ABSTRACT The Cu + pump ATP7B plays an irreplaceable role in the elimination of excess Cu + by the hepatocyte into the bile. The trafficking and site of action of ATP7B are subjects of controversy. One current proposal is that an increase in intracellular Cu + results in the translocation of ATP7B to the lysosomes and excretion of excess Cu + through lysosomal-mediated exocytosis at the bile canaliculus. Here, we show that ATP7B is transported from the trans-Golgi network (TGN) to the bile canaliculus by basolateral sorting and endocytosis, and microtubule-mediated transcytosis through the subapical compartment. Trafficking ATP7B is not incorporated into lysosomes, and addition of Cu + does not cause relocalization of lysosomes and the appearance of lysosome markers in the bile canaliculus. Our data reveal the pathway of the Cu + -mediated transport of ATP7B from the TGN to the bile canaliculus and indicates that the bile canaliculus is the primary site of ATP7B action in the elimination of excess Cu + .

Beta-cleavage of the neurodegenerative disease-associated prion protein (PrP) protects cells from death induced by oxidative insults. The beta-cleavage event produces two fragments, designated N2 and C2. We investigated the role of the N2 fragment (residues 23-89) in cellular stress response, determining mechanisms involved and regions important for this reaction. The N2 fragment differentially modulated the reactive oxygen species (ROS) response induced by serum deprivation, with amelioration when copper bound. Amino acid residues 23-50 alone mediated a ROS reduction response. PrP23-50 ROS reduction was not due to copper binding or direct antioxidant activity, but was instead mediated through proteoglycan binding partners localised in or interacting with cholesterol-rich membrane domains. Furthermore, mutational analyses of both PrP23-50 and N2 showed that their protective capacity requires the sterically constraining double proline motif within the N-terminal polybasic region. Our findings show that N2 is a biologically active fragment that is able to modulate stress-induced intracellular ROS through interaction of its structurally defined N-terminal polybasic region with cell-surface proteoglycans.

Copper is an essential nutrient for a variety of biochemical processes; however, the redox properties of copper also make it potentially toxic in the free form. Consequently, the uptake and intracellular distribution of this metal is strictly regulated. This raises the issue of whether specific pathophysiological conditions can promote adaptive changes in intracellular copper distribution. In this study, we demonstrate that oxygen limitation promotes a series of striking alterations in copper homeostasis in RAW264.7 macrophage cells. Hypoxia was found to stimulate copper uptake and to increase the expression of the copper importer, CTR1. This resulted in increased copper delivery to the ATP7A copper transporter and copper-dependent trafficking of ATP7A to cytoplasmic vesicles. Significantly, the ATP7A protein was required to deliver copper into the secretory pathway to ceruloplasmin, a secreted copperdependent enzyme, the expression and activity of which were stimulated by hypoxia. However, the activities of the alternative targets of intracellular copper delivery, superoxide dismutase and cytochrome c oxidase, were markedly reduced in response to hypoxia. Collectively, these findings demonstrate that copper delivery into the biosynthetic secretory pathway is regulated by oxygen availability in macrophages by a selective increase in copper transport involving ATP7A.

The cellular prion protein (PrP C ) is essential for the pathogenesis and transmission of prion diseases. Although PrP C is known to be located in detergent-insoluble lipid rafts at the surface of neuronal cells, the mechanism of its internalisation is unclear, with both raft/caveolae-based and clathrin-mediated processes being proposed. We have investigated the mechanism of copper-induced internalisation of PrP C in neuronal cells by immunofluorescence microscopy, surface biotinylation assays and buoyant sucrose density gradient centrifugation in the presence of Triton X-100. Clathrin-mediated endocytosis was selectively blocked with tyrphostin A23, which disrupts the interaction between tyrosine motifs in the cytosolic domains of integral membrane proteins and the adaptor complex AP2, and a dominant-negative mutant of the adaptor protein AP180. Both these agents inhibited the copper-induced endocytosis of PrP C . Copper caused PrP C to move laterally out of detergent-insoluble lipid rafts into detergent-soluble regions of the plasma membrane. Using mutants of PrP C that lack either the octapeptide repeats or the N-terminal polybasic region, and a construct with a transmembrane anchor, we show that copper binding to the octapeptide repeats promotes dissociation of PrP C from lipid rafts, whereas the N-terminal polybasic region mediates its interaction with a transmembrane adaptor protein that engages the clathrin endocytic machinery. Our results provide an experimental basis for reconciling the apparently contradictory observations that the prion protein undergoes clathrin-dependent endocytosis despite being localised in lipid rafts. In addition, we have been able to assign distinct functions in the endocytic process to separate regions of the protein.

Non-classical protein release independent of the ER-Golgi pathway has been reported for an increasing number of proteins lacking an N-terminal signal sequence. The export of FGF1 and IL-1α, two pro-angiogenic polypeptides, provides two such examples. In both cases, export is based on the Cu 2+ -dependent formation of multiprotein complexes containing the S100A13 protein and might involve translocation of the protein across the membrane as a `molten globule'. FGF1 and IL-1α are involved in pathological processes such as restenosis and tumor formation. Inhibition of their export by Cu 2+ chelators is thus an effective strategy for treatment of several diseases.

Copper is involved in the promotion of angiogenic and inflammatory events in vivo and, although recent clinical data has demonstrated the potential of Cu 2+ chelators for the treatment of cancer in man, the mechanism for this activity remains unknown. We have previously demonstrated that the signal peptide-less angiogenic polypeptide, FGF1, uses intracellular Cu 2+ to facilitate the formation of a multiprotein aggregate that enables the release of FGF1 in response to stress and that the expression of the precursor form but not the mature form of IL-1α represses the stress-induced export of FGF1 from NIH 3T3 cells. We report here that IL-1α is a Cu 2+ -binding protein and human U937 cells, like NIH 3T3 cells, release IL-1α in response to temperature stress in a Cu 2+ -dependent manner. We also report that the stress-induced export of IL-1α involves the intracellular association with the Cu 2+ -binding protein, S100A13. In addition, the expression of a S100A13 mutant lacking a sequence novel to this gene product functions as a dominant-negative repressor of IL-1α release, whereas the expression of wild-type S100A13 functions to eliminate the requirement for stress-induced transcription. Lastly, we present biophysical evidence that IL-1α may be endowed with molten globule character, which may facilitate its release through the plasma membrane. Because Cu 2+ chelation also represses the release of FGF1, the ability of Cu 2+ chelators to potentially serve as effective clinical anti-cancer agents may be related to their ability to limit the export of these proinflammatory and angiogenic signal peptide-less polypeptides into the extracellular compartment.