenol-pyruvate originating from cytosolic oxaloacetate, upper and lower bounds of mitochondrial pyruvate derived through malic enzyme) (Blank and Sauer, 2004) calculated using the mass isotopomer distribution of proteinogenic amino acids and the software FIAT FLUX (Zamboni et al., 2005). (B) Relative distribution of absolute carbon fluxes in wt and snf1Δ grown in 2% glucose in the presence or absence of 0.1 g/L methionine. All fluxes, given in the same order in each box, are normalized to the specific glucose uptake rate, which is shown in the top inset. Relative fluxes are reported in blue for the wt, cyan for the wt+M, red for snf1Δ and pink for snf1Δ+M. Supplementary Table Legends Table S1. Raw data of metabolic analysis, related to Fig. 2, S2 Table S2. List of the proteins common among wt and snf1Δ grown in the presence or absence of 0.1 g/L methionine whose differences were statistically significant according to ANOVA test (FDR 0.05). X indicates the proteins described as mitochondrial according to Yeast Mine software and (Morgenstern et al., 2017). Table S3. List of the proteins differentially expressed in the snf1Δ mutant in comparison with wt. X indicates the proteins described as mitochondrial according to Yeast Mine software and (Morgenstern et al., 2017) , Z the proteins described as mitochondrial according to (Morgenstern et al., 2017). Biological process enrichments and KEGG pathway enrichments for up-regulated and down-regulated proteins are shown. Table S4. List of the proteins differentially expressed in wt grown in the presence or absence of 0.1 g/L methionine. X indicates the proteins described as mitochondrial according to Yeast Mine software and (Morgenstern et al., 2017), Z the proteins described as mitochondrial according to (Morgenstern et al., 2017). Biological process enrichments and KEGG pathway enrichments for upregulated and down-regulated proteins are shown. Table S5. List of the proteins differentially expressed in snf1Δ cells grown in the presence or absence of 0.1 g/L methionine. X indicates the proteins described as mitochondrial according to Yeast Mine software and (Morgenstern et al., 2017), Z the proteins described as mitochondrial according to (Morgenstern et al., 2017). Biological process enrichments and KEGG pathway enrichments for up-regulated and down-regulated proteins are shown. Table S6. List of the proteins differentially expressed in the snf1Δ mutant in comparison with wt both grown in the presence of 0.1 g/L methionine. X indicates the proteins described as mitochondrial according to Yeast Mine software and (Morgenstern et al., 2017), Z the proteins described as mitochondrial according to (Morgenstern et al., 2017). Biological process enrichments and KEGG pathway enrichments for up-regulated and down-regulated proteins are shown. Supplementary Figures (for online