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

Polytopic copper (Cu)-ATPases are central regulators of the essential micronutrient copper in all organisms. In polarized epithelia, the vertebrate homologues ATP7A and ATP7B undergo copper-induced trafficking from the trans-Golgi network (TGN) to basolateral and apical membranes, respectively, to mediate efflux of excess copper. To probe (1) inter-domain interactions that drive trafficking and (2) the extent of divergence between homologous domains constituting Cu-ATPases, we replaced the copper-binding N-terminal (NT), nucleotide-binding (NBD) and/or C-terminal (CT) domains of ATP7B with those of ATP7A. The functionally active chimeras exhibited distinct trafficking phenotypes. Notably, the ATP7B-NT substitution led to constitutive basolateral membrane trafficking, whereas 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, might be essential for TGN localization. Interestingly, reciprocal replacement of the ATP7A-NBD and -NT with that from ATP7B did not rescue membrane localization, indicating that domain compatibility is restricted, suggesting greater evolutionary divergence of ATP7B domains. Analysing orthologous Cu-ATPase domain-sequences from diverse organisms, however, revealed similar evolutionary relationships between the NT and NBD, suggesting their co-evolution. We thus correlate the copper-responsive trafficking ability of Cu-ATPases with evolutionary stringency imparted onto Cu-ATPase domains.