Studies on Regulatory Intra- & Inter-molecular Interactions of Copper-ATPases

Jose, Sharon Mary (2026) Studies on Regulatory Intra- & Inter-molecular Interactions of Copper-ATPases. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Sharon Mary Jose (20RS044)) 20RS044.pdf - Submitted Version Restricted to Repository staff only Download (11MB)

Abstract

Copper-ATPases are essential multi-domain P-type ATPases that are central regulators of the copper in all organisms. In polarized epithelia, vertebrate copper-ATPase homologs ATP7A and ATP7B undergo copper-dependent trafficking from Trans-Golgi network (TGN) to basolateral and apical membranes, respectively, for efflux of excess copper. We studied the molecular cues that govern the trafficking of the mammalian copper transporters by investigating intramolecular domain communication and intermolecular interactions—with adaptor protein (AP) complexes, in the polarized epithelial model, MDCK cell line. For understanding the intramolecular cues, we exchanged three domains previously implicated in trafficking between the two homologs ̶ the copper-binding amino-terminal (NT), Nucleotide-Binding (NBD) and/or C-terminal and tested the copper-dependent cellular localization and catalytic activity of the chimeric copper-ATPases generated. We found that the 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 sister domains, as confirmed by in-vitro NT-NBD binding studies, may be essential for TGN-localization, but not sufficient for copper-dependent TGN-exit. However, reciprocal substitution of ATP7A-NBD and NT with that of ATP7B, did not rescue membrane localization, indicating that domain compatibility was restricted, possibly due to greater evolutionary divergence of ATP7B domains. For unravelling the inter-molecular cues that govern trafficking of these transporters, we probed the roles of AP-3 and AP-1, previously identified by our group as trafficking regulators of ATP7A and ATP7B. We found that AP-3 is involved in the steady-state TGN localization of copper-ATPases in polarized MDCK, along with the already reported AP-1, under basal conditions. However, in unpolarized stage, we found that only AP-1, and not AP-3 is required for steady-state TGN localization; while the copper-dependent trafficking to the cell membrane is dependent on AP-1, and not AP-3, in both stages of polarization, thus unravelling a gradual change in regulation of these transporters with epithelial cell polarization.

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