publication) Supplementary Figure S1 Click here to download Supplementary Material (for online publication): Supplementary Figures REVISIONE BBA A * 250 * MDT (min) 200 * 150 100 50 0 B * wt wt+M snf1Δ snf1Δ+M Budding Index 87±4% 80±4% 83±2% 68±4% Supplementary Figure S2 A B Glycolysis and Trehalose Cycle wt + M / wt snf1Δ / WT snf1Δ + M / wt snf1Δ + M / wt + M Dihydroxyacetone phosphate -0.53 -0.70 -1.39 -0.86 Fructose-1,6-diphosphate 0.03 -0.58 -0.62 -0.65 Glucose-6-phosphate -0.72 -0.96 -1.64 -0.92 Pyruvic acid 0.11 0.58 1.14 1.03 Trehalose 0.85 2.32 7.22 6.37 Pentose phosphate pathway 6-Phosphogluconic acid Ribose-5-phosphate wt + M / wt snf1Δ / WT snf1Δ + M / wt snf1Δ + M / wt + M -0.49 -0.98 -0.98 -0.49 -0.33 -0.81 -1.28 -0.95 Amino acids Alanine Aspartic acid Glutamic acid Glutamine Glycine Homoserine Isoleucine Phenylalanine Serine Succinylhomoserine Threonine Valine Lysine wt + M / wt snf1Δ / WT snf1Δ + M / wt snf1Δ + M / wt + M 0.14 0.21 0.36 0.22 -0.40 1.19 2.14 2.54 -1.34 0.77 1.21 2.55 -2.58 1.63 0.68 3.27 -2.57 -0.18 -1.04 1.53 -0.80 0.51 0.48 1.28 -2.25 0.53 -0.50 1.75 0.31 0.41 0.58 0.27 -1.08 0.65 0.36 1.44 -0.18 1.06 2.13 2.31 -1.20 0.70 0.79 2.00 -3.32 0.77 -0.96 2.36 0.46 -0.52 0.45 -0.01 Glycerol and lipids allo-Inositol Capric acid Glycerol Glycerol 1-phosphate Linoleic acid methyl Oleate myo-Inositol Palmitic acid Stearic acid Stearoyl-Glycerol wt + M / wt snf1Δ / WT snf1Δ + M / wt snf1Δ + M / wt + M 0.39 0.33 0.16 -0.23 0.14 0.23 0.43 0.29 -0.99 0.77 0.71 1.70 -0.20 1.05 1.75 1.95 0.15 0.25 0.47 0.32 0.80 0.58 0.42 -0.38 -0.41 0.26 0.72 1.13 0.08 0.23 0.59 0.51 0.20 0.26 0.69 0.49 1.06 0.70 1.48 0.42 TCA Cycle Citric acid Fumaric acid Malic acid wt + M / wt snf1Δ / WT snf1Δ + M / wt snf1Δ + M / wt + M 0.18 0.80 2.10 1.92 -1.11 -0.23 0.42 1.53 -0.68 -0.10 0.89 1.57 Glyoxylate Cycle Glyoxylic acid wt + M / wt snf1Δ / WT snf1Δ + M / wt snf1Δ + M / wt + M 0.02 0.14 0.34 0.32 Others 1,3-Propanediol 9H-purine-6-amine Acetanilide Acetyl-glutamic acid Benzoic acid Citraconic acid Cysteinylglycine Cytosine Dihydrouracil Dihydroxyphenylglycine Malonic Acid Nicotinic acid Oxalic acid Phenethylamine Phosphoric acid Pyroglutamic acid wt + M / wt snf1Δ / WT snf1Δ + M / wt snf1Δ + M / wt + M 0.05 0.18 0.26 0.21 0.45 0.36 0.30 -0.15 0.06 0.18 0.27 0.21 -1.02 0.64 1.11 2.12 -0.19 -0.75 0.08 0.26 0.49 0.71 0.89 0.40 0.12 0.20 0.34 0.23 -0.06 0.06 0.28 0.34 -0.26 0.20 0.33 0.58 0.22 1.43 2.12 1.90 -1.05 0.70 0.40 1.44 -0.13 0.76 1.40 1.53 0.11 0.28 0.40 0.29 -2.10 -0.15 -0.67 1.43 -0.21 0.03 0.31 0.52 -1.16 0.80 0.58 1.74 Urea Cycle N-acetyl-Ornithine 2 Ornithine Citrulline wt + M / wt snf1Δ / WT snf1Δ + M / wt snf1Δ + M / wt + M -0.90 0.30 0.44 1.34 -0.81 0.90 1.09 1.90 -0.74 0.65 0.70 1.44 Methionine Cycle Methionine Homocysteine wt + M / wt snf1Δ / WT snf1Δ + M / wt snf1Δ + M / wt + M 7.32 0.38 9.75 2.42 6.17 1.11 7.55 1.38 Supplementary Figure S3 Supplementary Figure S4 1.8 Lipid Droplets Content (a.u.) 