Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1997 Jun;17(6):2994–3004. doi: 10.1128/mcb.17.6.2994

The STK2 gene, which encodes a putative Ser/Thr protein kinase, is required for high-affinity spermidine transport in Saccharomyces cerevisiae.

M Kaouass 1, M Audette 1, D Ramotar 1, S Verma 1, D De Montigny 1, I Gamache 1, K Torossian 1, R Poulin 1
PMCID: PMC232151  PMID: 9154797

Abstract

Eukaryotic polyamine transport systems have not yet been characterized at the molecular level. We have used transposon mutagenesis to identify genes controlling polyamine transport in Saccharomyces cerevisiae. A haploid yeast strain was transformed with a genomic minitransposon- and lacZ-tagged library, and positive clones were selected for growth resistance to methylglyoxal bis(guanylhydrazone) (MGBG), a toxic polyamine analog. A 747-bp DNA fragment adjacent to the lacZ fusion gene rescued from one MGBG-resistant clone mapped to chromosome X within the coding region of a putative Ser/Thr protein kinase gene of previously unknown function (YJR059w, or STK2). A 304-amino-acid stretch comprising 11 of the 12 catalytic subdomains of Stk2p is approximately 83% homologous to the putative Pot1p/Kkt8p (Stk1p) protein kinase, a recently described activator of low-affinity spermine uptake in yeast. Saturable spermidine transport in stk2::lacZ mutants had an approximately fivefold-lower affinity and twofold-lower Vmax than in the parental strain. Transformation of stk2::lacZ cells with the STK2 gene cloned into a single-copy expression vector restored spermidine transport to wild-type levels. Single mutants lacking the catalytic kinase subdomains of STK1 exhibited normal parameters for the initial rate of spermidine transport but showed a time-dependent decrease in total polyamine accumulation and a low-level resistance to toxic polyamine analogs. Spermidine transport was repressed by prior incubation with exogenous spermidine. Exogenous polyamine deprivation also derepressed residual spermidine transport in stk2::lacZ mutants, but simultaneous disruption of STK1 and STK2 virtually abolished high-affinity spermidine transport under both repressed and derepressed conditions. On the other hand, putrescine uptake was also deficient in stk2::lacZ mutants but was not repressed by exogenous spermidine. Interestingly, stk2::lacZ mutants showed increased growth resistance to Li+ and Na+, suggesting a regulatory relationship between polyamine and monovalent inorganic cation transport. These results indicate that the putative STK2 Ser/Thr kinase gene is an essential determinant of high-affinity polyamine transport in yeast whereas its close homolog STK1 mostly affects a lower-affinity, low-capacity polyamine transport activity.

Full Text

The Full Text of this article is available as a PDF (356.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ask A., Persson L., Heby O. Increased survival of L1210 leukemic mice by prevention of the utilization of extracellular polyamines. Studies using a polyamine-uptake mutant, antibiotics and a polyamine-deficient diet. Cancer Lett. 1992 Sep 14;66(1):29–34. doi: 10.1016/0304-3835(92)90276-2. [DOI] [PubMed] [Google Scholar]
  2. Balasundaram D., Tabor C. W., Tabor H. Spermidine or spermine is essential for the aerobic growth of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5872–5876. doi: 10.1073/pnas.88.13.5872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bisson L. F., Fraenkel D. G. Involvement of kinases in glucose and fructose uptake by Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1983 Mar;80(6):1730–1734. doi: 10.1073/pnas.80.6.1730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boeke J. D., Trueheart J., Natsoulis G., Fink G. R. 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 1987;154:164–175. doi: 10.1016/0076-6879(87)54076-9. [DOI] [PubMed] [Google Scholar]
  5. Bolton B. J., Reischl U., Höltke H. J., Schmitz G. G., Jarsch M., Kessler C. EclXI, a novel isoschizomer of XmaIII from Enterobacter cloacae 590 recognizing 5'-C/GGCCG-3'. Nucleic Acids Res. 1990 Jan 25;18(2):381–381. doi: 10.1093/nar/18.2.381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Burns N., Grimwade B., Ross-Macdonald P. B., Choi E. Y., Finberg K., Roeder G. S., Snyder M. Large-scale analysis of gene expression, protein localization, and gene disruption in Saccharomyces cerevisiae. Genes Dev. 1994 May 1;8(9):1087–1105. doi: 10.1101/gad.8.9.1087. [DOI] [PubMed] [Google Scholar]
  7. Byers T. L., Kameji R., Rannels D. E., Pegg A. E. Multiple pathways for uptake of paraquat, methylglyoxal bis(guanylhydrazone), and polyamines. Am J Physiol. 1987 Jun;252(6 Pt 1):C663–C669. doi: 10.1152/ajpcell.1987.252.6.C663. [DOI] [PubMed] [Google Scholar]
  8. Byers T. L., Pegg A. E. Properties and physiological function of the polyamine transport system. Am J Physiol. 1989 Sep;257(3 Pt 1):C545–C553. doi: 10.1152/ajpcell.1989.257.3.C545. [DOI] [PubMed] [Google Scholar]
  9. Celenza J. L., Carlson M. A yeast gene that is essential for release from glucose repression encodes a protein kinase. Science. 1986 Sep 12;233(4769):1175–1180. doi: 10.1126/science.3526554. [DOI] [PubMed] [Google Scholar]
  10. Dujon B., Alexandraki D., André B., Ansorge W., Baladron V., Ballesta J. P., Banrevi A., Bolle P. A., Bolotin-Fukuhara M., Bossier P. Complete DNA sequence of yeast chromosome XI. Nature. 1994 Jun 2;369(6479):371–378. doi: 10.1038/369371a0. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Galan J. M., Moreau V., Andre B., Volland C., Haguenauer-Tsapis R. Ubiquitination mediated by the Npi1p/Rsp5p ubiquitin-protein ligase is required for endocytosis of the yeast uracil permease. J Biol Chem. 1996 May 3;271(18):10946–10952. doi: 10.1074/jbc.271.18.10946. [DOI] [PubMed] [Google Scholar]
  13. Galibert F., Alexandraki D., Baur A., Boles E., Chalwatzis N., Chuat J. C., Coster F., Cziepluch C., De Haan M., Domdey H. Complete nucleotide sequence of Saccharomyces cerevisiae chromosome X. EMBO J. 1996 May 1;15(9):2031–2049. [PMC free article] [PubMed] [Google Scholar]
  14. Grenson M. Inactivation-reactivation process and repression of permease formation regulate several ammonia-sensitive permeases in the yeast Saccharomyces cerevisiae. Eur J Biochem. 1983 Jun 1;133(1):135–139. doi: 10.1111/j.1432-1033.1983.tb07438.x. [DOI] [PubMed] [Google Scholar]
  15. Hamana K., Matsuzaki S., Hosaka K., Yamashita S. Interconversion of polyamines in wild-type strains and mutants of yeasts and the effects of polyamines on their growth. FEMS Microbiol Lett. 1989 Oct 1;52(1-2):231–236. doi: 10.1016/0378-1097(89)90202-4. [DOI] [PubMed] [Google Scholar]
  16. Hanks S. K., Quinn A. M. Protein kinase catalytic domain sequence database: identification of conserved features of primary structure and classification of family members. Methods Enzymol. 1991;200:38–62. doi: 10.1016/0076-6879(91)00126-h. [DOI] [PubMed] [Google Scholar]
  17. Hayashi S., Murakami Y., Matsufuji S. Ornithine decarboxylase antizyme: a novel type of regulatory protein. Trends Biochem Sci. 1996 Jan;21(1):27–30. [PubMed] [Google Scholar]
  18. Hessels J., Kingma A. W., Ferwerda H., Keij J., van den Berg G. A., Muskiet F. A. Microbial flora in the gastrointestinal tract abolishes cytostatic effects of alpha-difluoromethylornithine in vivo. Int J Cancer. 1989 Jun 15;43(6):1155–1164. doi: 10.1002/ijc.2910430632. [DOI] [PubMed] [Google Scholar]
  19. Igual J. C., Matallaná E., Gonzalez-Bosch C., Franco L., Pérez-Ortin J. E. A new glucose-repressible gene identified from the analysis of chromatin structure in deletion mutants of yeast SUC2 locus. Yeast. 1991 May-Jun;7(4):379–389. doi: 10.1002/yea.320070408. [DOI] [PubMed] [Google Scholar]
  20. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jauniaux J. C., Vandenbol M., Vissers S., Broman K., Grenson M. Nitrogen catabolite regulation of proline permease in Saccharomyces cerevisiae. Cloning of the PUT4 gene and study of PUT4 RNA levels in wild-type and mutant strains. Eur J Biochem. 1987 May 4;164(3):601–606. doi: 10.1111/j.1432-1033.1987.tb11169.x. [DOI] [PubMed] [Google Scholar]
  22. Kakinuma Y., Hoshino K., Igarashi K. Characterization of the inducible polyamine transporter in bovine lymphocytes. Eur J Biochem. 1988 Sep 15;176(2):409–414. doi: 10.1111/j.1432-1033.1988.tb14297.x. [DOI] [PubMed] [Google Scholar]
  23. Kakinuma Y., Maruyama T., Nozaki T., Wada Y., Ohsumi Y., Igarashi K. Cloning of the gene encoding a putative serine/threonine protein kinase which enhances spermine uptake in Saccharomyces cerevisiae. Biochem Biophys Res Commun. 1995 Nov 22;216(3):985–992. doi: 10.1006/bbrc.1995.2717. [DOI] [PubMed] [Google Scholar]
  24. Kakinuma Y., Masuda N., Igarashi K. Proton potential-dependent polyamine transport by vacuolar membrane vesicles of Saccharomyces cerevisiae. Biochim Biophys Acta. 1992 Jun 11;1107(1):126–130. doi: 10.1016/0005-2736(92)90337-l. [DOI] [PubMed] [Google Scholar]
  25. Kano K., Oka T. Polyamine transport and metabolism in mouse mammary gland. General properties and hormonal regulation. J Biol Chem. 1976 May 10;251(9):2795–2800. [PubMed] [Google Scholar]
  26. Ko C. H., Buckley A. M., Gaber R. F. TRK2 is required for low affinity K+ transport in Saccharomyces cerevisiae. Genetics. 1990 Jun;125(2):305–312. doi: 10.1093/genetics/125.2.305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ko C. H., Gaber R. F. TRK1 and TRK2 encode structurally related K+ transporters in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Aug;11(8):4266–4273. doi: 10.1128/mcb.11.8.4266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lessard M., Zhao C., Singh S. M., Poulin R. Hormonal and feedback regulation of putrescine and spermidine transport in human breast cancer cells. J Biol Chem. 1995 Jan 27;270(4):1685–1694. [PubMed] [Google Scholar]
  29. Maruyama T., Masuda N., Kakinuma Y., Igarashi K. Polyamine-sensitive magnesium transport in Saccharomyces cerevisiae. Biochim Biophys Acta. 1994 Sep 14;1194(2):289–295. doi: 10.1016/0005-2736(94)90311-5. [DOI] [PubMed] [Google Scholar]
  30. Matsufuji S., Matsufuji T., Miyazaki Y., Murakami Y., Atkins J. F., Gesteland R. F., Hayashi S. Autoregulatory frameshifting in decoding mammalian ornithine decarboxylase antizyme. Cell. 1995 Jan 13;80(1):51–60. doi: 10.1016/0092-8674(95)90450-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mendoza I., Rubio F., Rodriguez-Navarro A., Pardo J. M. The protein phosphatase calcineurin is essential for NaCl tolerance of Saccharomyces cerevisiae. J Biol Chem. 1994 Mar 25;269(12):8792–8796. [PubMed] [Google Scholar]
  32. Mitchell J. L., Diveley R. R., Jr, Bareyal-Leyser A. Feedback repression of polyamine uptake into mammalian cells requires active protein synthesis. Biochem Biophys Res Commun. 1992 Jul 15;186(1):81–88. doi: 10.1016/s0006-291x(05)80778-8. [DOI] [PubMed] [Google Scholar]
  33. Mitchell J. L., Judd G. G., Bareyal-Leyser A., Ling S. Y. Feedback repression of polyamine transport is mediated by antizyme in mammalian tissue-culture cells. Biochem J. 1994 Apr 1;299(Pt 1):19–22. doi: 10.1042/bj2990019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nozaki T., Nishimura K., Michael A. J., Maruyama T., Kakinuma Y., Igarashi K. A second gene encoding a putative serine/threonine protein kinase which enhances spermine uptake in Saccharomyces cerevisiae. Biochem Biophys Res Commun. 1996 Nov 12;228(2):452–458. doi: 10.1006/bbrc.1996.1681. [DOI] [PubMed] [Google Scholar]
  35. Ozcan S., Johnston M. Three different regulatory mechanisms enable yeast hexose transporter (HXT) genes to be induced by different levels of glucose. Mol Cell Biol. 1995 Mar;15(3):1564–1572. doi: 10.1128/mcb.15.3.1564. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Pegg A. E. Polyamine metabolism and its importance in neoplastic growth and a target for chemotherapy. Cancer Res. 1988 Feb 15;48(4):759–774. [PubMed] [Google Scholar]
  38. Pegg A. E., Poulin R., Coward J. K. Use of aminopropyltransferase inhibitors and of non-metabolizable analogs to study polyamine regulation and function. Int J Biochem Cell Biol. 1995 May;27(5):425–442. doi: 10.1016/1357-2725(95)00007-c. [DOI] [PubMed] [Google Scholar]
  39. Persson L., Holm I., Ask A., Heby O. Curative effect of DL-2-difluoromethylornithine on mice bearing mutant L1210 leukemia cells deficient in polyamine uptake. Cancer Res. 1988 Sep 1;48(17):4807–4811. [PubMed] [Google Scholar]
  40. Porter C. W., Miller J., Bergeron R. J. Aliphatic chain length specificity of the polyamine transport system in ascites L1210 leukemia cells. Cancer Res. 1984 Jan;44(1):126–128. [PubMed] [Google Scholar]
