Jose, S. M.; Maji, S.; Paul, M.; Mallick Gupta, A.; Gupta, A.

Polytopic transmembrane Copper-ATPases are central regulators of the essential, redox-active micronutrient copper in all organisms. The vertebrate Cu-ATPase homologs ATP7A and ATP7B facilitate excess copper efflux, in addition to metalating cuproproteins. ATP7A and ATP7B exhibit copper-responsive basolateral and apical trafficking polarity respectively, in polarized epithelia. To elucidate the role of conserved functional domains in copper-responsive trafficking and copper transport, we substituted the copper-binding amino-terminal, Nucleotide-Binding (NBD) and C-terminal domain of ATP7B with that of ATP7A either singly or in combinations. The six chimeric Cu-ATPases generated, although functionally active, exhibited altered trafficking phenotypes. Notably, the N-terminal-substituted ATP7B exhibited constitutive trafficking to the basolateral membrane; however, the N-terminal and NBD double-substituted chimera gained steady-state TGN localization, indicating a putative interaction between the two domains critical for its TGN-localization. We analyzed orthologous Cu-ATPase domain sequences from diverse organisms and found that the N-terminal domain and NBD showed similar evolutionary relationships, unlike that of C-terminal domain, suggesting their co-evolution. This study for the first time correlates evolutionary stringency imparted onto Cu-ATPase domains with their significance in proper trafficking regulation.