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 * * Supplementary Figure S5 A cytOAA from cytPYR mtOAA from anaplerosis mean 0.906 ± 0.028 0.932 ± 0.027 0.919 ± 0.029 0.886 ± 0.026 mean 0.954 ± 0.017 0.785 ± 0.016 0.708 ± 0.018 0.530 ± 0.016 wt wt + M snf1Δ snf1Δ + M malic enzyme lower malic enzyme upper bound bound mean 0.027 ± 0.016 0.061 ± 0.015 0.022 ± 0.016 0.064 ± 0.016 PEP from cytOAA mean 0.387 ± 0.236 0.419 ± 0.076 0.070 ± 0.052 0.235 ± 0.267 mean 0.045 ± 0.013 0.050 ± 0.040 0.076 ± 0.014 0.032 ± 0.016 B Medium GLUCOSE Cytosol wt 100% = 23.23 mmol/(g*h) wt+M 100% = 20.85 mmol/(g*h) snf1Δ 100% = 14.11 mmol/(g*h) snf1Δ+M 100% = 12.69 mmol/(g*h) NADPH 3.40 3.26 4.54 3.62 BIOMASS Glucose-6-P 3.53 3.17 8.08 3.62 93.07 95.11 87.38 94.48 Fructose-6-P Erythrose-4-P 0.65 0.53 1.98 0.63 ATP 1.12 1.01 2.62 1.18 Pentose-5-P 0.60 0.62 0.85 0.63 94.83 95.11 91.92 94.48 Triose 3P Sedoheptulose-7-P 1.12 1.01 2.62 1.18 BIOMASS 1.03 1.01 1.35 1.10 3-P glycerate 2.37 2.30 3.19 2.52 183.64 186.14 178.10 186.52 ATP NADH CO2 8.44 9.83 14.67 6.30 0.39 0.38 0.50 0.39 6.03 1.58 6.87 2.44 NADH GLYCEROL 0.65 0.62 0.85 0.71 NADH ETHANOL P-enol-pyruvate ATP 191.09 194.96 191.35 191.73 ATP 167.28 166.38 159.89 157.92 8.44 7.00 9.85 10.09 PYRUVATE 17.05 19.90 20.98 14.58 6.28 8.30 10.28 12.53 Acetaldehyde Mitochondrion 0.73 1.49 2.48 1.89 4.52 6.09 0.78 3.86 CO2 NADH 2.54 2.45 3.40 2.68 0.30 0.29 0.35 0.32 AcetylCoA 3.83 3.74 5.17 4.10 Citrate Isocitrate 7.02 9.30 5.60 7.64 6.63 10.36 5.46 11.90 0.34 2.54 2.34 6.15 Malate 4.86 8.63 3.12 10.01 NADH Fumarate 4.86 8.63 3.12 10.01 1.76 1.73 2.34 1.89 CO2 NADH 0.5 ATP Succinate ATP 2.50 2.45 3.40 2.68 BIOMASS CO2 NADPH α-ketoglutarate 4.86 8.63 3.12 10.01 O2 + 2 NADH 2 ATP Acetyl-CoA CO2 NADPH Oxaloacetate Oxaloacetate ACETATE NADPH 6.93 10.65 5.88 12.21 6.63 10.36 5.46 11.90 5.9 4.55 6.45 7.41 Acetate CO2 Pyruvate CO2 ATP 158.89 159.38 149.75 154.14 BIOMASS 21.39 31.99 20.91 40.35 Table S1 Click here to download Supplementary Material (for online publication): Table S1_Metabolomics.xlsx Compoundwt2A [5950] L-alan9.548866 [867] maloni7.478615 [5962] L-lysi13.05655 [70914] N-ace 16.02912 [311] citric acid 12.72457 [1 [791] DL-isol11.01494 [328] DL-4-hyd 5.747741 [597] cytosin10.20228 [985] palmiti14.07733 [750] glycine12.91456 [ [6288] L-threoni 11.53283 [91493] 6-pho 9.922213 [65270] cystein 9.437399 [5960] aspar13.85409 [439579] L-homo 5.947633 [12647] L-homo 8.591826 [6262] L-orni11.52903 [892] myo-in8.430027 [904] acetan14.57129 [5281] stear15.89922 [33032] L-gluta 13.94971 [7427] D-(+)7.757687 [24699] 1-stear 10.01086 [6137] L-me11.93181 [5951] L-seri13.47065 [5280450] li12.99612 [243] benzo 13.2907 [439167] D-r9.778563 [2969] capri10.52322 [971] oxalic acid 6.705238 [7 [994] Phenyl5.453515 [938] nicotini9.460487 [738] L-gluta12.05924 [5960] aspar12.78528 [1060] pyruvi8.901827 [10267] fruc6.090731 [92824] D-ma 8.907558 [439232] N- 11.1338 [638129] citr8.362603 [10442] 1,3-10.77517 [439958] D-g11.64856 [754] glycero9.105211 [892] allo-ino7.493501 [443586] 3,56.687738 [760] glyoxyl10.23754 [649] 5,6-dihyd 15.04297 [33032] L-gluta 13.64562 [668] dihydr8.208009 [1004] phosph 14.35853 wtB 8.930075 6.853664 12.60086 14.69884 11.67382 9.830399 4.738308 8.852299 13.40899 11.73319 11.53806 8.856834 8.905895 13.16653 5.030704 7.590035 11.20758 7.214488 13.91649 15.22218 12.69174 6.972048 9.184942 10.68636 12.74963 12.41145 12.20857 8.86767 9.781584 6.161531 4.65935 8.343735 10.88788 10.74884 8.005096 6.252999 7.538001 9.486794 8.124504 10.1392 10.78916 7.83145 6.771367 6.221756 9.647776 14.0637 13.08451 7.105441 13.42785 wtC 8.898563 6.96526 12.70614 14.62217 11.76415 9.990864 5.238764 8.807695 12.08698 11.74455 11.7869 9.146925 8.912449 13.26074 4.903676 7.923353 11.08132 7.71946 13.87782 13.74679 12.78355 7.230052 8.497473 10.80899 12.85502 12.37187 12.48434 9.162344 9.807175 5.943542 5.352079 8.599093 11.4881 10.89669 8.157452 4.968256 7.679023 9.365047 7.700724 10.1116 10.96511 7.780546 9.00119 5.7753 9.363968 14.21313 12.8704 7.323817 13.59234 wtD 8.805892 6.991457 12.84879 14.876 11.90613 9.955319 5.504828 8.807996 13.42477 11.95168 11.75227 9.356637 8.80709 13.24407 5.218043 7.982308 11.76983 7.586292 13.8192 15.32808 12.83867 7.183782 11.45408 10.90564 12.90185 12.44687 12.6332 9.358911 10.01041 6.101486 5.083129 8.673839 11.37614 11.12463 8.134126 6.658807 7.709544 9.754441 8.284537 10.07462 11.54069 7.914153 7.662946 6.445354 9.56005 14.17358 13.17358 7.547369 13.64763 wtE 8.818842 6.476352 12.6808 15.08025 12.02221 10.02017 5.061005 8.833857 13.40085 12.0074 11.79215 9.494328 8.896872 13.06951 4.807577 7.700054 11.4785 7.398889 13.9529 15.2602 13.09004 7.341092 9.456961 10.94911 12.48064 12.49737 12.7822 9.429152 10.02015 6.260603 4.748458 8.581425 10.58505 11.49143 8.506537 7.742334 7.819492 10.37804 8.407906 10.15803 11.93806 8.223545 6.859564 6.761925 9.701389 14.2047 13.21179 7.760975 13.92335 wt+A 9.059486 6.053428 13.38929 14.01597 12.30239 7.837225 5.544423 8.864298 12.0543 9.532492 10.64357 8.548975 8.998928 13.45056 11.28717 7.197647 10.66391 7.525226 13.98131 14.03113 11.99398 8.161732 8.627205 12.95597 12.03461 12.52268 12.4202 8.548975 9.908701 6.066575 5.638798 8.594143 9.008822 11.24862 8.428444 5.347035 7.37578 9.029093 8.210753 10.20518 10.50642 7.920092 9.050776 6.181258 9.407194 14.18281 11.97056 6.952872 13.50151 wt+B 8.9934 5.189891 12.53192 13.141 11.2854 7.201081 5.529748 8.895675 13.5764 8.960822 9.625159 8.302884 8.944282 12.38728 10.07708 6.552747 9.804477 6.715354 13.99648 15.52341 10.61809 7.580984 11.73563 11.97455 10.55458 12.56889 12.13962 8.346555 10.13129 6.247668 5.350328 