1887

Abstract

The Ctr1 family of proteins mediates high-affinity copper (Cu) acquisition in eukaryotic organisms. In the fission yeast , Cu uptake is carried out by a heteromeric complex formed by the Ctr4 and Ctr5 proteins. Unlike human and Ctr1 proteins, Ctr4 and Ctr5 are unable to function independently in Cu acquisition. Instead, both proteins physically interact with each other to form a Ctr4–Ctr5 heteromeric complex, and are interdependent for secretion to the plasma membrane and Cu transport activity. In this study, we used mutants that are defective in high-affinity Cu uptake to dissect the relative contribution of Ctr4 and Ctr5 to the Cu transport function. Functional complementation and localization assays show that the conserved Met-X-Met motif in transmembrane domain 2 of the Ctr5 protein is dispensable for the functionality of the Ctr4–Ctr5 complex, whereas the Met-X-Met motif in the Ctr4 protein is essential for function and for localization of the hetero-complex to the plasma membrane. Moreover, Ctr4/Ctr5 chimeric proteins reveal unique properties found either in Ctr4 or in Ctr5, and are sufficient for Cu uptake on the cell surface of cells. Functional chimeras contain the Ctr4 central and Ctr5 carboxyl-terminal domains (CTDs). We propose that the Ctr4 central domain mediates Cu transport in this hetero-complex, whereas the Ctr5 CTD functions in the regulation of trafficking of the Cu transport complex to the cell surface.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.046854-0
2011-04-01
2019-10-19
Loading full text...

Full text loading...

/deliver/fulltext/micro/157/4/1021.html?itemId=/content/journal/micro/10.1099/mic.0.046854-0&mimeType=html&fmt=ahah

