Copper is essential for the growth and development of mammalian cells. The key role in the intracellular distribution of
copper belongs to the recently discovered family of
metallochaperones and to
copper-transporting
P-type ATPases. The mutations in the
ATPase ATP7B, the
Wilson's disease protein (WNDP), lead to intracellular accumulation of
copper and severe hepatic and neurological abnormalities. Several of these mutations were shown to disrupt the
protein-
protein interactions between WNDP and the
metallochaperone Atox1, suggesting that these interactions are important for normal
copper homeostasis. To understand the functional consequences of the Atox1-WNDP interaction at the molecular level, we produced recombinant Atox1 and characterized its effects on WNDP. We demonstrate that Atox1 transfers
copper to the purified amino-terminal domain of WNDP (N-WNDP) in a dose-dependent and saturable manner. A maximum of six
copper atoms can be transferred to N-WNDP by the chaperone. Furthermore, the incubation of
copper Atox1 with the full-length WNDP leads to the stimulation of the WNDP catalytic activity, providing strong evidence for the direct effect of Atox1 on the function of this transporter. Our data also suggest that Atox1 can regulate the
copper occupancy of WNDP. The incubation with apo-Atox1 results in the removal of
copper from the metalated N-WNDP and apparent down-regulation of WNDP activity. Interestingly, at least one
copper atom remains tightly bound to N-WNDP even in the presence of excess apo-Atox1. We suggest that this incomplete reversibility reflects the functional non-equivalency of the
metal-binding sites in WNDP and speculate about the intracellular consequences of the reversible Atox1-mediated
copper transfer.