Skip to main content
SpringerLink
Log in

Dimerization of yeast transcription factors Ino2 and Ino4 is regulated by precursors of phospholipid biosynthesis mediated by Opi1 repressor

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

Abstract

Structural genes of phospholipid biosynthesis in the yeast S. cerevisiae are activated by the heterodimeric transcription factor Ino2 + Ino4, binding to ICRE (inositol/choline-responsive element) promoter motifs. In the presence of phospholipid precursors inositol and choline, Ino2-dependent activation is inhibited by the Opi1 repressor which interacts with Ino2. In this work, we systematically investigated the importance of regulatory mechanisms possibly affecting ICRE-dependent gene expression. Autoregulatory expression of INO2, INO4 and OPI1 was abolished by promoter exchange experiments, showing that autoregulation of regulators contributes to the degree of differential gene expression but is not responsible for it. Using GFP fusion proteins, Ino2 and Ino4 were found to localize to the nucleus under conditions of repression and derepression. Interestingly, nuclear localization of Ino2 required a functional INO4 gene. Targeting of a lexA–Ino2 fusion to a heterologous promoter containing lexA operator motifs revealed a constitutive gene activation which was not influenced by phospholipid precursors. We could show that Ino2-dependent activation of a lexA–Ino4 fusion is affected by inositol and choline. Since gene activation required interaction of Ino2 and Ino4 mediated by their helix–loop–helix domains, formation/dissociation of the heterodimer must be considered as an important step of target gene regulation.

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

Access this article

Log in via an institution

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

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Ambroziak J, Henry SA (1994) INO2 and INO4 gene products, positive regulators of phospholipid biosynthesis in Saccharomyces cerevisiae, form a complex that binds to the INO1 promoter. J Biol Chem 269:15344–15349

    PubMed  CAS  Google Scholar 

  • Ashburner BP, Lopes JM (1995) Autoregulated expression of the yeast INO2 and INO4 helix–loop–helix activator genes effects cooperative regulation on their target genes. Mol Cell Biol 15:1709–1715

    PubMed  CAS  Google Scholar 

  • Bailis AM, Lopes JM, Kohlwein SD, Henry SA (1992) Cis and trans regulatory elements required for regulation of the CHO1 gene of Saccharomyces cerevisiae. Nucleic Acids Res 20:1411–1418

    Article  PubMed  CAS  Google Scholar 

  • Benezra R, Davis RL, Lockshon D, Turner DL, Weintraub H (1990) The protein Id: a negative regulator of helix–loop–helix DNA binding proteins. Cell 61:49–59

    Article  PubMed  CAS  Google Scholar 

  • Bhat PJ, Murthy TV (2001) Transcriptional control of the GAL/MEL regulon of yeast Saccharomyces cerevisiae: mechanism of galactose-mediated signal transduction. Mol Microbiol 40:1059–1066

    Article  PubMed  CAS  Google Scholar 

  • Brickner JH, Walter P (2004) Gene recruitment of the activated INO1 locus to the nuclear membrane. PLoS Biol 2:e342

    Article  PubMed  Google Scholar 

  • Chen M, Lopes JM (2007) Multiple basic helix–loop–helix proteins regulate expression of the ENO1 gene of Saccharomyces cerevisiae. Eukaryot Cell 6:786–796

    Article  PubMed  CAS  Google Scholar 

  • Chen M, Hancock LC, Lopes JM (2007) Transcriptional regulation of yeast phospholipid biosynthetic genes. Biochim Biophys Acta 1771:310–321

    PubMed  CAS  Google Scholar 

  • Crameri A, Whitehorn EA, Tate E, Stemmer WP (1996) Improved green fluorescent protein by molecular evolution using DNA shuffling. Nat Biotechnol 14:315–319

    Article  PubMed  CAS  Google Scholar 

  • Dietz M, Heyken WT, Hoppen J, Geburtig S, Schüller HJ (2003) TFIIB and subunits of the SAGA complex are involved in transcriptional activation of phospholipid biosynthetic genes by the regulatory protein Ino2 in the yeast Saccharomyces cerevisiae. Mol Microbiol 48:1119–1130

    Article  PubMed  CAS  Google Scholar 

  • Falvey E, Schibler U (1991) How are the regulators regulated? FASEB J 5:309–314

    PubMed  CAS  Google Scholar 

  • Fürst P, Hamer D (1989) Cooperative activation of a eukaryotic transcription factor: interaction between Cu(I) and yeast ACE1 protein. Proc Natl Acad Sci USA 86:5267–5271

    Article  PubMed  Google Scholar 

  • Gardenour KR, Levy J, Lopes JM (2004) Identification of novel dominant INO2 c mutants with an Opi− phenotype. Mol Microbiol 52:1271–1280

    Article  PubMed  CAS  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

    Article  PubMed  CAS  Google Scholar 

  • Golemis EA, Brent R (1992) Fused protein domains inhibit DNA binding by LexA. Mol Cell Biol 12:3006–3014

    PubMed  CAS  Google Scholar 

  • Greenberg ML, Goldwasser P, Henry SA (1982) Characterization of a yeast regulatory mutant constitutive for synthesis of inositol-1-phosphate synthase. Mol Gen Genet 186:157–163

    Article  PubMed  CAS  Google Scholar 

  • Griggs DW, Johnston M (1991) Regulated expression of the GAL4 activator gene in yeast provides a sensitive genetic switch for glucose repression. Proc Natl Acad Sci USA 88:8597–8601

    Article  PubMed  CAS  Google Scholar 

  • Hanes SD, Brent R (1989) DNA specificity of the bicoid activator protein is determined by homeodomain recognition helix residue 9. Cell 57:1275–1283

    Article  PubMed  CAS  Google Scholar 

  • Heyken WT, Repenning A, Kumme J, Schüller HJ (2005) Constitutive expression of yeast phospholipid biosynthetic genes by variants of Ino2 activator defective for interaction with Opi1 repressor. Mol Microbiol 56:696–707

    Article  PubMed  CAS  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

    Article  PubMed  CAS  Google Scholar 

  • Hinnebusch AG (1984) Evidence for translational regulation of the activator of general amino acid control in yeast. Proc Natl Acad Sci USA 81:6442–6446

    Article  PubMed  CAS  Google Scholar 

  • Hollenberg SM, Sternglanz R, Cheng PF, Weintraub H (1995) Identification of a new family of tissue-specific basic helix–loop–helix proteins with a two-hybrid system. Mol Cell Biol 15:3813–3822

    PubMed  CAS  Google Scholar 

  • Hoppen J, Repenning A, Albrecht A, Geburtig S, Schüller HJ (2005) Comparative analysis of promoter regions containing binding sites of the heterodimeric transcription factor Ino2/Ino4 involved in yeast phospholipid biosynthesis. Yeast 22:601–613

    Article  PubMed  CAS  Google Scholar 

  • Hosaka K, Nikawa JI, Kosaki T, Yamashita S (1994) Cloning and characterization of the SCS1 gene required for the expression of genes in yeast phospholipid synthesis. J Biochem 115:131–136

    PubMed  CAS  Google Scholar 

  • Hoshizaki DK, Hill JE, Henry SA (1990) The Saccharomyces cerevisiae INO4 gene encodes a small, highly basic protein required for derepression of phospholipid biosynthesis enzymes. J Biol Chem 265:4736–4745

    PubMed  CAS  Google Scholar 

  • Jesch SA, Zhao X, Wells MT, Henry SA (2005) Genome-wide analysis reveals inositol, not choline, as the major effector of Ino2p–Ino4p and unfolded protein response target gene expression in yeast. J Biol Chem 280:9106–9118

    Article  PubMed  CAS  Google Scholar 

  • Johnston M, Flick JS, Pexton T (1994) Multiple mechanisms provide rapid and stringent glucose repression of GAL gene expression in Saccharomyces cerevisiae. Mol Cell Biol 14:3834–3841

    PubMed  CAS  Google Scholar 

  • Kadosh D, Struhl K (1997) Repression by Ume6 involves recruitment of a complex containing Sin3 corepressor and Rpd3 histone deacetylase to target promoters. Cell 89:365–371

    Article  PubMed  CAS  Google Scholar 

  • Littlewood TD, Evan GI (1998) Helix–loop–helix transcription factors, 3rd edn. Oxford University Press, Oxford

    Google Scholar 

  • Lo WS, Gamache ER, Henry KW, Yang D, Pillus L, Berger SL (2005) Histone H3 phosphorylation can promote TBP recruitment through distinct promoter-specific mechanisms. EMBO J 24:997–1008

    Article  PubMed  CAS  Google Scholar 

  • Loewen CJ, Roy A, Levine TP (2003) A conserved ER targeting motif in three families of lipid binding proteins and in Opi1p binds VAP. EMBO J 22:2025–2035

    Article  PubMed  CAS  Google Scholar 

  • Loewen CJ, Gaspar ML, Jesch SA, Delon C, Ktistakis NT, Henry SA, Levine TP (2004) Phospholipid metabolism regulated by a transcription factor sensing phosphatidic acid. Science 304:1644–1647

    Article  PubMed  CAS  Google Scholar 

  • Lopes JM, Henry SA (1991) Interaction of trans and cis regulatory elements in the INO1 promoter of Saccharomyces cerevisiae. Nucleic Acids Res 19:3987–3994

    Article  PubMed  CAS  Google Scholar 

  • Ma J, Ptashne M (1987) The carboxy-terminal 30 amino acids of GAL4 are recognized by GAL80. Cell 50:137–142

    Article  PubMed  CAS  Google Scholar 

  • Moll T, Tebb G, Surana U, Robitsch H, Nasmyth K (1991) The role of phosphorylation and the CDC28 protein kinase in cell cycle-regulated nuclear import of the S. cerevisiae transcription factor SWI5. Cell 66:743–758

    Article  PubMed  CAS  Google Scholar 

  • Mumberg D, Müller R, Funk M (1994) Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression. Nucleic Acids Res 22:5767–5768