7.967837 7.814986 9.962876 7.846849 5.206958 6.481913 8.224179 8.623206 10.20393 9.845544 7.720878 7.769261 6.037915 9.605592 13.80712 10.53225 6.342217 13.21352 wt+C 9.37414 6.99103 14.06714 15.04866 12.95198 8.766426 5.683978 9.26166 13.83565 10.36131 11.60604 9.947588 9.296968 14.10605 12.8468 8.015196 11.53026 7.890443 14.26211 15.79301 13.3365 8.868601 11.99827 13.65359 12.93794 12.8732 12.8354 9.872774 10.50875 6.523795 5.494621 9.295187 9.593386 12.35226 9.143993 7.378145 8.103207 10.14431 8.986523 10.47639 11.8057 8.531666 8.03162 7.368023 9.910625 14.4406 13.11682 7.791546 13.93939 Table S2 Click here to download Supplementary Material (for online publication): Table S2_Anova.xls Protein ID P38631 P32621 P53171 P41921 P21954 P32466 P28241 P30605 P06208 P42838 P40513 P17505 Q12285 P40185 P33201 P10507 Q07938 P49954 P32340 P32860 Q12428 P07257 P37299 P00128 P07256 P0CX41 P36528 P20435 Q12487 Q00711 P21825 Q99287 P00445 P00447 P0CF17 P08067 P32316 P32317 P23180 Q01976 Q04728 P07251 Q12165 P05626 P00830 P32451 P14066 Gene FKS1_YEAST GDA1_YEAST GEP7_YEAST GSHR_YEAST IDHP_YEAST HXT3_YEAST IDH2_YEAST ITR1_YEAST LEU1_YEAST LEM3_YEAST MAM33_YEAST MDHM_YEAST MDY2_YEAST MMF1_YEAST MRT4_YEAST MPPB_YEAST MTAP_YEAST NIT3_YEAST NDI1_YEAST NFU1_YEAST PRPD_YEAST QCR2_YEAST QCR10_YEAST QCR7_YEAST QCR1_YEAST RL23A_YEAST RM17_YEAST RPAB2_YEAST RM23_YEAST SDHA_YEAST SEC62_YEAST SEY1_YEAST SODC_YEAST SODM_YEAST U5072_YEAST UCRI_YEAST ACH1_YEAST AFG1_YEAST AIM17_YEAST ADPP_YEAST ARGJ_YEAST ATPA_YEAST ATPD_YEAST ATPF_YEAST ATPB_YEAST BIOB_YEAST CBS1_YEAST Protein names Length 1,3-beta-glucan synthase component FKS1 (EC 2.4.1 1876 Guanosine-diphosphatase (GDPase) (EC 3.6.1.42) 518 Genetic interactor of prohibitin 7, mitochondrial 287 Glutathione reductase (GR) (GRase) (EC 1.8.1.7) 483 Isocitrate dehydrogenase [NADP], mitochondrial (IDH) 428 Low-affinity glucose transporter HXT3 567 Isocitrate dehydrogenase [NAD] subunit 2, mitocho 369 Myo-inositol transporter 1 584 2-isopropylmalate synthase (EC 2.3.3.13) (Alpha-IPM s 619 Alkylphosphocholine resistance protein LEM3 (Bref 414 Mitochondrial acidic protein MAM33 266 Malate dehydrogenase, mitochondrial (EC 1.1.1.37 334 Ubiquitin-like protein MDY2 (Golgi to ER traffic pro 212 Protein MMF1, mitochondrial (Isoleucine biosynthesis a 145 Ribosome assembly factor MRT4 (mRNA turnover pr 236 Mitochondrial-processing peptidase subunit beta (EC 462 S-methyl-5'-thioadenosine phosphorylase (EC 2.4.2 337 Probable hydrolase NIT3 (EC 3.5.-.-) 291 Rotenone-insensitive NADH-ubiquinone oxidoreduc 513 NifU-like protein, mitochondrial 256 Probable 2-methylcitrate dehydratase (2-MC dehydr 516 Cytochrome b-c1 complex subunit 2, mitochondrial 368 Cytochrome b-c1 complex subunit 10 (Complex III subuni 77 Cytochrome b-c1 complex subunit 7 (Complex III subuni 127 Cytochrome b-c1 complex subunit 1, mitochondrial 457 60S ribosomal protein L23-A (L17a) (Large ribosoma 137 54S ribosomal protein L17, mitochondrial (Mitocho 281 DNA-directed RNA polymerases I, II, and III subunit R 155 54S ribosomal protein L23, mitochondrial (Mitocho 163 Succinate dehydrogenase [ubiquinone] flavoprotein subun 640 Translocation protein SEC62 (Sec62/63 complex 30 274 Protein SEY1 (EC 3.6.5.