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Published on: February 21, 2019
Molecular events initiating exit of a copper-transporting ATPase ATP7B from the trans-Golgi network
Nesrin M Hasan1, Arnab Gupta, Elena Polishchuk
1Department of Physiology, Johns Hopkins University, Baltimore, Maryland 21205, USA.
Abstract:
The copper-transporting ATPase ATP7B has a dual intracellular localization: the trans-Golgi network (TGN) and cytosolic vesicles. Changes in copper levels, kinase-mediated phosphorylation, and mutations associated with Wilson disease alter the steady-state distribution of ATP7B between these compartments. To identify a primary molecular event that triggers ATP7B exit from the TGN, we characterized the folding, activity, and trafficking of the ATP7B variants with mutations within the regulatory N-terminal domain (N-ATP7B). We found that structural changes disrupting the inter-domain contacts facilitate ATP7B exit from the TGN. Mutating Ser-340/341 in the N-ATP7B individually or together to Ala, Gly, Thr, or Asp produced active protein and shifted the steady-state localization of ATP7B to vesicles, independently of copper levels. The Ser340/341G mutant had a lower kinase-mediated phosphorylation under basal conditions and no copper-dependent phosphorylation. Thus, negative charges introduced by copper-dependent phosphorylation are not obligatory for ATP7B trafficking from the TGN. The Ser340/341A mutation did not alter the overall fold of N-ATP7B, but significantly decreased interactions with the nucleotide-binding domain, mimicking consequences of copper binding to N-ATP7B. We propose that structural changes that specifically alter the inter-domain contacts initiate exit of ATP7B from the TGN, whereas increased phosphorylation may be needed to maintain an open interface between the domains.
Insights
Structural changes disrupting inter-domain contacts, not copper levels, trigger copper-transporting ATPase ATP7B exit from the trans-Golgi network (TGN). This finding clarifies ATP7B trafficking in Wilson disease.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The copper-transporting ATPase ATP7B is crucial for copper homeostasis.
- ATP7B exhibits dual localization between the trans-Golgi network (TGN) and cytosolic vesicles.
- Altered copper levels, phosphorylation, and Wilson disease mutations affect ATP7B distribution.
Purpose of the Study:
- To pinpoint the primary molecular trigger for ATP7B's departure from the TGN.
- To investigate the role of the N-terminal regulatory domain (N-ATP7B) in ATP7B trafficking.
- To understand how structural changes influence ATP7B localization.
Main Methods:
- Characterization of ATP7B variants with mutations in the N-ATP7B domain.
- Analysis of protein folding, enzymatic activity, and intracellular trafficking.
- Site-directed mutagenesis of Ser-340/341 to various amino acids (Ala, Gly, Thr, Asp).
- Assessment of kinase-mediated phosphorylation under basal and copper-dependent conditions.
Main Results:
- Structural alterations disrupting inter-domain contacts facilitate ATP7B exit from the TGN.
- Mutations at Ser-340/341 shifted ATP7B localization to vesicles, independent of copper levels.
- The Ser340/341G mutation showed reduced phosphorylation, indicating it's not obligatory for TGN exit.
- The Ser340/341A mutation decreased inter-domain interactions, mimicking copper binding effects.
Conclusions:
- Disruption of inter-domain contacts within ATP7B is the key event initiating TGN exit.
- Copper-dependent phosphorylation may stabilize the open conformation rather than trigger initial exit.
- Understanding these mechanisms offers insights into Wilson disease pathogenesis and ATP7B regulation.
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