Cu is an essential cofactor of cellular proteins but is toxic in its free state. The hepatic Cu-ATPase ATP7B has two functions in Cu homeostasis: it loads Cu+ onto newly synthesized apoceruloplasmin in the secretory pathway, thereby activating the plasma protein; and it participates in the excretion of excess Cu+ into the bile. To carry out these two functions, the membrane protein responds to changes in intracellular Cu levels by cycling between the Golgi and apical region. We used polarized hepatic WIF-B cells and high-resolution confocal microscopy to map the itinerary of endogenous and exogenous ATP7B under different Cu conditions. In Cu-depleted cells, ATP7B resided in a post-trans-Golgi network compartment that also contained syntaxin 6, whereas in Cu-loaded cells, the protein relocated to unique vesicles very near to the apical plasma membrane as well as the membrane itself. To determine the role of ATP7B's cytoplasmic NH2 terminus in regulating its intracellular movements, we generated seven mutations/deletions in this large [approximately 650 amino acid (AA)] domain and analyzed the Cu-dependent behavior of the mutant ATP7B proteins in WIF-B cells. Truncation of the ATP7B NH2 terminus up to the fifth copper-binding domain (CBD5) yielded an active ATPase that was insensitive to cellular Cu levels and constitutively trafficked to the opposite (basolateral) plasma membrane domain. Fusion of the NH2-terminal 63 AA of ATP7B to the truncated protein restored both its Cu responsiveness and correct intracellular targeting. These results indicate that important targeting information is contained in this relatively short sequence, which is absent from the related CuATPase, ATP7A.

.-We report for the first time on the copper-dependent behavior of endogenous ATP7A in two types of polarized intestinal epithelia, rat enterocytes in vivo and filter-grown Caco-2 cells, an accepted in vitro model of human small intestine. We used high-resolution, confocal immunofluorescence combined with quantitative cell surface biotinylation and found that the vast majority of endogenous ATP7A was localized intracellularly under all copper conditions. In copper-depleted cells, virtually all of the ATP7A localized to a post-TGN compartment, with Ͻ3% of the total protein detectable at the basolateral cell surface. When copper levels were elevated, ATP7A dispersed to the cell periphery in punctae whose pattern did not overlap with the steady-state distributions of post-Golgi, endosomal, or basolateral membrane markers; only ϳ8 -10% of the recovered ATP7A was detected at the basolateral cell surface. These results raise several questions regarding prevailing models of ATP7A dynamics and the mechanism of copper efflux.

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.

We examined the role that lipid rafts play in regulating apical protein trafficking in polarized hepatic cells. Rafts are postulated to form in the trans-Golgi network where they recruit newly synthesized apical residents and mediate their direct transport to the apical plasma membrane. In hepatocytes, single transmembrane and glycolipid-anchored apical proteins take the "indirect" route. They are transported from the trans-Golgi to the basolateral plasma membrane where they are endocytosed and transcytosed to the apical surface. Do rafts sort hepatic apical proteins along this circuitous pathway? We took two approaches to answer this question. First, we determined the detergent solubility of selected apical proteins and where in the biosynthetic pathway insolubility was acquired. Second, we used pharmacological agents to deplete raft components and assessed their effects on basolateral-to-apical transcytosis. We found that cholesterol and glycosphingolipids are required for delivery from basolateral early endosomes to the subapical compartment. In contrast, fluid phase uptake and clathrin-mediated internalization of recycling receptors were only mildly impaired. Apical protein solubility did not correlate with raft depletion or impaired transcytosis, suggesting other factors contribute to apical protein insolubility. Examination of apical proteins in Fao cells also revealed that raft-dependent sorting does not require the polarized cell context.