References

  1. Alfa, C., Fantes, P., Hyams, J., McLeod, M. & Warbrick, E. ( 1993; ). Experiments with Fission Yeasts: Laboratory Course Manual. Cold Spring Harbor, NY. : Cold Spring Harbor Laboratory.
    [Google Scholar]
  2. Aller, S. G. & Unger, V. M. ( 2006; ). Projection structure of the human copper transporter CTR1 at 6 Å resolution reveals a compact trimer with a novel channel-like architecture. Proc Natl Acad Sci U S A 103, 3627–3632.[CrossRef]
    [Google Scholar]
  3. Aller, S. G., Eng, E. T., De Feo, C. J. & Unger, V. M. ( 2004; ). Eukaryotic CTR copper uptake transporters require two faces of the third transmembrane domain for helix packing, oligomerization, and function. J Biol Chem 279, 53435–53441.[CrossRef]
    [Google Scholar]
  4. Balamurugan, K. & Schaffner, W. ( 2006; ). Copper homeostasis in eukaryotes: teetering on a tightrope. Biochim Biophys Acta 1763, 737–746.[CrossRef]
    [Google Scholar]
  5. Beaudoin, J. & Labbé, S. ( 2001; ). The fission yeast copper-sensing transcription factor Cuf1 regulates the copper transporter gene expression through an Ace1/Amt1-like recognition sequence. J Biol Chem 276, 15472–15480.[CrossRef]
    [Google Scholar]
  6. Beaudoin, J., Laliberté, J. & Labbé, S. ( 2006; ). Functional dissection of Ctr4 and Ctr5 amino-terminal regions reveals motifs with redundant roles in copper transport. Microbiology 152, 209–222.[CrossRef]
    [Google Scholar]
  7. Bezanilla, M., Forsburg, S. L. & Pollard, T. D. ( 1997; ). Identification of a second myosin-II in Schizosaccharomyces pombe: Myp2p is conditionally required for cytokinesis. Mol Biol Cell 8, 2693–2705.[CrossRef]
    [Google Scholar]
  8. Dancis, A., Haile, D., Yuan, D. S. & Klausner, R. D. ( 1994a; ). The Saccharomyces cerevisiae copper transport protein (Ctr1p). Biochemical characterization, regulation by copper, and physiologic role in copper uptake. J Biol Chem 269, 25660–25667.
    [Google Scholar]
  9. Dancis, A., Yuan, D. S., Haile, D., Askwith, C., Eide, D., Moehle, C., Kaplan, J. & Klausner, R. D. ( 1994b; ). Molecular characterization of a copper transport protein in S. cerevisiae: an unexpected role for copper in iron transport. Cell 76, 393–402.[CrossRef]
    [Google Scholar]
  10. De Feo, C. J., Aller, S. G., Siluvai, G. S., Blackburn, N. J. & Unger, V. M. ( 2009; ). Three-dimensional structure of the human copper transporter hCTR1. Proc Natl Acad Sci U S A 106, 4237–4242.[CrossRef]
    [Google Scholar]
  11. Georgatsou, E., Mavrogiannis, L. A., Fragiadakis, G. S. & Alexandraki, D. ( 1997; ). The yeast Fre1p/Fre2p cupric reductases facilitate copper uptake and are regulated by the copper-modulated Mac1p activator. J Biol Chem 272, 13786–13792.[CrossRef]
    [Google Scholar]
  12. Halliwell, B. & Gutteridge, J. M. ( 1984; ). Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem J 219, 1–14.
    [Google Scholar]
  13. Hassett, R. & Kosman, D. J. ( 1995; ). Evidence for Cu(II) reduction as a component of copper uptake by Saccharomyces cerevisiae. J Biol Chem 270, 128–134.[CrossRef]
    [Google Scholar]
  14. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. & Pease, L. R. ( 1989; ). Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59.[CrossRef]
    [Google Scholar]
  15. Ioannoni, R., Beaudoin, J., Mercier, A. & Labbé, S. ( 2010; ). Copper-dependent trafficking of the Ctr4-Ctr5 copper transporting complex. PLoS ONE 5, e11964.[CrossRef]
    [Google Scholar]
  16. Jiang, J., Nadas, I. A., Kim, M. A. & Franz, K. J. ( 2005; ). A Mets motif peptide found in copper transport proteins selectively binds Cu(I) with methionine-only coordination. Inorg Chem 44, 9787–9794.[CrossRef]
    [Google Scholar]
  17. Kaiser, C., Michaelis, S. & Mitchell, A. ( 1994; ). Methods in Yeast Genetics. Cold Spring Harbor, NY. : Cold Spring Harbor Laboratory.
    [Google Scholar]
  18. Keeney, J. B. & Boeke, J. D. ( 1994; ). Efficient targeted integration at leu1-32 and ura4-294 in Schizosaccharomyces pombe. Genetics 136, 849–856.
    [Google Scholar]
  19. Kim, B. E., Nevitt, T. & Thiele, D. J. ( 2008; ). Mechanisms for copper acquisition, distribution and regulation. Nat Chem Biol 4, 176–185.[CrossRef]
    [Google Scholar]
  20. Klomp, A. E., Juijn, J. A., van der Gun, L. T., van den Berg, I. E., Berger, R. & Klomp, L. W. ( 2003; ). The N-terminus of the human copper transporter 1 (hCTR1) is localized extracellularly, and interacts with itself. Biochem J 370, 881–889.[CrossRef]
    [Google Scholar]
  21. Knight, S. A., Labbé, S., Kwon, L. F., Kosman, D. J. & Thiele, D. J. ( 1996; ). A widespread transposable element masks expression of a yeast copper transport gene. Genes Dev 10, 1917–1929.[CrossRef]
    [Google Scholar]
  22. Labbé, S., Zhu, Z. & Thiele, D. J. ( 1997; ). Copper-specific transcriptional repression of yeast genes encoding critical components in the copper transport pathway. J Biol Chem 272, 15951–15958.[CrossRef]
    [Google Scholar]
  23. Labbé, S., Peña, M. M., Fernandes, A. R. & Thiele, D. J. ( 1999; ). A copper-sensing transcription factor regulates iron uptake genes in Schizosaccharomyces pombe. J Biol Chem 274, 36252–36260.[CrossRef]
    [Google Scholar]
  24. Lee, J., Peña, M. M., Nose, Y. & Thiele, D. J. ( 2002; ). Biochemical characterization of the human copper transporter Ctr1. J Biol Chem 277, 4380–4387.[CrossRef]
    [Google Scholar]
  25. Linder, M. C. ( 1991; ). Biochemistry of Copper. New York. : Plenum Press.
    [Google Scholar]
  26. Liu, J., Sitaram, A. & Burd, C. G. ( 2007; ). Regulation of copper-dependent endocytosis and vacuolar degradation of the yeast copper transporter, Ctr1p, by the Rsp5 ubiquitin ligase. Traffic 8, 1375–1384.[CrossRef]
    [Google Scholar]
  27. Macomber, L. & Imlay, J. A. ( 2009; ). The iron-sulfur clusters of dehydratases are primary intracellular targets of copper toxicity. Proc Natl Acad Sci U S A 106, 8344–8349.[CrossRef]
    [Google Scholar]
  28. Martins, L. J., Jensen, L. T., Simon, J. R., Keller, G. L. & Winge, D. R. ( 1998; ). Metalloregulation of FRE1 and FRE2 homologs in Saccharomyces cerevisiae. J Biol Chem 273, 23716–23721.[CrossRef]
    [Google Scholar]
  29. Mumberg, D., Müller, R. & Funk, M. ( 1995; ). Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156, 119–122.[CrossRef]
    [Google Scholar]
  30. Ooi, C. E., Rabinovich, E., Dancis, A., Bonifacino, J. S. & Klausner, R. D. ( 1996; ). Copper-dependent degradation of the Saccharomyces cerevisiae plasma membrane copper transporter Ctr1p in the apparent absence of endocytosis. EMBO J 15, 3515–3523.
    [Google Scholar]
  31. Peña, M. M., Koch, K. A. & Thiele, D. J. ( 1998; ). Dynamic regulation of copper uptake and detoxification genes in Saccharomyces cerevisiae. Mol Cell Biol 18, 2514–2523.
    [Google Scholar]
  32. Peña, M. M., Puig, S. & Thiele, D. J. ( 2000; ). Characterization of the Saccharomyces cerevisiae high affinity copper transporter Ctr3. J Biol Chem 275, 33244–33251.[CrossRef]
    [Google Scholar]
  33. Puig, S. & Thiele, D. J. ( 2002; ). Molecular mechanisms of copper uptake and distribution. Curr Opin Chem Biol 6, 171–180.[CrossRef]
    [Google Scholar]
  34. Puig, S., Lee, J., Lau, M. & Thiele, D. J. ( 2002; ). Biochemical and genetic analyses of yeast and human high affinity copper transporters suggest a conserved mechanism for copper uptake. J Biol Chem 277, 26021–26030.[CrossRef]
    [Google Scholar]
  35. Rees, E. M., Lee, J. & Thiele, D. J. ( 2004; ). Mobilization of intracellular copper stores by the Ctr2 vacuolar copper transporter. J Biol Chem 279, 54221–54229.[CrossRef]
    [Google Scholar]
  36. Rubino, J. T., Riggs-Gelasco, P. & Franz, K. J. ( 2010; ). Methionine motifs of copper transport proteins provide general and flexible thioether-only binding sites for Cu(I) and Ag(I). J Biol Inorg Chem 15, 1033–1049.[CrossRef]
    [Google Scholar]
  37. Sambrook, J., Fritsch, E. F. & Maniatis, T. ( 1989; ). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY. : Cold Spring Harbor Laboratory.
    [Google Scholar]
  38. Wu, X., Sinani, D., Kim, H. & Lee, J. ( 2009; ). Copper transport activity of yeast Ctr1 is down-regulated via its C terminus in response to excess copper. J Biol Chem 284, 4112–4122.[CrossRef]
    [Google Scholar]
  39. Zhou, H. & Thiele, D. J. ( 2001; ). Identification of a novel high affinity copper transport complex in the fission yeast Schizosaccharomyces pombe. J Biol Chem 276, 20529–20535.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.046854-0
Loading
/content/journal/micro/10.1099/mic.0.046854-0
Loading

Data & Media loading...

Supplements

[PDF](33 KB)

PDF

The Met residue is essential for Ctr445 functionality. [PDF](62 KB)

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error