-) (Synthetic enhancer of YO 776 Superoxide dismutase [Cu-Zn] (EC 1.15.1.1) 154 Superoxide dismutase [Mn], mitochondrial (EC 1.15 233 UPF0507 protein YML002W 737 Cytochrome b-c1 complex subunit Rieske, mitocho 215 Acetyl-CoA hydrolase (EC 3.1.2.1) (Acetyl-CoA deac 526 Protein AFG1 509 Probable oxidoreductase AIM17 (EC 1.14.11.-) (Alter 465 ADP-ribose pyrophosphatase (EC 3.6.1.13) (ADP-ribo 231 Arginine biosynthesis bifunctional protein ArgJ, mit 441 ATP synthase subunit alpha, mitochondrial 545 ATP synthase subunit delta, mitochondrial (F-ATPase delt160 ATP synthase subunit 4, mitochondrial 244 ATP synthase subunit beta, mitochondrial (EC 3.6.3 511 Biotin synthase, mitochondrial (EC 2.8.1.6) 375 Cytochrome b translational activator protein CBS1, mi 229 Table S3 Click here to download Supplementary Material (for online publication): Table S3_snf1-wt.xlsx Protein ID Gene Δ N1NWR5 N1P175 N1P9Y0 N1P4D6 N1P520 N1P1I4 N1P2V2 N1P9X9 N1P227 N1P1U0 N1P7X1 N1PAC3 N1NVJ9 N1NZ71 N1P1X3 N1P797 N1P7M3 N1P998 N1NYB6 P32366 N1P2G2 Q99380 P34166 P54114 Q3E752 P40089 Q3E795 Q12306 Q06139 P22943 P38841 P10663 P27999 P26755 P38155 P40045 P38783 Q12127 SIL1 AIM25 PDX1 ZIM17 QCR6 SEC72 ZRT1 CRH1 AIM23 CCW14 ATP16 PST1 RPL43A YBT1 GEP7 ECM11 ADE1 TMA108 APS1 VMA6 SDP1 OST4 MFA2 ALD3 YPR036W-A LSM5 YLR361C-A SMT3 YLR346C HSP12 YHR138C MRP2 RPB9 RFA3 PAU24 TDA2 FYV4 CCW12 Table S4 Click here to download Supplementary Material (for online publication): Table S4_wt+M-wt.xlsx Protein ID Gene Protein names GEP7 GFA1 GCV1 GGC1 IDP1 HSP60 IDH2 IDH1 ILV3 LEU4 MAM33 MDH1 MMF1 MIX23 MSC6 MIR1 MRH1 MAS2 MAS1 MRM1 MRH4 NDI1 NFU1 PEX28 PDH1 QCR2 QCR10 QCR7 COR1 MRPL17 MRP49 MRPL35 SDH1 SET1 TOF2 RIP1 ADH3 Genetic interactor of prohibitin 7, mitochondrial Glutamine--fructose-6-phosphate aminotransferase [isomerizing] (GFA Aminomethyltransferase, mitochondrial (Glycine cleavage system T Mitochondrial GTP/GDP carrier protein 1 Isocitrate dehydrogenase [NADP], mitochondrial (IDH) (IDP) (NADP(+ Heat shock protein 60, mitochondrial (CPN60) (P66) (Stimulator factor Isocitrate dehydrogenase [NAD] subunit 2, mitochondrial (Isocitric dehydr Isocitrate dehydrogenase [NAD] subunit 1, mitochondrial (Isocitric dehydr Dihydroxy-acid dehydratase, mitochondrial (DAD) (2,3-dihydroxy acid 2-isopropylmalate synthase (Alpha-IPM synthase) (Alpha-isopropylm Mitochondrial acidic protein MAM33 Malate dehydrogenase, mitochondrial Protein MMF1, mitochondrial (Isoleucine biosynthesis and maintena Mitochondrial intermembrane