Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1998 Sep 15;17(18):5400–5408. doi: 10.1093/emboj/17.18.5400

Identification of a copper-induced intramolecular interaction in the transcription factor Mac1 from Saccharomyces cerevisiae.

L T Jensen 1, D R Winge 1
PMCID: PMC1170865  PMID: 9736617

Abstract

Mac1 mediates copper (Cu)-dependent expression of genes involved in high-affinity uptake of copper ions in Saccharomyces cerevisiae. Mac1 is a transcriptional activator in Cu-deficient cells, but is inhibited in Cu-replete cells. Mac1 resides within the nucleus in both Cu-deficient and Cu-loaded cells. Cu inhibition of Mac1 appears to result from binding of eight copper ions within a C-terminal segment consisting of two Cys-rich motifs. In addition, two zinc ions are bound within the N-terminal DNA-binding domain. Only 4-5 mol. eq. Cu are bound to a mutant Mac1 (His279Gln substitution) that is impervious to Cu inhibition. The CuMac1 complex is luminescent, indicative of copper bound in the Cu(I) state. Cu binding induces a molecular switch resulting in an intramolecular interaction in Mac1 between the N-terminal DNA-binding domain and the C-terminal activation domain. This allosteric interaction is Cu dependent and is not observed when Mac1 contained the mutant His279Gln substitution. Fusion of the minimal DNA-binding domain of Mac1 (residues 1-159) to the minimal Cu-binding activation domain (residues 252-341) yields a functional Cu-regulated transcriptional activator. These results suggest that Cu repression of Mac1 arises from a Cu-induced intramolecular interaction that inhibits both DNA binding and transactivation activities.

Full Text

The Full Text of this article is available as a PDF (304.1 KB).

