Conserved inter-domain interactions drive trans-Golgi network localisation and trafficking of homologous copper-ATPases

Polytopic copper-ATPases are central regulators of the essential micronutrient copper in all organisms. In polarized epithelia, vertebrate homologs ATP7A and ATP7B undergo copper-induced trafficking from TGN to basolateral and apical membranes, respectively, for efflux of excess copper. To probe (i) inter-domain interactions that drive trafficking and (ii) extent of divergence between homologous domains constituting copper-ATPases, we substituted the copper-binding amino-terminal (NT), Nucleotide-Binding (NBD) and/or C-terminal of ATP7B with those of ATP7A. The functionally active chimeras exhibited distinct trafficking phenotypes. Notably, ATP7B-NT substitution led to constitutive basolateral membrane trafficking, while simultaneous NT-NBD substitution led to steady-state TGN localization, suggesting that interaction between the two domains, as confirmed by in-vitro NT-NBD binding studies, may be essential for TGN-localization. Interestingly, reciprocal substitution of ATP7A-NBD and NT with that of ATP7B did not rescue membrane localization, indicating that domain compatibility was restricted, suggesting greater evolutionary divergence of ATP7B domains. Analyzing orthologous copper-ATPase domain-sequences from diverse organisms however, revealed similar evolutionary relationships between NT and NBD, suggesting their co-evolution. We thus correlate the copper-responsive trafficking ability of copper-ATPases with evolutionary stringency imparted onto Cu-ATPase domains.