space cysteine motif-containing prote Meiotic sister-chromatid recombination protein 6, mitochondrial Mitochondrial phosphate carrier protein (Mitochondrial import receptor) Protein MRH1 (Membrane protein related to HSP30) Mitochondrial-processing peptidase subunit alpha (Alpha-MPP) Mitochondrial-processing peptidase subunit beta (Beta-MPP) (PEP) rRNA methyltransferase 1, mitochondrial (21S rRNA (guanosine(2270 ATP-dependent RNA helicase MRH4, mitochondrial (Mitochondrial R Rotenone-insensitive NADH-ubiquinone oxidoreductase, mitochondr NifU-like protein, mitochondrial Peroxisomal membrane protein PEX28 (Peroxin-28) Probable 2-methylcitrate dehydratase (2-MC dehydratase) ((2S,3S)-2-m Cytochrome b-c1 complex subunit 2, mitochondrial (Complex III subun Cytochrome b-c1 complex subunit 10 (Complex III subunit 10) (Compl Cytochrome b-c1 complex subunit 7 (Complex III subunit 7) (Complex Cytochrome b-c1 complex subunit 1, mitochondrial (Complex III subun 54S ribosomal protein L17, mitochondrial (Mitochondrial large ribosom 54S ribosomal protein MRP49, mitochondrial (Mitochondrial large ribos 54S ribosomal protein L35, mitochondrial (Mitochondrial large ribosom Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochon Histone-lysine N-methyltransferase, H3 lysine-4 specific (COMPASS c Topoisomerase 1-associated factor 2 Cytochrome b-c1 complex subunit Rieske, mitochondrial (Complex III Alcohol dehydrogenase 3, mitochondrial (Alcohol dehydrogenase III) Up-regulated in wt+M P53171 P14742 P48015 P38988 P21954 P19882 P28241 P28834 P39522 P06208 P40513 P17505 P40185 P38162 Q08818 P23641 Q12117 P11914 P10507 P25270 P53166 P32340 P32860 P38848 Q12428 P07257 P37299 P00128 P07256 P36528 P32388 Q06678 Q00711 P38827 Q02208 P08067 P07246 Table S5 Click here to download Supplementary Material (for online publication): Table S5_snf1+M-snf1.xlsx Protein ID Gene Up-regulated in snf1Δ+M P36141 P33893 P00360 P53171 P39726 P41921 P38715 P32191 P21954 P28241 P33416 Q6Q560 P36775 P06208 P40513 P17505 Q12230 P36112 P32787 Q99257 P25573 P38341 P50945 Q03104 P40185 P40364 Q08818 P10507 Q07938 P40215 P32340 P32860 P38921 P40530 P35999 P41903 Q12428 P07257 P37299 P00128 P07256 FMP46 PET112 TDH1 GEP7 GCV3 GLR1 GRE3 GUT2 IDP1 IDH2 HSP78 ISD11 PIM1 LEU4 MAM33 MDH1 LSP1 MIC60 MGM101 MEX67 MGR1 MIC12 MIC27 MSC1 MMF1 MPM1 MSC6 MAS1 MEU1 NDE1 NDI1 NFU1 PET8 PKP1 OCT1 TES1 PDH1 QCR2 QCR10 QCR7 COR1 Table S6 Click here to download Supplementary Material (for online publication): Table S6_snf1+M-wt+M.xlsx Protein ID P38631 P32621 P16474 P47042 P27810 P42838 Q12404 P33201 P42934 P33333 P31382 P53131 P25560 P10964 P21825 Q08199 Q99287 P53165 P35209 Q12133 Q12513 P36017 P21576 P21147 P53730 P15703 P41810 O13547 P24871 P32891 P25340 P32462 P53337 P43555 P32476 Q12452 Q05040 P43613 P32339 P27476 P38837 P46964 Q99380 P53224 P25343 Q03529 Gene FKS1 GDA1 KAR2 IKS1 KTR1 LEM3 MPD1 MRT4 PMT6 SLC1 PMT2 PRP43 RER1 RPA190 SEC62 SIL1 SEY1 SGF73 SPT21 SPC3 TMA17 VPS21 VPS1 OLE1 ALG12 BGL2 SEC26 CCW14 CLB4 DLD1 ERG4 ERG24 ERV29 EMP47 ERG1 ERG27 FAR8 ERJ5 HMX1 NSR1 NSG1 OST2 OST4 ORM1 RVS161 SCS7 Protein names Down-regulated in snf1 Δ+M 1,3-beta-glucan synthase component FKS1 (1,3-beta-D-glucan-UDP Guanosine-diphosphatase (GDPase) 78 kDa glucose-regulated protein homolog (GRP-78) (Immunoglobul Probable serine/threonine-protein kinase IKS1 (IRA1 kinase suppres Alpha-1,2 mannosyltransferase KTR1 Alkylphosphocholine resistance protein LEM3 (Brefeldin-A sensitivi Protein disulfide-isomerase MPD1 Ribosome assembly factor MRT4 (mRNA turnover protein 4) Dolichyl-phosphate-mannose--protein mannosyltransferase 6 Probable 1-acyl-sn-glycerol-3-phosphate acyltransferase (1-AGP acylt Dolichyl-phosphate-mannose--protein mannosyltransferase 2 Pre-mRNA-splicing factor ATP-dependent RNA helicase PRP43 (Heli Protein RER1 (Retention of ER proteins 1) DNA-directed RNA polymerase I subunit RPA190 (EC 2.7.7.6) (DNATranslocation protein SEC62 (Sec62/63 complex 30 kDa subunit) Nucleotide exchange factor SIL1 (Protein SLS1) Protein SEY1 ( (Synthetic enhancer of YOP1 protein) SAGA-associated factor 73 (73 kDa SAGA-associated factor) (SAGA hi Protein with a role in transcriptional silencing; required for normal tr Signal peptidase complex subunit SPC3 (Microsomal signal peptida Translation machinery-associated protein 17 (ATPase-dedicated cha Vacuolar protein sorting-associated protein 21 (GTP-binding protei Vacuolar protein sorting-associated protein 1 Acyl-CoA desaturase 1 (Delta 9 fatty acid desaturase) (Fatty acid desatu Dol-P-Man:Man(7)GlcNAc(2)-PP-Dol alpha-1,6-mannosyltransferase Glucan 1,3-beta-glucosidase (Exo-1,3-beta-glucanase) (GP29) (Solubl Coatomer subunit beta (Beta-coat protein) (Beta-COP) Covalently-linked cell wall protein 14 (Inner cell wall protein) G2/mitotic-specific cyclin-4 D-lactate dehydrogenase [cytochrome] 1, mitochondrial (D-lactate f Delta(24(24(1)))-sterol reductase (C-24(28) sterol reductase) (Stero Delta(14)-sterol reductase (C-14 sterol reductase) (Sterol C14-reduc ER-derived vesicles protein ERV29 Protein EMP47 (47 kDa endomembrane protein) (Endosomal P44 pr Squalene monooxygenase (Squalene epoxidase) (SE) 3-keto-steroid reductase Factor arrest protein 8 ER-localized J domain-containing protein 5 Heme-binding protein HMX1 Nuclear localization sequence-binding protein (p67) Protein involved in regulation of sterol biosynthesis Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subuni Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subuni Protein that mediates sphingolipid homeostasis Reduced viability upon starvation protein 161 Ceramide very long chain fatty acid hydroxylase SCS7 (Ceramide VL *Conflict of Interest Click here to download Conflict of Interest: coi_disclosure.pdf