Selected References

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

  1. Andersson S. G., Kurland C. G. Codon preferences in free-living microorganisms. Microbiol Rev. 1990 Jun;54(2):198–210. doi: 10.1128/mr.54.2.198-210.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bai C., Elledge S. J. Gene identification using the yeast two-hybrid system. Methods Enzymol. 1997;283:141–156. doi: 10.1016/s0076-6879(97)83013-3. [DOI] [PubMed] [Google Scholar]
  3. Bonner J. J., Heyward S., Fackenthal D. L. Temperature-dependent regulation of a heterologous transcriptional activation domain fused to yeast heat shock transcription factor. Mol Cell Biol. 1992 Mar;12(3):1021–1030. doi: 10.1128/mcb.12.3.1021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  5. Cho H. S., Liu C. W., Damberger F. F., Pelton J. G., Nelson H. C., Wemmer D. E. Yeast heat shock transcription factor N-terminal activation domains are unstructured as probed by heteronuclear NMR spectroscopy. Protein Sci. 1996 Feb;5(2):262–269. doi: 10.1002/pro.5560050210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dancis A., Haile D., Yuan D. S., Klausner R. D. The Saccharomyces cerevisiae copper transport protein (Ctr1p). Biochemical characterization, regulation by copper, and physiologic role in copper uptake. J Biol Chem. 1994 Oct 14;269(41):25660–25667. [PubMed] [Google Scholar]
  7. Dancis A., Roman D. G., Anderson G. J., Hinnebusch A. G., Klausner R. D. Ferric reductase of Saccharomyces cerevisiae: molecular characterization, role in iron uptake, and transcriptional control by iron. Proc Natl Acad Sci U S A. 1992 May 1;89(9):3869–3873. doi: 10.1073/pnas.89.9.3869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dancis A., Yuan D. S., Haile D., Askwith C., Eide D., Moehle C., Kaplan J., Klausner R. D. Molecular characterization of a copper transport protein in S. cerevisiae: an unexpected role for copper in iron transport. Cell. 1994 Jan 28;76(2):393–402. doi: 10.1016/0092-8674(94)90345-x. [DOI] [PubMed] [Google Scholar]
  9. Farr S. B., Kogoma T. Oxidative stress responses in Escherichia coli and Salmonella typhimurium. Microbiol Rev. 1991 Dec;55(4):561–585. doi: 10.1128/mr.55.4.561-585.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Finegold A. A., Shatwell K. P., Segal A. W., Klausner R. D., Dancis A. Intramembrane bis-heme motif for transmembrane electron transport conserved in a yeast iron reductase and the human NADPH oxidase. J Biol Chem. 1996 Dec 6;271(49):31021–31024. doi: 10.1074/jbc.271.49.31021. [DOI] [PubMed] [Google Scholar]
  11. Fridovich I. The biology of oxygen radicals. Science. 1978 Sep 8;201(4359):875–880. doi: 10.1126/science.210504. [DOI] [PubMed] [Google Scholar]
  12. Fürst P., Hu S., Hackett R., Hamer D. Copper activates metallothionein gene transcription by altering the conformation of a specific DNA binding protein. Cell. 1988 Nov 18;55(4):705–717. doi: 10.1016/0092-8674(88)90229-2. [DOI] [PubMed] [Google Scholar]
  13. Georgatsou E., Mavrogiannis L. A., Fragiadakis G. S., Alexandraki D. The yeast Fre1p/Fre2p cupric reductases facilitate copper uptake and are regulated by the copper-modulated Mac1p activator. J Biol Chem. 1997 May 23;272(21):13786–13792. doi: 10.1074/jbc.272.21.13786. [DOI] [PubMed] [Google Scholar]
  14. Graden J. A., Winge D. R. Copper-mediated repression of the activation domain in the yeast Mac1p transcription factor. Proc Natl Acad Sci U S A. 1997 May 27;94(11):5550–5555. doi: 10.1073/pnas.94.11.5550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hahn S. Structure(?) and function of acidic transcription activators. Cell. 1993 Feb 26;72(4):481–483. doi: 10.1016/0092-8674(93)90064-w. [DOI] [PubMed] [Google Scholar]
  16. Hassett R., Kosman D. J. Evidence for Cu(II) reduction as a component of copper uptake by Saccharomyces cerevisiae. J Biol Chem. 1995 Jan 6;270(1):128–134. doi: 10.1074/jbc.270.1.128. [DOI] [PubMed] [Google Scholar]
  17. Imlay J. A., Linn S. DNA damage and oxygen radical toxicity. Science. 1988 Jun 3;240(4857):1302–1309. doi: 10.1126/science.3287616. [DOI] [PubMed] [Google Scholar]
  18. Jakobsen B. K., Pelham H. R. A conserved heptapeptide restrains the activity of the yeast heat shock transcription factor. EMBO J. 1991 Feb;10(2):369–375. doi: 10.1002/j.1460-2075.1991.tb07958.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Johansen F. E., Prywes R. Identification of transcriptional activation and inhibitory domains in serum response factor (SRF) by using GAL4-SRF constructs. Mol Cell Biol. 1993 Aug;13(8):4640–4647. doi: 10.1128/mcb.13.8.4640. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jungmann J., Reins H. A., Lee J., Romeo A., Hassett R., Kosman D., Jentsch S. MAC1, a nuclear regulatory protein related to Cu-dependent transcription factors is involved in Cu/Fe utilization and stress resistance in yeast. EMBO J. 1993 Dec 15;12(13):5051–5056. doi: 10.1002/j.1460-2075.1993.tb06198.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Knight S. A., Labbé S., Kwon L. F., Kosman D. J., Thiele D. J. A widespread transposable element masks expression of a yeast copper transport gene. Genes Dev. 1996 Aug 1;10(15):1917–1929. doi: 10.1101/gad.10.15.1917. [DOI] [PubMed] [Google Scholar]
  22. Kornitzer D., Raboy B., Kulka R. G., Fink G. R. Regulated degradation of the transcription factor Gcn4. EMBO J. 1994 Dec 15;13(24):6021–6030. doi: 10.1002/j.1460-2075.1994.tb06948.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kussie P. H., Gorina S., Marechal V., Elenbaas B., Moreau J., Levine A. J., Pavletich N. P. Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation domain. Science. 1996 Nov 8;274(5289):948–953. doi: 10.1126/science.274.5289.948. [DOI] [PubMed] [Google Scholar]
