Skip to main content
Springer Nature Link
Log in

The GEF1 gene of Saccharomyces cerevisiae encodes an integral membrane protein; mutations in which have effects on respiration and iron-limited growth

  • Original Articles
  • Published:
Molecular and General Genetics MGG Aims and scope Submit manuscript

Abstract

We have isolated a new class of respiration-defective, i.e petite, mutants of the yeast Saccharomyces cerevisiae. Mutations in the GEF1 gene cause cells to grow slowly on rich media containing carbon sources utilized by respiration. This phenotype is suppressed by adding high concentrations of iron to the growth medium. Gef1 − mutants also fail to grow on a fermentable carbon source, glucose, when iron is reduced to low concentrations in the medium, suggesting that the GEF1 gene is required for efficient metabolism of iron during growth on fermentable as well as respired carbon sources. However, activity of the iron uptake system appears to be unaffected in gef1 − mutants. Fe(II) transporter activity and regulation is normal in gef1 − mutants. Fe(III) reductase induction during iron-limited growth is disrupted, but this appears to be a secondary effect of growth rate alterations. The wild-type GEF1 gene was cloned and sequenced; it encodes a protein of 779 amino acids, 13 possible transmembrane domains, and significant similarity to chloride channel proteins from fish and mammals, suggesting that GEF1 encodes an integral membrane protein. A gef1 − deletion mutation generated in vitro and introduced into wild-type haploid strains by gene transplacement was not lethal. Oxygen consumption by intact gef1 − cells and by mitochondrial fractions isolated from gef1 − mutants was reduced 25–50% relative to wild type, indicating that mitochondrial function is defective in these mutants. We suggest that GEF1 encodes a transport protein that is involved in intracellular iron metabolism.

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

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

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

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Google Scholar 

  • Burkholder AC, Hartwell LH (1985) The yeast alpha-factor receptor: Structural properties deduced from the sequence of the STE2 gene. Nucleic Acids Res 13:8463–8475

    Google Scholar 

  • Carle GF, Olson MV (1985) An electrophoretic karyotype for yeast. Proc Natl Acad Sci USA 82:3756–3760

    Google Scholar 

  • Carlson M, Botstein D (1982) Two differentially regulated mRNAs with different 5′ ends encode secreted and intracellular forms of yeast invertase. Cell 28:145–154

    Google Scholar 

  • Celenza JL, Marshall-Carlson L, Carlson M (1988) The yeast SNF3 gene encodes a glucose transporter homologous to the mammalian protein. Proc Natl Acad Sci USA 85:2130–2134

    Google Scholar 

  • Chappell JB (1964) The oxidation of citrate, isocitrate, and cis-aconitate by isolated mitochondria. Biochem J 90:225–237

    Google Scholar 

  • Dancis A, Klausner RD, Hinnebusch AG, Barriocanal JG (1990) Genetic evidence that ferric reductase is required for iron uptake in Saccharomyces cerevisiae. Mol Cell Biol 10:2294–2301

    Google Scholar 

  • Dancis A, Roman DG, Anderson GJ, Hinnebusch AG, Klausner RD (1992) Ferric reductase of Saccharomyces cerevisiae: Molecular characterization, role in iron uptake, and transcriptional control by iron. Proc Natl Acad Sci USA 89:3869–3873

    Google Scholar 

  • Dayhoff MO, Schwartz RM, Orcutt BC (1978) Atlas of Protein Sequence and Structure. National Biomedical Research Foundation, Silver Spring, Md., pp. 345–352

    Google Scholar 

  • Devereux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387–395

    Google Scholar 

  • Eide D, Guarente L (1992) Increased dosage of a transcriptional activator gene enhances iron-limited growth of Saccharomyces cerevisiae. J Gen Microbiol 138:347–354

    Google Scholar 

  • Eide D, Davis-Kaplan S, Jordan I, Sipe D, Kaplan J (1992) Regulation of iron uptake in Saccharomyces cerevisiae: the ferrireductase and Fe(II) transporter are regulated independently. J Biol Chem 267:20774–20781

    Google Scholar 

  • Forsburg SL, Guarente L (1988) Mutational analysis of upstream activation sequence 2 of the CYC1 gene of Saccharomyces cerevisiae: a HAP2-HAP3- responsive site. Mol Cell Biol 8:647–654

    Google Scholar 

  • Hagen DC, McCaffrey G, Sprague GF (1986) Evidence the yeast STE3 gene encodes a receptor for the peptide pheromone a-factor: Gene sequence and implications for the structure of the presumed receptor. Proc Natl Acad Sci USA 83:1418–1422

    Google Scholar 

  • Henikoff S (1984) Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28:351–359

    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

    Google Scholar 

  • Hill RL, Bradshaw RA (1969) Fumarase. Methods Enzymol 13:91–99

    Google Scholar 

  • Hirata R, Ohsumi Y, Nakano A, Kawasaki H, Suzuki K, Anraku Y (1990) Molecular structure of a gene, VMA1, encoding the catalytic subunit of H+-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. J Biol Chem 265:6726–6733

    Google Scholar 

  • Hoffman W (1985) Molecular characterization of the CAN1 locus in Saccharomyces cerevisiae: a transmembrane protein without N-terminal hydrophobic signal sequence. J Biol Chem 260:11831–11837

    Google Scholar 

  • Huisman O, Raymond W, Froehlich K-U, Errada P, Kleckner N, Botstein D, Hoyt MA (1987) A Tn10-1acZ-kanR-URA3 gene fusion transposon for insertion mutagenesis and fusion analysis of yeast and bacterial genes. Genetics 116:191–199

