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. 1992 Apr;11(4):1631–1640. doi: 10.1002/j.1460-2075.1992.tb05209.x

Gene amplification at a locus encoding a putative Na+/H+ antiporter confers sodium and lithium tolerance in fission yeast.

Z P Jia 1, N McCullough 1, R Martel 1, S Hemmingsen 1, P G Young 1
PMCID: PMC556613  PMID: 1314171

Abstract

We have identified a new locus, sodium 2 (sod2) based on selection for increased LiCl tolerance in fission yeast, Schizosaccharomyces pombe. Tolerant strains have enhanced pH-dependent Na+ export capacity and sodium transport experiments suggest that the gene encodes an Na+/H+ antiport. The predicted sod2 gene product can be placed in the broad class of transporters which possess 12 hydrophobic transmembrane domains. The protein shows some sequence similarity to the human and bacterial Na+/H+ antiporters. Overexpression of sod2 increased Na+ export capacity and conferred sodium tolerance. Osmotolerance was not affected and sod2 cells were unaffected for growth in K+. In a sod2 disruption strain cells were incapable of exporting sodium. They were hypersensitive to Na+ or Li+ and could not grow under conditions that approximate pH7. The sod2 gene amplification could be selected stepwise and the degree of such amplification correlated with the level of Na+ or Li+ tolerance.

