Handling Editor: Mahak Sharma
ABSTRACT
The Wilson disease protein, ATP7B maintains copper (herein referring to the Cu+ ion) homeostasis in the liver. ATP7B traffics from trans-Golgi network to endolysosomes to export excess copper. Regulation of ATP7B trafficking to and from endolysosomes is not well understood. We investigated the fate of ATP7B after copper export. At high copper levels, ATP7B traffics primarily to acidic, active hydrolase (cathepsin-B)-positive endolysosomes and, upon subsequent copper chelation, returns to the trans-Golgi network (TGN). At high copper, ATP7B colocalizes with endolysosomal markers and with a core member of retromer complex, VPS35. Knocking down VPS35 did not abrogate the copper export function of ATP7B or its copper-responsive anterograde trafficking to vesicles; rather upon subsequent copper chelation, ATP7B failed to relocalize to the TGN, which was rescued by overexpressing wild-type VPS35. Overexpressing mutants of the retromer complex-associated proteins Rab7A and COMMD1 yielded a similar non-recycling phenotype of ATP7B. At high copper, VPS35 and ATP7B are juxtaposed on the same endolysosome and form a large complex that is stabilized by in vivo photoamino acid labeling and UV-crosslinking. We demonstrate that retromer regulates endolysosome to TGN trafficking of copper transporter ATP7B in a manner that is dependent upon intracellular copper.
Footnotes
Competing interests
The authors declare no competing or financial interests.
Author contributions
Conceptualization: A.G., S.D.; Methodology: A.G., S.D., R.R., I.B., T.S., N.N.; Software: S.M.; Validation: A.G., R.R.; Formal analysis: A.G., S.D., S.M.; Investigation: A.G., S.D., T.S.; Resources: A.G., S.D.; Data curation: S.D., R.R., N.N.; Writing - original draft: A.G., S.D.; Writing - review & editing: A.G., S.D.; Visualization: R.R.; Supervision: A.G.; Project administration: A.G.; Funding acquisition: A.G.
Funding
This work was supported by a The Wellcome Trust DBT India Alliance Fellowship (IA/I/16/1/502369) and Early Career Research Award (ECR/2015/000220) from Department of Science and Technology, Ministry of Science and Technology, India (SERB) and IISER K intramural funding to A.G. S.M. and I.B. was supported by Pre-doctoral fellowship from Council of Scientific and Industrial Research, India. T.S. was supported by National Postdoctoral Fellowship, SERB, India. The pre-doctoral fellowship for S.D. is supported by Welcome Trust DBT India Alliance. The predoctoral fellowship for Ruturaj is supported by Intramural Institute funding (IISER-K). The post-doctoral fellowship for N.N. is funded by SINP Intramural funding.
Supplementary information
Supplementary information available online at https://jcs.biologists.org/lookup/doi/10.1242/jcs.246819.supplemental
- Received March 29, 2020.
- Accepted November 19, 2020.
- © 2020. Published by The Company of Biologists Ltd
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