Molecular events initiating exit of a copper-transporting ATPase ATP7B from the trans-Golgi network.
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.
Department of Physiology, Johns Hopkins University, Baltimore, Maryland 21205, USA.
Nesrin M
NM
Hasan
Arnab
A
Gupta
Elena
E
Polishchuk
Corey H
CH
Yu
Roman
R
Polishchuk
Oleg Y
OY
Dmitriev
Svetlana
S
Lutsenko
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D000073840:Copper-Transporting ATPases; D057809:HEK293 Cells; D006527:Hepatolenticular Degeneration / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020125:Mutation, Missense; D010766:Phosphorylation / Q000502:physiology; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D022161:Transport Vesicles / Q000235:genetics / Q000378:metabolism*; D021601:trans-Golgi Network / Q000235:genetics / Q000378:metabolism*
D027682:Cation Transport Proteins; D000251:Adenosine Triphosphatases; C000612464:ATP7B protein, human; D000073840:Copper-Transporting ATPases