Skip to main content
Log in

Isocitrate lyase of the yeast Kluyveromyces lactis is subject to glucose repression but not to catabolite inactivation

  • Research Article
  • Published:
Current Genetics Aims and scope Submit manuscript

An Erratum to this article was published on 20 February 2004

Abstract

KlICL1, encoding the isocitrate lyase of Kluyveromyces lactis, was isolated by complementation of the Saccharomyces cerevisiae icl1 deletion mutant. Sequence analysis revealed an open reading frame of 1,626 nucleotides encoding a protein with 542 amino acids. The deduced protein shows extensive homologies to isocitrate lyases from various organisms, with an overall identity of 69% to the enzyme from S. cerevisiae. The KlICL1 gene has two major transcription start-points, located at −113 bp and −95 bp relative to the ATG translation start codon. The gene is expressed on ethanol medium only in respiratory-competent cells. Transcription is repressed by glucose. Mutants carrying a Klcat8 deletion lack the ability to derepress KlICL1 transcription. A Klicl1 deletion mutant does not grow on ethanol medium and lacks any isocitrate lyase activity. A strain lacking the gene KlFBP1, which encodes the gluconeogenic enzyme fructose 1,6-bisphosphatase, lacks the ability to grow on non-fermentable carbon sources. This implies that K. lactis does not contain additional isoenzymes catalyzing either of the reactions. Enzyme assays revealed that neither KlIcl1p nor KlFbp1p are subject to catabolite inactivation. However, the respective enzymes from S. cerevisiae are efficiently inactivated when expressed in K. lactis. Thus, despite the extensive sequence similarities of the enzymes involved, non-fermentative carbohydrate metabolism in the two yeasts displays distinct regulatory properties.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1A–C
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Amaral FC, Van Dijck P, Nicoli JR, Thevelein JM (1997) Molecular cloning of the neutral trehalase gene from Kluyveromyces lactis and the distinction between neutral and acid trehalases. Arch Microbiol 167:202–208

    Article  CAS  PubMed  Google Scholar 

  • Beeching JR (1989) High sequence conservation between isocitrate lyase from Escherichia coli and Ricinus communis. Protein Seq Data Anal 2:463–466

    CAS  PubMed  Google Scholar 

  • Berben G, Dumont J, Gilliquet V, Bolle P-A, Hilger F (1991) The YDp plasmids: a uniform set of vectors bearing versatile gene disruption cassettes for Saccharomyces cerevisiae. Yeast 7:475–477

    CAS  PubMed  Google Scholar 

  • Bianchi MM, Falcone C, Chen XJ, Wésolowski-Louvel M, Fontali L, Fukuhara H (1987) Transformation of the yeast Kluyveromyces lactis by new vectors derived from the 1.6-μm circular plasmid pKD1. Curr Genet 12:185–192

    Google Scholar 

  • Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523

    CAS  PubMed  Google Scholar 

  • Bojunga N, Kötter P, Entian K-D (1998) The succinate/fumarate transporter Acr1p of Saccharomyces cerevisiae is part of the gluconeogenic pathway and its expression is regulated by Cat8p. Mol Gen Genet 260:453–461

    Article  PubMed  Google Scholar 

  • Breunig KD, Kuger P (1987) Functional homology between the yeast regulatory proteins GAL4 and LAC9: LAC9-mediated transcriptional activation in Kluyveromyces lactis involves protein binding to a regulatory sequence homologous to the GAL4 protein-binding site. Mol Cell Biol 7:4400–4406

    CAS  PubMed  Google Scholar 

  • Breunig KD, Bolotin-Fukuhara M, Bianchi MM, Bourgarel D, Falcone C, Ferrero II, Frontali L, Goffrini P, Krijger JJ, Mazzoni C, Milkowski C, Steensma HY, Wesolowski-Louvel M, Zeeman AM (2000) Regulation of primary carbon metabolism in Kluyveromyces lactis. Enzyme Microb Technol 26:771–780

    Article  CAS  PubMed  Google Scholar 

  • Broach JR, Strathern JN, Hicks JB (1979) Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene. Gene 8:121–123

    CAS  PubMed  Google Scholar 

  • Burnette WN (1981) ″Western blotting″: electrophoretic transfer of proteins from sodium dodecyl sulfate–polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112:195–203

    CAS  PubMed  Google Scholar 

  • Carlson M (1999) Glucose repression in yeast. Curr Opin Microbiol 2:202–207

    Google Scholar 

  • Chen XJ (1996) Low- and high-copy-number shuttle vectors for replication in the budding yeast Kluyveromyces lactis. Gene 172:131–136

    CAS  PubMed  Google Scholar 

  • Chen XJ, Clark-Walker GD (1993) Mutations in MGI genes convert Kluyveromyces lactis into a petite-positive yeast. Genetics 133:517–525

    CAS  PubMed  Google Scholar 

  • Chen XJ, Hansbro PM, Clark-Walker GD (1998) Suppression of ρ0 lethality by mitochondrial ATP synthase F1 mutations in Kluyveromyces lactis occurs in the absence of F0. Mol Gen Genet 259:457–467

    Article  CAS  PubMed  Google Scholar 

  • De Mesquita JF, Zaragoza O, Gancedo JM (1998) Functional analysis of upstream activating elements in the promoter of the FBP1 gene from Saccharomyces cerevisiae. Curr Genet 33:406–411

    Article  PubMed  Google Scholar 

  • Dixon GH, Kornberg HL (1959) Assay methods for key enzymes of the glyoxylate cycle. Biochem J 72:3P

    Google Scholar 

  • Dohmen RJ, Strasser AWM, Höner CB, Hollenberg CP (1991) An efficient transformation procedure enabling long-term storage of competent cells of various yeast genera. Yeast 7:691–692

    CAS  PubMed  Google Scholar 

  • Entian KD, Schüller HJ (1997). Glucose repression (Carbon catabolite repression) in yeast. In: Zimmermann FK, Entian K-D (eds) Yeast sugar metabolism. Technomic, Lancaster, Pa., pp 409–434

  • Fernández E, Moreno F, Rodicio R (1992) The ICL1 gene from Saccharomyces cerevisiae. Eur J Biochem 204:983–990

    PubMed  Google Scholar 

  • Fernández E, Fernández M, Moreno F, Rodicio R (1993) Transcriptional regulation of the isocitrate lyase encoding gene in Saccharomyces cerevisiae. FEBS Lett 333:238–242

    Article  CAS  PubMed  Google Scholar 

  • Flores CL, Rodriguez C, Petit T, Gancedo C (2000) Carbohydrate and energy-yielding metabolism in non-conventional yeasts. FEMS Microbiol Rev 24:507–529

    Google Scholar 

  • Gamo FJ, Navas MA, Blazquez MA, Gancedo C, Gancedo JM (1994) Catabolite inactivation of heterologous fructose-1,6-bisphosphatases and fructose-1,6-bisphosphatase-beta-galactosidase fusion proteins in Saccharomyces cerevisiae. Eur J Biochem 222:879–884

    CAS  PubMed  Google Scholar 

  • Gancedo C (1971) Inactivation of fructose-1,6-diphosphatase by glucose in yeast. J Bacteriol 107:401–405

    CAS  PubMed  Google Scholar 

  • Gancedo C, Serrano R (1989) Energy-yielding metabolism. In: Rose AH, Harrison JS (eds) The yeasts. Metabolism and physiology of yeasts. Academic Press, London, pp 205–259

  • Gancedo JM (1998) Yeast carbon catabolite repression. Microbiol Mol Biol Rev 62:334–361

    CAS  PubMed  Google Scholar 

  • Gancedo JM, Gancedo C (1997) Gluconeogenesis and catabolite inactivation in yeast. In: Zimmermann FK, Entian K-D (ed) Yeast sugar metabolism. Technomic, Lancaster, Pa., pp 409–434

  • Gancedo JM, Mazon MJ, Gancedo C (1983) Fructose 2,6-bisphosphate activates the cAMP-dependent phosphorylation of yeast fructose-1,6-bisphosphatase in vitro. J Biol Chem 258:5998–5999

    CAS  PubMed  Google Scholar 

  • Georis I, Krijger JJ, Breunig KD, Vandenhaute J (2000) Differences in regulation of yeast gluconeogenesis revealed by Cat8p-independent activation of PCK1 and FBP1 genes in Kluyveromyces lactis. Mol Gen Genet 264:193–203

    CAS  PubMed  Google Scholar 

  • Gietz RD, Sugino A (1988) New yeast–Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene 74:527–534

    CAS  PubMed  Google Scholar 

  • Goffrini P, Ficarelli A, Donnini C, Lodi T, Puglisi PP, Ferrero I (1996) FOG1 and FOG2 genes, required for the transcriptional activation of glucose-repressible genes of Kluyveromyces lactis, are homologous to GAL83 and SNF1 of Saccharomyces cerevisiae. Curr Genet 29:316–326

    Google Scholar 

  • Gonzalez-Siso MI, Freire-Picos MA, Ramil E, Gonzalez-Dominguez M, Rodriguez Torres A, Cerdan ME (2000) Respirofermentative metabolism in Kluyveromyces lactis: insights and perspectives. Enzyme Microb Technol 26:699–705

    Article  CAS  PubMed  Google Scholar 

  • Gubler U, Hoffman BJ (1983) A simple and very efficient method for generating cDNA libraries. Gene 25: 263–269

    CAS  PubMed  Google Scholar 

  • Hammerle M, Bauer J, Rose M, Szallies A, Thumm M, Dusterhus S, Mecke D, Entian KD, Wolf DH (1998) Proteins of newly isolated mutants and the amino-terminal proline are essential for ubiquitin-proteasome-catalyzed catabolite degradation of fructose-1,6-bisphosphatase of Saccharomyces cerevisiae. J Biol Chem 273:25000–25005

    Article  CAS  PubMed  Google Scholar 

  • Hanahan D (1985) Techniques for transformation of E. coli. In: Glover DM (ed) DNA cloning: a practical approach, 2nd edn. IRL Press, Oxford, pp 1–36

  • Hedges D, Proft M, Entian KD (1995) CAT8, a new zinc cluster-encoding gene necessary for derepression of gluconeogenic enzymes in the yeast Saccharomyces cerevisiae. Mol Cell Biol 15:1915–1922

    CAS  PubMed  Google Scholar 

  • Heinisch J (1993) PFK2, ISP42, ERG2 and RAD14 are located on the right arm of chromosome XIII. Yeast 9:1103–1105

    CAS  PubMed  Google Scholar 

  • Heinisch J, Kirchrath L, Liesen T, Vogelsang K, Hollenberg CP (1993) Molecular genetics of phosphofructokinase in the yeast Kluyveromyces lactis. Mol Microbiol 8:559–570

    CAS  PubMed  Google Scholar 

  • Heinisch JJ, Valdes E, Alvarez J, Rodicio R (1996) Molecular genetics of ICL2, encoding a non-functional isocitrate lyase in Saccharomyces cerevisiae. Yeast 12:1285–1295

    Article  CAS  PubMed  Google Scholar 

  • Hill JE, Myers AM, Koerner TJ, Tzagoloff A (1986) Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast 2:163–167

    CAS  PubMed  Google Scholar 

  • Hoffman CS, Winston F (1987) A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57:267–272

    CAS  PubMed  Google Scholar 

  • Jacoby J, Hollenberg CP, Heinisch JJ (1993) Transaldolase mutants in the yeast Kluyveromyces lactis provide evidence that glucose can be metabolized through the pentose phosphate pathway. Mol Microbiol 10:867–876

    CAS  PubMed  Google Scholar 

  • Kanai T, Takeshita S, Atomi H, Umemura K, Ueda M. Tanaka A (1998) A regulatory factor, Fil1p, involved in derepression of the isocitrate lyase gene in Saccharomyces cerevisiae. A possible mitochondrial protein necessary for protein synthesis in mitochondria. Eur J Biochem 256:212–220

    Article  CAS  PubMed  Google Scholar 

  • Kirchrath L, Lorberg A, Schmitz HP, Gengenbacher U, Heinisch JJ (2000) Comparative genetic and physiological studies of the MAP kinase Mpk1p from Kluyveromyces lactis and Saccharomyces cerevisiae. J Mol Biol 300:743–758

    Article  CAS  PubMed  Google Scholar 

  • Klebe RJ, Harriss JV, Sharp ZD, Douglas MG (1983) A general method for polyethylene-glycol-induced genetic transformation of bacteria and yeast. Gene 25:333–334

    CAS  PubMed  Google Scholar 

  • Klein CJ, Rasmussen JJ, Ronnow B, Olsson L, Nielsen J (1999) Investigation of the impact of MIG1 and MIG2 on the physiology of Saccharomyces cerevisiae. J Biotechnol 68:197–212

    Google Scholar 

  • Kolodner R, Fishel RA, Howard M (1985) Genetic recombination of bacterial plasmid DNA. Effect of RecF pathway mutations on plasmid recombination in E. coli. J Bacteriol 163:1060–1066

    CAS  PubMed  Google Scholar 

  • Lodi T, Saliola M, Donnini C, Goffrini P (2001) Three target genes for the transcriptional activator Cat8p of Kluyveromyces lactis: acetyl coenzyme A synthetase genes KlACS1 and KlACS2 and lactate permease gene KlJEN1. J Bacteriol 183:5257–5261

    Article  CAS  PubMed  Google Scholar 

  • López-Boado YS, Herrero P, Gascon S, Moreno F (1987) Catabolite inactivation of isocitrate lyase from Saccharomyces cerevisiae. Arch Microbiol 147:231–234

    PubMed  Google Scholar 

  • López-Boado YS, Herrero P, Fernandez T, Fernandez R, Moreno F (1988) Glucose-stimulated phosphorylation of yeast isocitrate lyase in vivo. J Gen Microbiol 134:2499–2505

    PubMed  Google Scholar 

  • Lorberg A, Kirchrath L, Ernst JF, Heinisch JJ (1999) Genetic and biochemical characterization of phosphofructokinase from the opportunistic pathogenic yeast Candida albicans. Eur J Biochem 260:217–226

    Article  CAS  PubMed  Google Scholar 

  • Lorberg A, Schmitz HP, Gengenbacher U, Heinisch JJ (2003) KlROM2 encodes an essential GEF homologue in Kluyveromyces lactis. Yeast 20:611–624

    CAS  PubMed  Google Scholar 

  • Mazón MJ, Gancedo JM, Gancedo C (1982) Inactivation of yeast fructose 1,6-bisphosphatase. In vivo phosphorylation of the enzyme. J Biol Chem 257:1128–1130

    PubMed  Google Scholar 

  • Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

  • Myers AM, Tzagoloff A, Kinney DM, Lusty CJ (1986) Yeast shuttle and integrative vectors with multiple cloning sites suitable for construction of lacZ fusions. Gene 45:299–310

    CAS  PubMed  Google Scholar 

  • Ordiz I, Herrero P, Rodicio R, Moreno F (1995) Glucose-induced inactivation of isocitrate lyase in Saccharomyces cerevisiae is mediated by an internal decapeptide sequence. FEBS Lett 367:219–222

    Article  CAS  PubMed  Google Scholar 

  • Ordiz I, Herrero P, Rodicio R, Moreno F (1996) Glucose-induced inactivation of isocitrate lyase in Saccharomyces cerevisiae is mediated by the cAMP-dependent protein kinase catalytic subunits Tpk1 and Tpk2. FEBS Lett 385:43–46

    Article  CAS  PubMed  Google Scholar 

  • Ordiz I, Herrero P, Rodicio R, Gancedo JM, Moreno F (1998) A 27 kDa protein binds to a positive and a negative regulatory sequence in the promoter of the ICL1 gene from Saccharomyces cerevisiae. Biochem J 329:383–388

    CAS  PubMed  Google Scholar 

  • Parikh VS, Morgan MM, Scott R, Clements IS, Butow RA (1987) The mitochondrial genotype can influence nuclear gene expression in yeast. Science 235:576–580

    CAS  PubMed  Google Scholar 

  • Pohlig G, Wingender-Drissen R, Noda T, Holzer H (1983) Cyclic AMP and fructose-2,6-bisphosphate stimulated in vitro phosphorylation of yeast fructose-1,6-bisphosphatase. Biochem Biophys Res Commun 115:317–324

    CAS  PubMed  Google Scholar 

  • Rahner A, Schöler A, Martens E, Gollwitzer B, Schüller HJ (1996) Dual influence of the yeast Cat1p (Snf1p) protein kinase on carbon-source dependent transcriptional activation of gluconeogenic genes by the regulatory gene CAT8. Nucleic Acids Res 24:2331–2337

    Article  CAS  PubMed  Google Scholar 

  • Rahner A, Hiesinger M, Schüller H-J (1999) Deregulation of gluconeogenic structural genes by variants of the transcriptional activator Cat8p of the yeast Saccharomyces cerevisiae. Mol Microbiol 34:146–156

    Article  CAS  PubMed  Google Scholar 

  • Randez-Gil F, Bojunga N, Proft M, Entian K-D (1997) Glucose derepression of gluconeogenic enzymes in Saccharomyces cerevisiae correlates with phosphorylation of the gene activator Cat8p. Mol Cell Biol 17:2502–2510

    CAS  PubMed  Google Scholar 

  • Redruello B, Valdés E, López ML, Rodicio R (1999) Multiple regulatory elements control the expression of the yeast ACR1 gene. FEBS Lett 445:246–250

    Article  CAS  PubMed  Google Scholar 

  • Rittenhouse J, Moberly L, Marcus F (1987) Phosphorylation in vivo of yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase at the cyclic AMP-dependent site. J Biol Chem 262:10114–10119

    CAS  PubMed  Google Scholar 

  • Robzyk K, Kassir Y (1992) A simple and highly efficient procedure for rescuing autonomous plasmids from yeast. Nucleic Acids Res 20:3790

    CAS  PubMed  Google Scholar 

  • Rose M, Entian KD, Hofmann L, Vogel RD, Mecke D (1988) Irreversible inactivation of Saccharomyces cerevisiae fructose-1,6-bisphosphatase independent of protein phosphorylation at Ser11. FEBS Lett 241:55–59

    Article  CAS  PubMed  Google Scholar 

  • Rothstein RJ, Sherman F (1980) Genes affecting the expression of cytochrome c in yeast: genetic mapping and genetic interactions. Genetics 94:871–889

    Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

    Google Scholar 

  • Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    PubMed  Google Scholar 

  • Scherman F, Fink GR, Hicks JB (1986) Laboratory course manual for methods in yeast genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

  • Schöler A, Schüller HJ (1993) Structure and regulation of the isocitrate lyase gene ICL1 from the yeast Saccharomyces cerevisiae. Curr Genet 23:375–381

    Google Scholar 

  • Schöler A, Schüller HJ (1994) A carbon source-responsive promoter element necessary for activation of the isocitrate lyase gene ICL1 is common to genes of the gluconeogenic pathway in the yeast Saccharomyces cerevisiae. Mol Cell Biol 14:3613–3622

    PubMed  Google Scholar 

  • Schüller HJ (2003) Transcriptional control of nonfermentative metabolism in the yeast Saccharomyces cerevisiae. Curr Genet 43:139–160

    PubMed  Google Scholar 

  • Shyjan AW, Butow RA (1993) Intracellular dialogue. Curr Biol 3:398–400

    CAS  Google Scholar 

  • Thomas PS (1980) Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci 77:5201–5205

    CAS  PubMed  Google Scholar 

  • Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680

    PubMed  Google Scholar 

  • Toyoda Y, Sy J (1984) Purification and phosphorylation of fructose-1,6-bisphosphatase from Kluyveromyces fragilis. J Biol Chem 259:8718–8723

    CAS  PubMed  Google Scholar 

  • Vieira J, Messing J (1991) New pUC-derived cloning vectors with different selectable markers and DNA replication origins. Gene 100:189–194

    CAS  PubMed  Google Scholar 

  • Vincent O, Carlson M (1998) Sip4, a Snf1 kinase-dependent transcriptional activator, binds to the carbon source-responsive element of gluconeogenic genes. EMBO J 17:7002–7008

    Article  CAS  PubMed  Google Scholar 

  • Vincent O, Gancedo JM (1995) Analysis of positive elements sensitive to glucose in the promoter of the FBP1 gene from yeast. J Biol Chem 270:12832–12838

    Article  CAS  PubMed  Google Scholar 

  • Wésolowski-Louvel M, Tanguy-Rougeau C, Fukuhara H (1988) A nuclear gene required for the expression of the linear DNA-associated killer system in the yeast Kluyveromyces lactis. Yeast 4:71–81

    PubMed  Google Scholar 

  • Zamenhof S (1957) Preparation and assay of deoxyribonucleic acid from animal tissue. Methods Enzymol 3:696–704

    Google Scholar 

  • Zaror I, Marcus F, Moyer DL, Tung J, Shuster JR (1993) Fructose-1,6-bisphosphatase of the yeast Kluyveromyces lactis. Eur J Biochem 212:193–199

    CAS  PubMed  Google Scholar 

Download references

Acknowledgement

This work was supported by the grant DGICYT BMC 2001-1690 C02-02.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Rosaura Rodicio.

Additional information

Communicated by K. Breunig

An erratum to this article can be found at http://dx.doi.org/10.1007/s00294-004-0489-5

Rights and permissions

Reprints and permissions

About this article

Cite this article

López, M.L., Redruello, B., Valdés, E. et al. Isocitrate lyase of the yeast Kluyveromyces lactis is subject to glucose repression but not to catabolite inactivation. Curr Genet 44, 305–316 (2004). https://doi.org/10.1007/s00294-003-0453-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00294-003-0453-9

Keywords

Navigation