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LIVER AND BILIARY TRACT

Apical targeting and Golgi retention signals reside within a 9-amino acid sequence in the copper-ATPase, ATP7B

¹Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, Maryland; ²National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland; ³GI Cell Biology Laboratory, The Children's Hospital, Harvard Medical School, Boston, Massachusetts; ⁴Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon

Submitted 12 August 2008 ; accepted in final form 21 November 2008

ATP7B is a copper-transporting P-type ATPase present predominantly in liver. In basal copper, hepatic ATP7B is in a post-trans-Golgi network (TGN) compartment where it loads cytoplasmic Cu(I) onto newly synthesized ceruloplasmin. When copper levels rise, the protein redistributes via unique vesicles to the apical periphery where it exports intracellular Cu(I) into bile. We want to understand the mechanisms regulating the copper-sensitive trafficking of ATP7B. Earlier, our laboratory reported the presence of apical targeting/TGN retention information within residues 1–63 of human ATP7B; deletion of these residues resulted in a mutant protein that was not efficiently retained in the post-TGN in low copper and constitutively trafficked to the basolateral membrane of polarized, hepatic WIF-B cells with and without copper (13). In this study, we used mutagenesis and adenovirus infection of WIF-B cells followed by confocal immunofluorescence microscopy analysis to identify the precise retention/targeting sequences in the context of full-length ATP7B. We also analyzed the expression of selected mutants in livers of copper-deficient and -loaded mice. Our combined results clearly demonstrate that nine amino acids, F₃₇AFDNVGYE₄₅, comprise an essential apical targeting determinant for ATP7B in elevated copper and participate in the TGN retention of the protein under low-copper conditions. The signal is novel, does not require phosphorylation, and is highly conserved in 24 species of ATP7B. Furthermore, N41S, which is part of the signal we identified, is the first and only Wilson disease-causing missense mutation in residues 1–63 of ATP7B. Expression of N41S-ATP7B in WIF-B cells severely disabled the targeting and retention of the protein. We present a working model of how this physiologically relevant signal might work.

Wilson protein; WIF-B cells; mutagenesis; in vivo; trans-Golgi network