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Abstract: As the trace element copper is essential, but extremely toxic in high concentrations, intracellular copper concentrations are tightly controlled. Once in the cell, copper is distributed by metallochaperones, including the small cytoplasmic protein ATOX1. ATOX1 plays an important role in the transfer of copper to the copper export P-type ATPases ATP7A and ATP7B to facilitate copper excretion. Recently, a novel function for Atox1 as a transcription factor (TF) regulating Ccnd1 was proposed. Crystal structures of ATOX1 reveal copper-dependent homodimerization of ATOX1. As many TFs regulate gene expression as a dimer and bind to DNA repeats, we investigated the promotor region of CCND1 and detected a direct repeat sequence in the Atox1 binding site (tentatively referred to as CABE, copper-responsive Atox1 binding element). We therefore propose copper-dependent homodimerization to be an essential step in the regulation of ATOX1-dependent transcription. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]