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  1. Biggin M. D., Gibson T. J., Hong G. F. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3963–3965. doi: 10.1073/pnas.80.13.3963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Burke J. F. High-sensitivity S1 mapping with single-stranded [32P]DNA probes synthesized from bacteriophage M13mp templates. Gene. 1984 Oct;30(1-3):63–68. doi: 10.1016/0378-1119(84)90105-7. [DOI] [PubMed] [Google Scholar]
  3. Clarke L., Amstutz H., Fishel B., Carbon J. Analysis of centromeric DNA in the fission yeast Schizosaccharomyces pombe. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8253–8257. doi: 10.1073/pnas.83.21.8253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Eilam Y., Othman M., Halachmi D. Transient increase in Ca2+ influx in Saccharomyces cerevisiae in response to glucose: effects of intracellular acidification and cAMP levels. J Gen Microbiol. 1990 Dec;136(12):2537–2543. doi: 10.1099/00221287-136-12-2537. [DOI] [PubMed] [Google Scholar]
  5. Eisenberg D., Schwarz E., Komaromy M., Wall R. Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J Mol Biol. 1984 Oct 15;179(1):125–142. doi: 10.1016/0022-2836(84)90309-7. [DOI] [PubMed] [Google Scholar]
  6. Fogel S., Welch J. W., Louis E. J. Meiotic gene conversion mediates gene amplification in yeast. Cold Spring Harb Symp Quant Biol. 1984;49:55–65. doi: 10.1101/sqb.1984.049.01.009. [DOI] [PubMed] [Google Scholar]
  7. Franchi A., Cragoe E., Jr, Pouysségur J. Isolation and properties of fibroblast mutants overexpressing an altered Na+/H+ antiporter. J Biol Chem. 1986 Nov 5;261(31):14614–14620. [PubMed] [Google Scholar]
  8. Gaber R. F., Styles C. A., Fink G. R. TRK1 encodes a plasma membrane protein required for high-affinity potassium transport in Saccharomyces cerevisiae. Mol Cell Biol. 1988 Jul;8(7):2848–2859. doi: 10.1128/mcb.8.7.2848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Giulotto E., Saito I., Stark G. R. Structure of DNA formed in the first step of CAD gene amplification. EMBO J. 1986 Sep;5(9):2115–2121. doi: 10.1002/j.1460-2075.1986.tb04474.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Goldberg E. B., Arbel T., Chen J., Karpel R., Mackie G. A., Schuldiner S., Padan E. Characterization of a Na+/H+ antiporter gene of Escherichia coli. Proc Natl Acad Sci U S A. 1987 May;84(9):2615–2619. doi: 10.1073/pnas.84.9.2615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Grinstein S., Rothstein A. Mechanisms of regulation of the Na+/H+ exchanger. J Membr Biol. 1986;90(1):1–12. doi: 10.1007/BF01869680. [DOI] [PubMed] [Google Scholar]
  12. Gros P., Croop J., Housman D. Mammalian multidrug resistance gene: complete cDNA sequence indicates strong homology to bacterial transport proteins. Cell. 1986 Nov 7;47(3):371–380. doi: 10.1016/0092-8674(86)90594-5. [DOI] [PubMed] [Google Scholar]
  13. Haro R., Garciadeblas B., Rodríguez-Navarro A. A novel P-type ATPase from yeast involved in sodium transport. FEBS Lett. 1991 Oct 21;291(2):189–191. doi: 10.1016/0014-5793(91)81280-l. [DOI] [PubMed] [Google Scholar]
  14. Hediger M. A., Coady M. J., Ikeda T. S., Wright E. M. Expression cloning and cDNA sequencing of the Na+/glucose co-transporter. 1987 Nov 26-Dec 2Nature. 330(6146):379–381. doi: 10.1038/330379a0. [DOI] [PubMed] [Google Scholar]
  15. Henikoff S. Unidirectional digestion with exonuclease III in DNA sequence analysis. Methods Enzymol. 1987;155:156–165. doi: 10.1016/0076-6879(87)55014-5. [DOI] [PubMed] [Google Scholar]
  16. Higgins C. F., Gallagher M. P., Mimmack M. L., Pearce S. R. A family of closely related ATP-binding subunits from prokaryotic and eukaryotic cells. Bioessays. 1988 Apr;8(4):111–116. doi: 10.1002/bies.950080406. [DOI] [PubMed] [Google Scholar]
  17. Hindley J., Phear G., Stein M., Beach D. Sucl+ encodes a predicted 13-kilodalton protein that is essential for cell viability and is directly involved in the division cycle of Schizosaccharomyces pombe. Mol Cell Biol. 1987 Jan;7(1):504–511. doi: 10.1128/mcb.7.1.504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ishikawa T., Hama H., Tsuda M., Tsuchiya T. Isolation and properties of a mutant of Escherichia coli possessing defective Na+/H+ antiporter. J Biol Chem. 1987 Jun 5;262(16):7443–7446. [PubMed] [Google Scholar]
  19. Juranka P. F., Zastawny R. L., Ling V. P-glycoprotein: multidrug-resistance and a superfamily of membrane-associated transport proteins. FASEB J. 1989 Dec;3(14):2583–2592. doi: 10.1096/fasebj.3.14.2574119. [DOI] [PubMed] [Google Scholar]
  20. Karpel R., Olami Y., Taglicht D., Schuldiner S., Padan E. Sequencing of the gene ant which affects the Na+/H+ antiporter activity in Escherichia coli. J Biol Chem. 1988 Jul 25;263(21):10408–10414. [PubMed] [Google Scholar]
  21. Klein P., Kanehisa M., DeLisi C. The detection and classification of membrane-spanning proteins. Biochim Biophys Acta. 1985 May 28;815(3):468–476. doi: 10.1016/0005-2736(85)90375-x. [DOI] [PubMed] [Google Scholar]
  22. Klionsky D. J., Herman P. K., Emr S. D. The fungal vacuole: composition, function, and biogenesis. Microbiol Rev. 1990 Sep;54(3):266–292. doi: 10.1128/mr.54.3.266-292.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kozak M. An analysis of vertebrate mRNA sequences: intimations of translational control. J Cell Biol. 1991 Nov;115(4):887–903. doi: 10.1083/jcb.115.4.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Krulwich T. A. Bioenergetics of alkalophilic bacteria. J Membr Biol. 1986;89(2):113–125. doi: 10.1007/BF01869707. [DOI] [PubMed] [Google Scholar]
  25. Krulwich T. A., Guffanti A. A., Fong M. Y., Falk L., Hicks D. B. Alkalophilic Bacillus firmus RAB generates variants which can grow at lower Na+ concentrations than the parental strain. J Bacteriol. 1986 Mar;165(3):884–889. doi: 10.1128/jb.165.3.884-889.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  27. Maiden M. C., Davis E. O., Baldwin S. A., Moore D. C., Henderson P. J. Mammalian and bacterial sugar transport proteins are homologous. Nature. 1987 Feb 12;325(6105):641–643. doi: 10.1038/325641a0. [DOI] [PubMed] [Google Scholar]