    Google Scholar 

  • Jentsch TJ, Steinmeyer K, Schwarz G (1990) Primary structure of Torpedo marmorata chloride channel isolated by expression cloning in Xenopus oocytes. Nature 348:510–514

    Google Scholar 

  • Jund R, Weber E, Chevallier M-R (1988) Primary structure of the uracil transport protein of Saccharomyces cerevisiae. Eur J Biochem 171:417–424

    Google Scholar 

  • Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132

    Google Scholar 

  • Lagunas R (1986) Misconceptions about the energy metabolism of Saccharomyces cerevisiae. Yeast 2:221–228

    Google Scholar 

  • Lesuisse E, Horion B, Labbe P, Hilger F (1991) The plasma membrane ferrireductase activity of Saccharomyces cerevisiae is partially controlled by cyclic AMP. Biochem J 280:545–548

    Google Scholar 

  • Lowry CV, Cerdan ME, Zitomer RS (1990) A hypoxic consensus operator and a constitutive activation region regulate the ANB1 gene of Saccharomyces cerevisiae. Mol Cell Biol 10:5921–5926

    Google Scholar 

  • Marck C (1988) “DNA Strider”: a “C” program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers. Nucleic Acids Res 16:1829–1836

    Google Scholar 

  • Mortimer RK, Schild D (1981) Genetic mapping in Saccharomyces cerevisiae. In: Strathern J, Jones E, Broach JR (eds) The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp 11–26

    Google Scholar 

  • Nakayama N, Miyajima A, Arai K (1985) Nucleotide sequences of STE2 and STE3, cell type-specific sterile genes from Saceharomyces cerevisiae. EMBO J 4:2643–2648

    Google Scholar 

  • Nelson H, Nelson N (1989) The progenitor of ATP synthases was closely related to the current vacuolar H+-ATPase. FEBS Lett 247:147–153

    Google Scholar 

  • Pearson WR, Lipman DJ (1988) Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 85:2444–2448

    Google Scholar 

  • Raguzzi F, Lesuisse E, Crichton RR (1988) Iron storage in Saccharomyces cerevisiae. FEBS Lett 231:253–258

    Google Scholar 

  • Rothstein R (1991) Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzymol 194:281–301

    Google Scholar 

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

    Google Scholar 

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

    Google Scholar 

  • Sherman F, Fink GR, Hicks JB (1986) Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • Shih C-K, Wagner R, Feinstein S, Kanik-Ennulat C, Neff N (1988) A dominant trifluoperazine resistance gene from Saccharomyces cerevisiae has homology with F0F1 ATP synthase and confers calcium-sensitive growth. Mol Cell Biol 8:3094–3103

    Google Scholar 

  • Tamanoi F (1988) Yeast RAS genes. Biochim Biophys Acta 948:1–15

    Google Scholar 

  • Tanaka J-I, Fink GR (1985) The histidine permease gene (HIP1) of Saccharomyces cerevisiae. Gene 38:205–214

    Google Scholar 

  • Thiemann A, Grunder S, Pusch M, Jentsch TJ (1992) A chloride channel widely expressed in epithelial and nonepithelial cells. Nature 356:57–60

    Google Scholar 

  • Tzagoloff A, Dieckmann CL (1990) PET genes of Saccharomyces cerevisiae. Microbiol Rev 54:211–225

    Google Scholar 

  • Umemoto N, Yoshihisa T, Hirata R, Anraku Y (1990) Roles of the VMA3 gene product, subunit c of the vacuolar membrane H+-ATPase on vacuolar acidification and protein transport. J Biol Chem 265:18447–18453

    Google Scholar 

  • Wada Y, Ohsumi Y, Anraku Y (1992) Chloride transport of yeast vacuolar membrane vesicles: a study of in vitro vacuolar acidification. Biochim Biophys Acta 1101:296–302

    Google Scholar 

  • Wickerham LJ (1946) A critical evaluation of the nitrogen assimilation tests commonly used in the classification of yeasts. J Bacteriol 52:293–301

    Google Scholar 

  • Yaffe MP (1991) Analysis of mitochondrial function and assembly. Methods Enzymol 194:627–643

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Schering-Plough Research Institute, 2015 Galloping Hill Rd., 07033, Kenilworth, NJ, USA

    Jonathan R. Greene, Nathaniel H. Brown & Beth J. DiDomenico

  2. Department of Pathology, University of Utah Medical Center, 50 N. Medical Drive, 84132, Salt Lake City, UT, USA

    Jerry Kaplan

  3. Department of Biochemistry and Molecular Biology, University of Minnesota-Duluth, 10 University Drive, 55812-2487, Duluth, MN, USA

    David J. Eide

Authors
  1. Jonathan R. Greene
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Nathaniel H. Brown
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Beth J. DiDomenico
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Jerry Kaplan
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. David J. Eide
    View author publications

    You can also search for this author inPubMed Google Scholar

Additional information

Communicated by W. Gajewski

Rights and permissions

Reprints and permissions

About this article

Cite this article

Greene, J.R., Brown, N.H., DiDomenico, B.J. et al. The GEF1 gene of Saccharomyces cerevisiae encodes an integral membrane protein; mutations in which have effects on respiration and iron-limited growth. Molec. Gen. Genet. 241, 542–553 (1993). https://doi.org/10.1007/BF00279896

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00279896

Key words