Copper-dependent protein-protein interactions studied by yeast two-hybrid analysis

Biochem Biophys Res Commun. 2004 Oct 22;323(3):789-95. doi: 10.1016/j.bbrc.2004.08.160.


An important step in copper homeostasis is delivery of copper to a specific P-type ATPase in the Golgi apparatus (Ccc2 in yeast, ATP7A and ATP7B in humans) by a small copper chaperone protein (Atx1 in yeast, ATOX1 in humans). Atx1 and ATOX1 both contain an MXCXXC motif that is also present in Ccc2 (two motifs) and ATP7A/B (six motifs). Protein-protein interactions probably require coordination of one Cu(I) by cysteines from both MXCXXC motifs. We applied yeast two-hybrid analysis to screen systematically all possible interactions between MXCXXC-containing domains in these proteins. We demonstrate that ATOX1 and Atx1 preferentially interact with domains 2 and 4 of ATP7B and that Atx1 interacts with both Ccc2 domains. All combinations show a remarkable bell-shaped dependency on copper concentration that is maximal just below normal copper levels. Our results suggest that yeast two-hybrid analysis can be used to study the intracellular copper status of a cell.

Publication types

  • Comparative Study
  • Evaluation Study

MeSH terms

  • Adenosine Triphosphatases / chemistry
  • Adenosine Triphosphatases / metabolism
  • Binding Sites
  • Cation Transport Proteins / chemistry*
  • Cation Transport Proteins / metabolism*
  • Copper / chemistry*
  • Copper / metabolism*
  • Copper-Transporting ATPases
  • Protein Binding
  • Protein Interaction Mapping / methods*
  • Two-Hybrid System Techniques*


  • Cation Transport Proteins
  • Copper
  • Adenosine Triphosphatases
  • ATP7B protein, human
  • Copper-Transporting ATPases