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Nikoloff DM, McGraw P, Henry SA (1992) The INO2 gene of Saccharomyces cerevisiae encodes a helix–loop–helix protein that is required for activation of phospholipid synthesis. Nucleic Acids Res 20:3253

    Article  PubMed  CAS  Google Scholar 

  • O’Neill EM, Kaffman A, Jolly ER, O’Shea EK (1996) Regulation of PHO4 nuclear localization by the PHO80–PHO85 cyclin-CDK complex. Science 271:209–212

    Article  PubMed  CAS  Google Scholar 

  • Olson KA, Nelson C, Tai G, Hung W, Yong C, Astell C, Sadowski I (2000) Two regulators of Ste12p inhibit pheromone-responsive transcription by separate mechanisms. Mol Cell Biol 20:4199–4209

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Rhee Y, Gurel F, Gafni Y, Dingwall C, Citovsky V (2000) A genetic system for detection of protein nuclear import and export. Nat Biotechnol 18:433–437

    Article  PubMed  CAS  Google Scholar 

  • Robinson KA, Lopes JM (2000) The promoter of the yeast INO4 regulatory gene: a model of the simplest yeast promoter. J Bacteriol 182:2746–2752

    Article  PubMed  CAS  Google Scholar 

  • Schüller HJ, Hahn A, Tröster F, Schütz A, Schweizer E (1992a) Coordinate genetic control of yeast fatty acid synthetase genes FAS1 and FAS2 by an upstream activation site common to genes involved in the membrane lipid biosysnthesis. EMBO J 11:107–114

    PubMed  Google Scholar 

  • Schüller HJ, Schorr R, Hoffmann B, Schweizer E (1992b) Regulatory gene INO4 of yeast phospholipid biosynthesis is positively autoregulated and functions as a trans-activator of fatty acid synthase genes FAS1 and FAS2 from Saccharomyces cerevisiae. Nucleic Acids Res 20:5955–5961

    Article  PubMed  Google Scholar 

  • Schüller HJ, Richter K, Hoffmann B, Ebbert R, Schweizer E (1995) DNA binding site of the yeast heteromeric Ino2p/Ino4p basic helix–loop–helix transcription factor: structural requirements as defined by saturation mutagenesis. FEBS Lett 370:149–152

    Article  PubMed  Google Scholar 

  • Schwank S, Ebbert R, Rautenstrauss K, Schweizer E, Schüller HJ (1995) Yeast transcriptional activator INO2 interacts as an Ino2p/Ino4p basic helix–loop–helix heteromeric complex with the inositol/choline-responsive element necessary for expression of phospholipid biosynthetic genes in Saccharomyces cerevisiae. Nucleic Acids Res 23:230–237

    Article  PubMed  CAS  Google Scholar 

  • Schwank S, Hoffmann B, Schüller HJ (1997) Influence of gene dosage and autoregulation of the regulatory genes INO2 and INO4 on inositol/choline-repressible gene transcription in the yeast Saccharomyces cerevisiae. Curr Genet 31:462–468

    Article  PubMed  CAS  Google Scholar 

  • Sikorski RS, Hieter P (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19–27

    PubMed  CAS  Google Scholar 

  • Taheri N, Köhler T, Braus GH, Mösch HU (2000) Asymmetrically localized Bud8p and Bud9p proteins control yeast cell polarity and development. EMBO J 19:6686–6696

    Article  PubMed  CAS  Google Scholar 

  • Wagner C, Blank M, Strohmann B, Schüller HJ (1999) Overproduction of the Opi1 repressor inhibits transcriptional activation of structural genes required for phospholipid biosynthesis in the yeast Saccharomyces cerevisiae. Yeast 15:843–854

    Article  PubMed  CAS  Google Scholar 

  • Wagner C, Dietz M, Wittmann J, Albrecht A, Schüller HJ (2001) The negative regulator Opi1 of phospholipid biosynthesis in yeast contacts the pleiotropic repressor Sin3 and the transcriptional activator Ino2. Mol Microbiol 41:155–166

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work has been supported by the Deutsche Forschungsgemeinschaft (DFG). We thank Prof. H. -U. Mösch, Prof. G. Braus and Dr. N. Taheri for several suggestions and invaluable support with fluorescence microscopy.

Author information

Authors and Affiliations

  1. Institut für Genetik und Funktionelle Genomforschung, Jahnstrasse 15a, 17487, Greifswald, Germany

    Jacqueline Kumme, Martin Dietz, Christian Wagner & Hans-Joachim Schüller

Authors
  1. Jacqueline Kumme
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Martin Dietz
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christian Wagner
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hans-Joachim Schüller
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hans-Joachim Schüller.

Additional information

Communicated by G. Braus.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kumme, J., Dietz, M., Wagner, C. et al. Dimerization of yeast transcription factors Ino2 and Ino4 is regulated by precursors of phospholipid biosynthesis mediated by Opi1 repressor. Curr Genet 54, 35–45 (2008). https://doi.org/10.1007/s00294-008-0197-7

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00294-008-0197-7

Keywords

Search

Navigation