Copper-dependent protein-protein interactions studied by yeast two-hybrid analysis.
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.
van Dongen
Elisabeth M W M
EM
Laboratory of Macromolecular and Organic Chemistry, Department of Biomedical Engineering, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands.
Klomp
Leo W J
LW
Merkx
Maarten
M
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D003300:Copper / Q000737:chemistry* / Q000378:metabolism*; D000073840:Copper-Transporting ATPases; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D020798:Two-Hybrid System Techniques
D027682:Cation Transport Proteins; D003300:Copper; D000251:Adenosine Triphosphatases; C000612464:ATP7B protein, human; D000073840:Copper-Transporting ATPases