  28. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. McLeod M., Stein M., Beach D. The product of the mei3+ gene, expressed under control of the mating-type locus, induces meiosis and sporulation in fission yeast. EMBO J. 1987 Mar;6(3):729–736. doi: 10.1002/j.1460-2075.1987.tb04814.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mertins P., Gallwitz D. Nuclear pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe strictly requires an intron-contained, conserved sequence element. EMBO J. 1987 Jun;6(6):1757–1763. doi: 10.1002/j.1460-2075.1987.tb02428.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nurse P. Genetic control of cell size at cell division in yeast. Nature. 1975 Aug 14;256(5518):547–551. doi: 10.1038/256547a0. [DOI] [PubMed] [Google Scholar]
  32. Nurse P., Thuriaux P., Nasmyth K. Genetic control of the cell division cycle in the fission yeast Schizosaccharomyces pombe. Mol Gen Genet. 1976 Jul 23;146(2):167–178. doi: 10.1007/BF00268085. [DOI] [PubMed] [Google Scholar]
  33. Padan E., Zilberstein D., Rottenberg H. The proton electrochemical gradient in Escherichia coli cells. Eur J Biochem. 1976 Apr 1;63(2):533–541. doi: 10.1111/j.1432-1033.1976.tb10257.x. [DOI] [PubMed] [Google Scholar]
  34. Padan E., Zilberstein D., Schuldiner S. pH homeostasis in bacteria. Biochim Biophys Acta. 1981 Dec;650(2-3):151–166. doi: 10.1016/0304-4157(81)90004-6. [DOI] [PubMed] [Google Scholar]
  35. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Proudfoot N. J., Brownlee G. G. 3' non-coding region sequences in eukaryotic messenger RNA. Nature. 1976 Sep 16;263(5574):211–214. doi: 10.1038/263211a0. [DOI] [PubMed] [Google Scholar]
  37. Rodriguez-Navarro A., Asensio J. An efflux mechanism determines the low net entry of lithium in yeasts. FEBS Lett. 1977 Mar 15;75(1):169–172. doi: 10.1016/0014-5793(77)80078-1. [DOI] [PubMed] [Google Scholar]
  38. Rodríguez-Navarro A., Ortega M. D. The mechanism of sodium efflux in yeast. FEBS Lett. 1982 Feb 22;138(2):205–208. doi: 10.1016/0014-5793(82)80442-0. [DOI] [PubMed] [Google Scholar]
  39. Rodríguez-Navarro A., Sancho E. D., Pérez-Lloveres C. Energy source for lithium efflux in yeast. Biochim Biophys Acta. 1981 Jan 8;640(1):352–358. doi: 10.1016/0005-2736(81)90558-7. [DOI] [PubMed] [Google Scholar]
  40. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sardet C., Counillon L., Franchi A., Pouysségur J. Growth factors induce phosphorylation of the Na+/H+ antiporter, glycoprotein of 110 kD. Science. 1990 Feb 9;247(4943):723–726. doi: 10.1126/science.2154036. [DOI] [PubMed] [Google Scholar]
  42. Sardet C., Franchi A., Pouysségur J. Molecular cloning, primary structure, and expression of the human growth factor-activatable Na+/H+ antiporter. Cell. 1989 Jan 27;56(2):271–280. doi: 10.1016/0092-8674(89)90901-x. [DOI] [PubMed] [Google Scholar]
  43. Seol W., Shatkin A. J. Escherichia coli kgtP encodes an alpha-ketoglutarate transporter. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3802–3806. doi: 10.1073/pnas.88.9.3802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Stark G. R., Debatisse M., Giulotto E., Wahl G. M. Recent progress in understanding mechanisms of mammalian DNA amplification. Cell. 1989 Jun 16;57(6):901–908. doi: 10.1016/0092-8674(89)90328-0. [DOI] [PubMed] [Google Scholar]
  45. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  46. Szkutnicka K., Tschopp J. F., Andrews L., Cirillo V. P. Sequence and structure of the yeast galactose transporter. J Bacteriol. 1989 Aug;171(8):4486–4493. doi: 10.1128/jb.171.8.4486-4493.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tse C. M., Ma A. I., Yang V. W., Watson A. J., Levine S., Montrose M. H., Potter J., Sardet C., Pouyssegur J., Donowitz M. Molecular cloning and expression of a cDNA encoding the rabbit ileal villus cell basolateral membrane Na+/H+ exchanger. EMBO J. 1991 Aug;10(8):1957–1967. doi: 10.1002/j.1460-2075.1991.tb07725.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tsuchiya T., Raven J., Wilson T. H. Co-transport of Na+ and methul-beta-D-thiogalactopyranoside mediated by the melibiose transport system of Escherichia coli. Biochem Biophys Res Commun. 1977 May 9;76(1):26–31. doi: 10.1016/0006-291x(77)91663-1. [DOI] [PubMed] [Google Scholar]
  49. Walker J. E., Saraste M., Runswick M. J., Gay N. J. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982;1(8):945–951. doi: 10.1002/j.1460-2075.1982.tb01276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wright A., Maundrell K., Heyer W. D., Beach D., Nurse P. Vectors for the construction of gene banks and the integration of cloned genes in Schizosaccharomyces pombe and Saccharomyces cerevisiae. Plasmid. 1986 Mar;15(2):156–158. doi: 10.1016/0147-619x(86)90051-x. [DOI] [PubMed] [Google Scholar]
  51. Yamaguchi A., Ono N., Akasaka T., Noumi T., Sawai T. Metal-tetracycline/H+ antiporter of Escherichia coli encoded by a transposon, Tn10. The role of the conserved dipeptide, Ser65-Asp66, in tetracycline transport. J Biol Chem. 1990 Sep 15;265(26):15525–15530. [PubMed] [Google Scholar]
  52. Yamato I., Ohsawa M., Anraku Y. Defective cation-coupling mutants of Escherichia coli Na+/proline symport carrier. Characterization and localization of mutations. J Biol Chem. 1990 Feb 15;265(5):2450–2455. [PubMed] [Google Scholar]
  53. Yoshida H., Bogaki M., Nakamura S., Ubukata K., Konno M. Nucleotide sequence and characterization of the Staphylococcus aureus norA gene, which confers resistance to quinolones. J Bacteriol. 1990 Dec;172(12):6942–6949. doi: 10.1128/jb.172.12.6942-6949.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 1983;100:468–500. doi: 10.1016/0076-6879(83)00074-9. [DOI] [PubMed] [Google Scholar]

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