Molecular Events Initiating Exit of a Copper-transporting ATPase ATP7B from the Trans-Golgi Network

Abstract

Background: ATP7B trafficking from Golgi to vesicles is essential for copper homeostasis. Results: Mutating Ser-340/341 alters the inter-domain contacts, diminishes protein phosphorylation, and shifts ATP7B localization to vesicles. Conclusion: Spatial arrangement of functional domains determines ATP7B readiness to traffic, whereas phosphorylation may maintain the trafficking-compatible state. Significance: The proposed mechanism explains how unrelated signals trigger similar trafficking responses from ATP7B.

Communication between the N and C Termini Is Required for Copper-stimulated Ser/Thr Phosphorylation of Cu(I)-ATPase (ATP7B)

“…Thus, our studies reached the unexpected conclusion that the competence of all six MBDs to bind copper was not required to observe copper-dependent apical targeting, suggesting the existence of another copper-sensing domain(s) in ATP7B (39,40) that regulates the apical targeting step in polarized cells. Our studies extend previous ones that have suggested that copper binding to MBDs triggers conformational changes that propagate structural changes to other domains of ATP7B and trigger its TGN exit via vesicles (13). We suggest an additional role for copper-stimulated conformational changes: perhaps those structural changes expose both the apical targeting motif and C-terminal Ser/Thr to TGN-resident kinases in a copperdependent manner.…”

“…they all showed increased ATP7B in vesicles. The conclusion of this study was that phosphorylation does not initiate ATP7B trafficking per se but rather maintains the protein in a trafficking-permissive state following a copper-induced conformational change (13). Overall, the relationship between copper-stimulated Ser/ Thr hyperphosphorylation and copper-dependent trafficking remains enigmatic, and the ATP7B residues phosphorylated physiologically in high copper remain undetermined.…”

“…An ATP7B mutant with all four of these Ser changed to Ala exhibited defective TGN exit, suggesting that phosphorylation at these sites is required for TGN exit (12). The conformation of a different Ser cluster (Ser-340 and Ser-341), located in the loop between MBD3 and MBD4 also appears to be important for TGN exit in HEK293TRex cells (13). Inactivating mutations (Ser-340 and Ser-341 to Ala) did not reduce basal phosphorylation of ATP7B.…”

“…Previous studies showed that copper-dependent TGN exit of ATP7B correlated with its hyperphosphorylation (9). ATP7B phosphorylation also occurs during its biosynthesis; these modifications have been referred to as "basal" phosphorylation and seem to affect protein stability, lifetime, and/or quality control (9,(12)(13)(14). Mass spectrometry (MS) analysis of recombinant ATP7B isolated from Sf9 cells revealed that the loop between metal binding domain 3 (MBD3) and MBD4 as well as 10 other N-terminal (N-term) peptides were phosphorylated following in vitro radiolabeling in the presence of mammalian cell extract (15).…”

Distinct phenotype of a Wilson disease mutation reveals a novel trafficking determinant in the copper transporter ATP7B

“…In addition to the cellular components required for copperdependent ATP7B trafficking, studies have shown that molecular signals within the protein, as well as intramolecular interactions, play important roles (30,40). Two well-characterized trafficking signals encoded within the primary sequences of both copper-ATPases have been identified: (i) F 37 -E 45 in ATP7B, which is required for retention of ATP7B in the TGN (−Cu) and targeting to the apical region in polarized hepatic cells (+Cu, anterograde trafficking) (11); and (ii) di-and trileucines in ATP7A and ATP7B's C-termini, which are needed for retrograde trafficking to the TGN (−Cu) (12,41,42).…”