  24. Labbé S., Zhu Z., Thiele D. J. Copper-specific transcriptional repression of yeast genes encoding critical components in the copper transport pathway. J Biol Chem. 1997 Jun 20;272(25):15951–15958. doi: 10.1074/jbc.272.25.15951. [DOI] [PubMed] [Google Scholar]
  25. Li X. Y., Green M. R. Intramolecular inhibition of activating transcription factor-2 function by its DNA-binding domain. Genes Dev. 1996 Mar 1;10(5):517–527. doi: 10.1101/gad.10.5.517. [DOI] [PubMed] [Google Scholar]
  26. Linder M. C., Hazegh-Azam M. Copper biochemistry and molecular biology. Am J Clin Nutr. 1996 May;63(5):797S–811S. doi: 10.1093/ajcn/63.5.797. [DOI] [PubMed] [Google Scholar]
  27. Lutsenko S., Kaplan J. H. Organization of P-type ATPases: significance of structural diversity. Biochemistry. 1995 Dec 5;34(48):15607–15613. doi: 10.1021/bi00048a001. [DOI] [PubMed] [Google Scholar]
  28. Mitchell P. J., Tjian R. Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science. 1989 Jul 28;245(4916):371–378. doi: 10.1126/science.2667136. [DOI] [PubMed] [Google Scholar]
  29. Momand J., Zambetti G. P., Olson D. C., George D., Levine A. J. The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. Cell. 1992 Jun 26;69(7):1237–1245. doi: 10.1016/0092-8674(92)90644-r. [DOI] [PubMed] [Google Scholar]
  30. Ptashne M. How eukaryotic transcriptional activators work. Nature. 1988 Oct 20;335(6192):683–689. doi: 10.1038/335683a0. [DOI] [PubMed] [Google Scholar]
  31. Rupp R. A., Snider L., Weintraub H. Xenopus embryos regulate the nuclear localization of XMyoD. Genes Dev. 1994 Jun 1;8(11):1311–1323. doi: 10.1101/gad.8.11.1311. [DOI] [PubMed] [Google Scholar]
  32. Sadowski I., Ma J., Triezenberg S., Ptashne M. GAL4-VP16 is an unusually potent transcriptional activator. Nature. 1988 Oct 6;335(6190):563–564. doi: 10.1038/335563a0. [DOI] [PubMed] [Google Scholar]
  33. Shatwell K. P., Dancis A., Cross A. R., Klausner R. D., Segal A. W. The FRE1 ferric reductase of Saccharomyces cerevisiae is a cytochrome b similar to that of NADPH oxidase. J Biol Chem. 1996 Jun 14;271(24):14240–14244. doi: 10.1074/jbc.271.24.14240. [DOI] [PubMed] [Google Scholar]
  34. Shen F., Triezenberg S. J., Hensley P., Porter D., Knutson J. R. Critical amino acids in the transcriptional activation domain of the herpesvirus protein VP16 are solvent-exposed in highly mobile protein segments. An intrinsic fluorescence study. J Biol Chem. 1996 Mar 1;271(9):4819–4826. doi: 10.1074/jbc.271.9.4819. [DOI] [PubMed] [Google Scholar]
  35. Vandromme M., Gauthier-Rouvière C., Lamb N., Fernandez A. Regulation of transcription factor localization: fine-tuning of gene expression. Trends Biochem Sci. 1996 Feb;21(2):59–64. [PubMed] [Google Scholar]
  36. Vulpe C. D., Packman S. Cellular copper transport. Annu Rev Nutr. 1995;15:293–322. doi: 10.1146/annurev.nu.15.070195.001453. [DOI] [PubMed] [Google Scholar]
  37. Wang D., Hu Y., Zheng F., Zhou K., Kohlhaw G. B. Evidence that intramolecular interactions are involved in masking the activation domain of transcriptional activator Leu3p. J Biol Chem. 1997 Aug 1;272(31):19383–19392. doi: 10.1074/jbc.272.31.19383. [DOI] [PubMed] [Google Scholar]
  38. Wen W., Meinkoth J. L., Tsien R. Y., Taylor S. S. Identification of a signal for rapid export of proteins from the nucleus. Cell. 1995 Aug 11;82(3):463–473. doi: 10.1016/0092-8674(95)90435-2. [DOI] [PubMed] [Google Scholar]
  39. Whiteside S. T., Goodbourn S. Signal transduction and nuclear targeting: regulation of transcription factor activity by subcellular localisation. J Cell Sci. 1993 Apr;104(Pt 4):949–955. doi: 10.1242/jcs.104.4.949. [DOI] [PubMed] [Google Scholar]
  40. Winge D. R., Dameron C. T., George G. N. The metallothionein structural motif in gene expression. Adv Inorg Biochem. 1994;10:1–48. [PubMed] [Google Scholar]
  41. Yamaguchi-Iwai Y., Serpe M., Haile D., Yang W., Kosman D. J., Klausner R. D., Dancis A. Homeostatic regulation of copper uptake in yeast via direct binding of MAC1 protein to upstream regulatory sequences of FRE1 and CTR1. J Biol Chem. 1997 Jul 11;272(28):17711–17718. doi: 10.1074/jbc.272.28.17711. [DOI] [PubMed] [Google Scholar]
  42. Yano K., Fukasawa T. Galactose-dependent reversible interaction of Gal3p with Gal80p in the induction pathway of Gal4p-activated genes of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1997 Mar 4;94(5):1721–1726. doi: 10.1073/pnas.94.5.1721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Zawel L., Reinberg D. Common themes in assembly and function of eukaryotic transcription complexes. Annu Rev Biochem. 1995;64:533–561. doi: 10.1146/annurev.bi.64.070195.002533. [DOI] [PubMed] [Google Scholar]
  44. Zhang L., Guarente L. Heme binds to a short sequence that serves a regulatory function in diverse proteins. EMBO J. 1995 Jan 16;14(2):313–320. doi: 10.1002/j.1460-2075.1995.tb07005.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Zhou K. M., Kohlhaw G. B. Transcriptional activator LEU3 of yeast. Mapping of the transcriptional activation function and significance of activation domain tryptophans. J Biol Chem. 1990 Oct 15;265(29):17409–17412. [PubMed] [Google Scholar]
  46. Zhu Z., Labbé S., Peña M. M., Thiele D. J. Copper differentially regulates the activity and degradation of yeast Mac1 transcription factor. J Biol Chem. 1998 Jan 16;273(3):1277–1280. doi: 10.1074/jbc.273.3.1277. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES