Colonies of Saccharomyces cerevisiae laboratory strains pass through specific developmental phases when growing on solid respiratory medium. During entry into the so-called alkali phase, in which ammonia signaling is initiated, 2 prominent cell types are formed within the colonies: U cells in upper colony regions, which have a longevity phenotype and activate the expression of a large number of metabolic genes, and L cells in lower regions, which die more quickly and exhibit a starvation phenotype. Here, we performed a detailed analysis of the activities of enzymes of central carbon metabolism in lysates of both cell types and determined several fermentation end products, showing that previously reported expression differences are reflected in the different enzymatic capabilities of each cell type. Hence, U cells, despite being grown on respiratory medium, behave as fermenting cells, whereas L cells rely on respiratory metabolism and possess active gluconeogenesis. Using a spectrum of different inhibitors, we showed that glycolysis is essential for the formation, and particularly, the survival of U cells. We also showed that β-1,3-glucans that are released from the cell walls of L cells are the most likely source of carbohydrates for U cells.

b Institute of Microbiology of the Academy of Sciences of the Czech Republic ; Prague Czech Republic

Department of Genetics and Microbiology ; Faculty of Science; Charles University Prague ; Prague Czech Republic

Zobrazit více v PubMed

Palkova Z, Janderova B, Gabriel J, Zikanova B, Pospisek M, Forstova J. Ammonia mediates communication between yeast colonies. Nature 1997; 390:532-6; PMID:9394006; http://dx.doi.org/10.1038/37398 PubMed DOI

Palkova Z, Forstova J. Yeast colonies synchronise their growth and development. J Cell Sci 2000; 113:1923-8; PMID:10806103 PubMed

Palkova Z, Devaux F, Ricicova M, Minarikova L, Le Crom S, Jacq C. Ammonia pulses and metabolic oscillations guide yeast colony development. Mol Biol Cell 2002; 13:3901-14; PMID:12429834; http://dx.doi.org/10.1091/mbc.E01-12-0149 PubMed DOI PMC

Palkova Z, Wilkinson D, Vachova L. Aging and differentiation in yeast populations: elders with different properties and functions. FEMS Yeast Res 2014; 14:96-108; PMID:24119061; http://dx.doi.org/10.1111/1567-1364.12103 PubMed DOI

Vachova L, Hatakova L, Cap M, Pokorna M, Palkova Z. Rapidly developing yeast microcolonies differentiate in a similar way to aging giant colonies. Oxid Med Cell Longev 2013; 2013:102485; PMID:23970946; http://dx.doi.org/10.1155/2013/102485 PubMed DOI PMC

Cap M, Stepanek L, Harant K, Vachova L, Palkova Z. Cell differentiation within a yeast colony: metabolic and regulatory parallels with a tumor-affected organism. Mol Cell 2012; 46:436-48; PMID:22560924; http://dx.doi.org/10.1016/j.molcel.2012.04.001 PubMed DOI

Vachova L, Kucerova H, Devaux F, Ulehlova M, Palkova Z. Metabolic diversification of cells during the development of yeast colonies. Environ Microbiol 2009; 11:494-504; PMID:19196279; http://dx.doi.org/10.1111/j.1462-2920.2008.01789.x PubMed DOI

Vachova L, Chernyavskiy O, Strachotova D, Bianchini P, Burdikova Z, Fercikova I, Kubinova L, Palkova Z. Architecture of developing multicellular yeast colony: Spatio-temporal expression of Ato1p ammonium exporter. Environ Microbiol 2009; 11:1866-77; PMID:19302539; http://dx.doi.org/10.1111/j.1462-2920.2009.01911.x PubMed DOI

DeBerardinis RJ, Cheng T. Q's next: The diverse functions of glutamine in metabolism, cell biology and cancer. Oncogene 2010; 29:313-24; PMID:19881548; http://dx.doi.org/10.1038/onc.2009.358 PubMed DOI PMC

Vachova L, Cap M, Palkova Z. Yeast colonies: A model for studies of aging, environmental adaptation, and longevity. Oxid Med Cell Longev 2012; 2012:601836; PMID:22928081; http://dx.doi.org/10.1155/2012/601836 PubMed DOI PMC

DeRisi JL, Iyer VR, Brown PO. Exploring the metabolic and genetic control of gene expression on a genomic scale. Science 1997; 278:680-6; PMID:9381177; http://dx.doi.org/10.1126/science.278.5338.680 PubMed DOI

Gasch AP, Spellman PT, Kao CM, Carmel-Harel O, Eisen MB, Storz G, Botstein D, Brown PO. Genomic expression programs in the response of yeast cells to environmental changes. Mol Biol Cell 2000; 11:4241-57; PMID:11102521; http://dx.doi.org/10.1091/mbc.11.12.4241 PubMed DOI PMC

van Meerloo J, Kaspers GJ, Cloos J. Cell sensitivity assays: The MTT assay. Methods Mol Biol 2011; 731:237-45; PMID:21516412; http://dx.doi.org/10.1007/978-1-61779-080-5_20 PubMed DOI

Chen W, Gueron M. The inhibition of bovine heart hexokinase by 2-deoxy-d-glucose-6-phosphate: characterization by 31P NMR and metabolic implications. Biochimie 1992; 74:867-73; PMID:1467345; http://dx.doi.org/10.1016/0300-9084(92)90070-U PubMed DOI

Wick AN, Drury DR, Nakada HI, Wolfe JB. Localization of the primary metabolic block produced by 2-deoxyglucose. J Biol Chem 1957; 224:963-9; PMID:13405925 PubMed

Williamson JR. Glycolytic control mechanisms III. Effects of iodoacetamide and fluoroacetate on glucose metabolism in the perfused rat heart. J Biol Chem 1967; 242:4476-85; PMID:4229046 PubMed

Dresel K. Über die Wirkung der arsenigen Säure auf Atmung und Gärung. Biochem Z 1926; 178:70-4

Huijing F, Slater E. The use of oligomycin as an inhibitor of oxidative phosphorylation. J Biochem 1961; 49:493-501; PMID:13716716 PubMed

Slater E. The mechanism of action of the respiratory inhibitor, antimycin. Biochim Biophys Acta 1973; 301:129-54; PMID:4358868; http://dx.doi.org/10.1016/0304-4173(73)90002-5 PubMed DOI

Ingledew WJ, Ohnishi T. The probable site of action of thenolytrifluoracetone on the respiratory chain. Biochem J 1977; 164:617-20; PMID:196591; http://dx.doi.org/10.1042/bj1640617 PubMed DOI PMC

Farrow BG, Dawson AP. Investigation of the interaction of triethyltin with rat liver mitochondria using binding studies and Mössbauer spectroscopy. Eur J Biochem 1978; 86:85-95; PMID:26563; http://dx.doi.org/10.1111/j.1432-1033.1978.tb12287.x PubMed DOI

Dombek KM, Ingram LO. Ethanol production during batch fermentation with Saccharomyces cerevisiae: changes in glycolytic enzymes and internal pH. Appl Environ Microbiol 1987; 53:1286-91; PMID:3300550 PubMed PMC

van Hoek P, de Hulster E, van Dijken JP, Pronk JT. Fermentative capacity in high-cell-density fed-batch cultures of baker's yeast. Biotechnol Bioeng 2000; 68:517-23; PMID:10797237; http://dx.doi.org/10.1002/(SICI)1097-0290(20000605)68:5<517::AID-BIT5>3.0.CO;2-O PubMed DOI

van Hoek P, van Dijken JP, Pronk JT. Regulation of fermentative capacity and levels of glycolytic enzymes in chemostat cultures of Saccharomyces cerevisiae. Enzyme Microb Technol 2000; 26:724-36; PMID:10862878; http://dx.doi.org/10.1016/S0141-0229(00)00164-2 PubMed DOI

Miller SM, Magasanik B. Role of NAD-linked glutamate dehydrogenase in nitrogen metabolism in Saccharomyces cerevisiae. J Bacteriol 1990; 172:4927-35; PMID:1975578 PubMed PMC

DeLuna A, Avendaño A, Riego L, González A. NADP-glutamate dehydrogenase isoenzymes of Saccharomyces cerevisiae : Purification, kinetic properties and physiological role. J Biol Chem 2001; 276:43775-83; PMID:11562373; http://dx.doi.org/10.1074/jbc.M107986200 PubMed DOI

Moore D. Effects of hexose analogues on fungi: Mechanisms of inhibition and of resistance. New Phytologist 1981; 87:487-515; http://dx.doi.org/10.1111/j.1469-8137.1981.tb03221.x DOI

Liu H, Lightfoot R, Stevens JL. Activation of heat shock factor by alkylating agents is triggered by glutathione depletion and oxidation of protein thiols. J Biol Chem 1996; 271:4805-12; PMID:8617749; http://dx.doi.org/10.1074/jbc.271.9.4805 PubMed DOI

Bonawitz ND, Chatenay-Lapointe M, Pan Y, Shadel GS. Reduced TOR signaling extends chronological life span via increased respiration and upregulation of mitochondrial gene expression. Cell Metab 2007; 5:265-77; PMID:17403371; http://dx.doi.org/10.1016/j.cmet.2007.02.009 PubMed DOI PMC

Lavoie H, Whiteway M. Increased respiration in the sch9D mutant is required for increasing chronological life span but not replicative life span. Eukaryot Cell 2008; 7:1127-35; PMID:18469137; http://dx.doi.org/10.1128/EC.00330-07 PubMed DOI PMC

Lin SJ, Kaeberlein M, Andalis AA, Sturtz LA, Defossez PA, Culotta VC, Fink GR, Guarente L. Calorie restriction extends Saccharomyces cerevisiae lifespan by increasing respiration. Nature 2002; 418:344-8; PMID:12124627; http://dx.doi.org/10.1038/nature00829 PubMed DOI

Pan Y, Shadel GS. Extension of chronological life span by reduced TOR signaling requires down-regulation of Sch9p and involves increased mitochondrial OXPHOS complex density. Aging 2009; 1:131-45; PMID:20157595 PubMed PMC

Traven A, Jänicke A, Harrison P, Swaminathan A, Seemann T, Beilharz TH. Transcriptional profiling of a yeast colony provides new insight into the heterogeneity of multicellular fungal communities. PloS One 2012; 7:e46243; PMID:23029448; http://dx.doi.org/10.1371/journal.pone.0046243 PubMed DOI PMC

Wei M, Fabrizio P, Madia F, Hu J, Ge H, Li LM, Longo VD. Tor1/Sch9-regulated carbon source substitution is as effective as calorie restriction in life span extension. PLoS Genet 2009; 5:8 PubMed PMC

Hachinohe M, Yamane M, Akazawa D, Ohsawa K, Ohno M, Terashita Y, Masumoto H. A reduction in age-enhanced gluconeogenesis extends lifespan. PloS One 2013; 8:e54011; PMID:23342062; http://dx.doi.org/10.1371/journal.pone.0054011 PubMed DOI PMC

Lin SS, Manchester JK, Gordon JI. Enhanced gluconeogenesis and increased energy storage as hallmarks of aging in Saccharomyces cerevisiae. J Biol Chem 2001; 276:36000-7; PMID:11461906; http://dx.doi.org/10.1074/jbc.M103509200 PubMed DOI

Adams DJ. Fungal cell wall chitinases and glucanases. Microbiology 2004; 150:2029-35; PMID:15256547; http://dx.doi.org/10.1099/mic.0.26980-0 PubMed DOI

Kuznetsov E, Kucerova H, Vachova L, Palkova Z. SUN family proteins Sun4p, Uth1p and Sim1p are secreted from Saccharomyces cerevisiae and produced dependently on oxygen level. PloS One 2013; 8:e73882; PMID:24040106; http://dx.doi.org/10.1371/journal.pone.0073882 PubMed DOI PMC

Brauer MJ, Saldanha AJ, Dolinski K, Botstein D. Homeostatic adjustment and metabolic remodeling in glucose-limited yeast cultures. Mol Biol Cell 2005; 16:2503-17; PMID:15758028; http://dx.doi.org/10.1091/mbc.E04-11-0968 PubMed DOI PMC

Masuko T, Minami A, Iwasaki N, Majima T, Nishimura S-I, Lee YC. Carbohydrate analysis by a phenol–sulfuric acid method in microplate format. Anal Biochem 2005; 339:69-72; PMID:15766712; http://dx.doi.org/10.1016/j.ab.2004.12.001 PubMed DOI

Goward CR, Hartwell R, Atkinson T, Scawen MD. The purification and characterization of glucokinase from the thermophile Bacillus stearothermophilus. Biochem J 1986; 237:415-20; PMID:3099754; http://dx.doi.org/10.1042/bj2370415 PubMed DOI PMC

Bergmeyer HU, Gawehn K, Grassl M. Methods of Enzymatic Analysis. New York, NY, USA: Academic Press, 1974:501-3

Clifton D, Weinstock SB, Fraenkel DG. Glycolysis mutants in Saccharomyces cerevisiae. Genetics 1978; 88:1-11; PMID:147195 PubMed PMC

Bergmeyer HU, Gawehn K, Grassl M. Methods of Enzymatic Analysis. New York, NY, USA: Academic Press, 1974:430

Krietsch WKG, Bücher T. Three-Phosphoglycerate kinase from rabbit sceletal muscle and yeast. Eur J Biochem 1970; 17:568-80; PMID:5493986; http://dx.doi.org/10.1111/j.1432-1033.1970.tb01202.x PubMed DOI