  41. Posas F., Camps M., Ariño J. The PPZ protein phosphatases are important determinants of salt tolerance in yeast cells. J Biol Chem. 1995 Jun 2;270(22):13036–13041. doi: 10.1074/jbc.270.22.13036. [DOI] [PubMed] [Google Scholar]
  42. Ramotar D., Popoff S. C., Gralla E. B., Demple B. Cellular role of yeast Apn1 apurinic endonuclease/3'-diesterase: repair of oxidative and alkylation DNA damage and control of spontaneous mutation. Mol Cell Biol. 1991 Sep;11(9):4537–4544. doi: 10.1128/mcb.11.9.4537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Rodríguez-Navarro A., Quintero F. J., Garciadeblás B. Na(+)-ATPases and Na+/H+ antiporters in fungi. Biochim Biophys Acta. 1994 Aug 30;1187(2):203–205. doi: 10.1016/0005-2728(94)90111-2. [DOI] [PubMed] [Google Scholar]
  44. Rogers S., Wells R., Rechsteiner M. Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis. Science. 1986 Oct 17;234(4774):364–368. doi: 10.1126/science.2876518. [DOI] [PubMed] [Google Scholar]
  45. Seiler N., Dezeure F. Polyamine transport in mammalian cells. Int J Biochem. 1990;22(3):211–218. doi: 10.1016/0020-711x(90)90332-w. [DOI] [PubMed] [Google Scholar]
  46. Seiler N., Sarhan S., Grauffel C., Jones R., Knödgen B., Moulinoux J. P. Endogenous and exogenous polyamines in support of tumor growth. Cancer Res. 1990 Aug 15;50(16):5077–5083. [PubMed] [Google Scholar]
  47. Skala J., Purnelle B., Crouzet M., Aigle M., Goffeau A. The open reading frame YCR101 located on chromosome III from Saccharomyces cerevisiae is a putative protein kinase. Yeast. 1991 Aug-Sep;7(6):651–655. doi: 10.1002/yea.320070614. [DOI] [PubMed] [Google Scholar]
  48. Stanbrough M., Magasanik B. Transcriptional and posttranslational regulation of the general amino acid permease of Saccharomyces cerevisiae. J Bacteriol. 1995 Jan;177(1):94–102. doi: 10.1128/jb.177.1.94-102.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Suzuki T., He Y., Kashiwagi K., Murakami Y., Hayashi S., Igarashi K. Antizyme protects against abnormal accumulation and toxicity of polyamines in ornithine decarboxylase-overproducing cells. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8930–8934. doi: 10.1073/pnas.91.19.8930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Tabor C. W., Tabor H. Polyamines. Annu Rev Biochem. 1984;53:749–790. doi: 10.1146/annurev.bi.53.070184.003533. [DOI] [PubMed] [Google Scholar]
  51. Tyagi A. K., Tabor C. W., Tabor H. Ornithine decarboxylase from Saccharomyces cerevisiae. Purification, properties, and regulation of activity. J Biol Chem. 1981 Dec 10;256(23):12156–12163. [PubMed] [Google Scholar]
  52. Vandenbol M., Jauniaux J. C., Grenson M. The Saccharomyces cerevisiae NPR1 gene required for the activity of ammonia-sensitive amino acid permeases encodes a protein kinase homologue. Mol Gen Genet. 1990 Jul;222(2-3):393–399. doi: 10.1007/BF00633845. [DOI] [PubMed] [Google Scholar]
  53. Vandenbol M., Jauniaux J. C., Vissers S., Grenson M. Isolation of the NPR1 gene responsible for the reactivation of ammonia-sensitive amino-acid permeases in Saccharomyces cerevisiae. RNA analysis and gene dosage effects. Eur J Biochem. 1987 May 4;164(3):607–612. doi: 10.1111/j.1432-1033.1987.tb11170.x. [DOI] [PubMed] [Google Scholar]
  54. Varela J. C., Mager W. H. Response of Saccharomyces cerevisiae to changes in external osmolarity. Microbiology. 1996 Apr;142(Pt 4):721–731. doi: 10.1099/00221287-142-4-721. [DOI] [PubMed] [Google Scholar]
  55. Vidal M., Buckley A. M., Yohn C., Hoeppner D. J., Gaber R. F. Identification of essential nucleotides in an upstream repressing sequence of Saccharomyces cerevisiae by selection for increased expression of TRK2. Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):2370–2374. doi: 10.1073/pnas.92.6.2370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Volland C., Garnier C., Haguenauer-Tsapis R. In vivo phosphorylation of the yeast uracil permease. J Biol Chem. 1992 Nov 25;267(33):23767–23771. [PubMed] [Google Scholar]
  57. Wiame J. M., Grenson M., Arst H. N., Jr Nitrogen catabolite repression in yeasts and filamentous fungi. Adv Microb Physiol. 1985;26:1–88. doi: 10.1016/s0065-2911(08)60394-x. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES