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

Abstract

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, F(37)AFDNVGYE(45), 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 approximately 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.

Distinct phenotype of a Wilson disease mutation reveals a novel trafficking determinant in the copper transporter ATP7B

“…evaluate the activity, stability, and trafficking of ATP7B and its mutants (11,12). In this study, we combined these assays with additional mutational analysis and computational studies to dissect the molecular phenotype of WD mutations found in a highly conserved region of ATP7B, G 621 -S 668 .…”

Cellular copper levels determine the phenotype of the Arg 875 variant of ATP7B/Wilson disease protein

“…To evaluate the transport activity of ATP7B-Arg 875 , we used Menkes fibroblasts transfected with tyrosinase (YSTT cells). These cells lack endogenous copper-ATPase and are unable to transport copper into the TGN, thus producing inactive tyrosinase (9,10). The expression of ATP7B-Gly 875 in YSTT cells restores copper delivery to the TGN and activates tyrosinase, as evident from the formation of black pigment [levo-3,4-dihydroxy-L-phenylalanine (L-DOPA) quinone].…”

“…YSTT cells were maintained in DMEM with 10% (vol/vol) FBS, 200 μg/mL G418, and 0.5 μg/mL puromycin. The pcTYR plasmid was described earlier (10). For localization studies, the N-terminal FLAG tag was added to ATP7B by PCR and the construct was cloned into the pcDNA5 FRT/TO plasmid (Invitrogen).…”

A stimulus needed for the study of membrane traffic in hepatocytes

“…The apical transporters studied to date appear to have unique and complex sorting signals, the recognition systems are only beginning to be identified, and the targeting mechanisms are still largely unknown. More study is needed, particularly of patient missense mutations whose protein phenotypes (in hepatic cells) may give important clues about these processes (eg, see Braiterman et al 5 ). Second, are several apical PM protein recognition systems missing from hepatocytes?…”