Bergmeyer HU, Gawehn K, Grassl M. Methods of Enzymatic Analysis. New York, NY, USA: Academic Press, 1974:509-10

Bergmeyer HU, Gawehn K, Grassl M. Methods of Enzymatic Analysis. New York, NY, USA: Academic Press, 1974:449

Postma E, Verduyn C, Scheffers WA, Van Dijken JP. Enzymic analysis of the crabtree effect in glucose-limited chemostat cultures of Saccharomyces cerevisiae. Appl Env Microbiol 1989; 55:468-77 PubMed PMC

Bostian KA, Betts GF. Rapid purification and properties of potassium-activated aldehyde dehydrogenase from Saccharomyces cerevisiae. Biochem J 1978; 173:773-86; PMID:213051; http://dx.doi.org/10.1042/bj1730773 PubMed DOI PMC

Dickinson FM. The purification and some properties of the Mg2+-activated cytosolic aldehyde dehydrogenase of Saccharomyces cerevisiae. Biochem J 1996; 315:393; PMID:8615805; http://dx.doi.org/10.1042/bj3150393 PubMed DOI PMC

Rippa M, Signorini M. Six-Phosphogluconate dehydrogenase from Candida utilis. Methods Enzymol 1975; 41:237-40; PMID:236443; http://dx.doi.org/10.1016/S0076-6879(75)41054-0 PubMed DOI

Van Schaftingen E, Hers HG. Inhibition of fructose-1, 6-bisphosphatase by fructose 2, 6-biphosphate. Proc Natl Acad Sci U S A 1981; 78:2861-3; PMID:6265919; http://dx.doi.org/10.1073/pnas.78.5.2861 PubMed DOI PMC

Srere PA. Citrate synthase: [EC 4.1.3.7. Citrate oxaloacetate-lyase (CoA-acetylating)] In: John ML, ed. Methods in Enzymology: Academic Press, London, UK, 1969:3-11

Morrison JF. The activation of aconitase by ferrous ions and reducing agents. Biochem J 1954; 58:685-92; PMID:13230022; http://dx.doi.org/10.1042/bj0580685 PubMed DOI PMC

Illingworth JA. Purification of yeast isocitrate dehydrogenase. Biochem J 1972; 129:1119-24; PMID:4571176; http://dx.doi.org/10.1042/bj1291119 PubMed DOI PMC

Loftus TM, Hall LV, Anderson SL, McAlister-Henn L. Isolation, characterization, and disruption of the yeast gene encoding cytosolic NADP-specific isocitrate dehydrogenase. Biochemistry 1994; 33:9661-7; PMID:8068643; http://dx.doi.org/10.1021/bi00198a035 PubMed DOI

Frieden C, Bock RM, Alberty RA. Studies of the enzyme fumarase. II.1 Isolation and physical properties of crystalline enzyme. J Am Chem Soc 1954; 76:2482-4; http://dx.doi.org/10.1021/ja01638a052 DOI

Bergmeyer HU, Gawehn K, Grassl M. Methods of Enzymatic Analysis. New York, NY, USA: Academic Press, 1974:485-6

Chell RM, Sundaram TK, Wilkinson AE. Isolation and characterization of isocitrate lyase from a thermophilic Bacillus sp. Biochem J 1978; 173:165-77; PMID:687365; http://dx.doi.org/10.1042/bj1730165 PubMed DOI PMC

Doherty D. L-glutamate dehydrogenases (yeast) In: Herbert Tabor CWT, ed. Methods in Enzymology: Academic Press, London, UK, 1970:850-6

Differential stability of Gcn4p controls its cell-specific activity in differentiated yeast colonies

The characteristics of differentiated yeast subpopulations depend on their lifestyle and available nutrients

Spatially structured yeast communities: Understanding structure formation and regulation with omics tools

Mitochondrial Retrograde Signaling Contributes to Metabolic Differentiation in Yeast Colonies

The Whi2p-Psr1p/Psr2p complex regulates interference competition and expansion of cells with competitive advantage in yeast colonies

Long Noncoding RNAs in Yeast Cells and Differentiated Subpopulations of Yeast Colonies and Biofilms

Transcriptome Remodeling of Differentiated Cells during Chronological Ageing of Yeast Colonies: New Insights into Metabolic Differentiation

Multilevel regulation of an α-arrestin by glucose depletion controls hexose transporter endocytosis

Mitochondria in aging cell differentiation