Journal Article

The Menkes Protein (ATP7A; MNK) Cycles via the Plasma Membrane Both in Basal and Elevated Extracellular Copper Using a C-Terminal Di-Leucine Endocytic Signal

Michael J. Petris and Julian F.B. Mercer

in Human Molecular Genetics

Volume 8, issue 11, pages 2107-2115
Published in print October 1999 | ISSN: 0964-6906
Published online October 1999 | e-ISSN: 1460-2083 | DOI: http://dx.doi.org/10.1093/hmg/8.11.2107
The Menkes Protein (ATP7A; MNK) Cycles via the Plasma Membrane Both in Basal and Elevated Extracellular Copper Using a C-Terminal Di-Leucine Endocytic Signal

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Menkes disease is an X-linked recessive copper deficiency disorder caused by mutations in the ATP7A (MNK) gene which encodes a coppertransporting P-type ATPase (MNK). MNK is normally localized predominantly in the trans-Golgi network (TGN); however, when cells are exposed to excessive copper it is rapidly relocalized to the plasma membrane where it functions in copper efflux. In this study, the c-myc epitope was introduced within the loop connecting the first and second transmembrane regions of MNK. This myc epitope allowed detection of the protein at the surface of living cells and provided the first experimental evidence supporting the common topological model. In cells stably expressing the tagged MNK protein (MNK-tag), extracellular antibodies were internalized to the perinuclear region, indicating that MNK-tag at the TGN constitutively cycles via the plasma membrane in basal copper conditions. Under elevated copper conditions, MNK-tag was recruited to the plasma membrane; however, internalization of MNK-tag was not inhibited and the protein continued to recycle through cytoplasmic membrane compartments. These findings suggest that copper stimulates exocytic movement of MNK to the plasma membrane ratherthan reducing MNK retrieval and indicate that MNK may remove copperfrom the cytoplasm by transporting copper into the vesicles through which it cycles. Newly internalized MNK-tag and transferrin were found to co-localize, suggesting that MNK-tag follows a clathrin-coated pit/endosomal pathway into cells. Mutation of the di-leucine, L1487 L1488, prevented uptake of anti-myc antibodies in both basal and elevated copper conditions, thereby identifying this sequence as an endocytic signal for MNK. Analysis of the effects of the di-leucine mutation in elevated copper provided further support for copperstimulated exocytic movement of MNK from the TGN to the plasma membrane.

Journal Article.  6645 words.  Illustrated.

Subjects: Genetics and Genomics

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