Intracellular Localization Of Atp7b

To accomplish their function, cellular proteins have to locate properly at specific intracellular sites. New information concerning the intracellular localization of ATP7B has emerged from studies with immunofluorescence and subcellular fractionation. These studies reveal that ATP7B is localized to the trans-Golgi network and to post-Golgi compartments, the latter likely being endosomes, in human hepatoblastoma cells and rat hepatocytes under basal conditions (12,30,34-36). Other studies suggest the presence of ATP7B in mitochondria and at the canalicular pole of hepatocytes (37,38). These results remain controversial and further studies are needed to resolve uncertainties.

ATP7B is known to behave differently depending on intracellular levels of copper. Exposure to copper induces a rapid movement of ATP7B from the Golgi compartments to as yet undefined post-Golgi vesicles (12). This phenomenon is reversible and does not require de novo synthesis of protein. Rapid trafficking of ATP7A from the trans-Golgi network to the plasma membrane induced by copper has been reported (39). Evidently, recycling of ATP7B between the trans-Golgi network and the post-Golgi compartments is regulated by the copper concentration, favoring the efficient discharge of cellular copper into the excretory compartments, yet the precise mechanism of the unique copper-induced protein trafficking remain to be determined.

5. BIOLOGICAL FUNCTION OF ATP7B

The properties of ATP7B described earlier strongly suggest that the protein plays a significant role in the intracellular transport of hepatic copper. The major export pathways for copper from hepatocytes consist of the secretion into blood following its incorporation into ceruloplasmin and the excretion into bile. The reconstitution of copper transport by the introduction of ATP7B cDNA into a yeast strain lacking Ccc2 and the subsequent reconstitution of copper incorporation into the copper protein Fet3 provide supporting evidence for the role of ATP7B in copper transport and ceruloplas-min biosynthesis (12).

To further investigate the role of ATP7B in copper transport in vivo, ATP7B cDNA was introduced into the LEC rat by adenoviral-mediated gene delivery (13). Transgene expression was observed in the liver and ATP7B was found to localize to the Golgi apparatus of hepatocytes. Secretion of holoCPN, the copper-bound form of the protein, was detected after viral infusion. These data indicate that ATP7B functions in copper export coupled with CPN synthesis and that the Golgi apparatus is the likely site for the incorporation of copper into CPN (40,41). In addition, the elevated levels of copper content in the hepatic lysosomal fractions and the increased biliary excretion of copper were observed after the introduction of ATP7B, suggesting that ATP7B participates in the excretory pathway of copper, involving a vesicular transport of copper to lysosomes for delivery to the apical, canalicular membrane (14,42). Together, these data provide the notion that ATP7B translocates copper into the Golgi compartments and into both the secretory and the excretory pathways for copper, thereby regulating the homeostasis of copper in liver cells.

Mutational analyses also support the requirement of ATP7B in hepatic copper transport, which explains the pathogenesis of Wilson's disease. Mutations of H1069Q in the SEHPL motif or N1270S in the MXGDGXNDXP sequence, reported in the patients, were shown to lose their copper-transport function by in vitro studies (12,15,43), leading to a disturbance of intracellular copper transport in the disease. In addition, mutations introduced into the critical sites in ATP7B, such as the DKTGT/S motif and the CPC motif, were also found to alter the copper-transport function of ATP7B (12,15). Regarding the copper-binding domains, although these domains are essential for copper transport, the sixth domain is sufficient to manifest the function (15,33). These observations imply that intact ATP7B is necessary for regulating copper homeostasis in the liver.

In the intracellular trafficking of copper, ATP7B may act as a gate that controls the copper flow through the secretory and excretory pathways in liver.

6. COPPER TRANSPORT AND ATP7B

Liver is the main site of copper metabolism in the body, because mobilization of copper into the systemic circulation and into bile take place via hepatocytes.

Only half of dietary copper ingested, 0.8-1.5 mg/d, is absorbed from the upper intestine (1). This process is probably mediated by hCTR1, identified by searching for proteins with homology to genes products associated with copper uptake in yeast (44). The yeast Ctr1, identified through its effects on iron metabolism, was found to be necessary for the high-affinity copper transport (45), and hCTR1 indeed replaced the function of Ctr1 in yeast (44). hCTR1 is predicted to be a membrane-associated

Fig. 2. Model of copper transport associated with ATP7B in hepatocyte. In the portal blood flow, copper is mostly bound with albumin via its N-terminal tripeptide (Asp-Ala-His) and then taken up into hepatocytes as a copper-histidine complex. This hepatic uptake is mediated by hCTRl. Subsequently, the cytosolic copper forms a complex with glutathione, metallothionein, or the copper chaperone. The copper chaperones hCOX17 and hCCS deliver copper to cytochrome-c oxidase in the mitochondria and the cytoplasmic copper/zinc superoxide dismutase, respectively. HAH1 distributes copper to ATP7B that resides in the trans-Golgi (GA) or in the post-Golgi compartments (post-GA), likely endosomes, and then copper is transported into these organelles by ATP7B. In the secretory pathway, after ceruloplasmin (CPN) incorporates copper at the Golgi, holoCPN (Cu-CPN) is secreted into blood through secretory vesicles (SV). In the excretory pathway, copper in the trans-Golgi or in the post-Golgi is excreted into bile through lysosomes (Lys). The recycling of ATP7B between the trans-Golgi and the post-Golgi is regulated by intracellular copper concentration. Under the basal condition, ATP7B resides in the trans-Golgi; however, it rapidly moves to the post-Golgi compartments when the level of intracellular copper is elevated.

Fig. 2. Model of copper transport associated with ATP7B in hepatocyte. In the portal blood flow, copper is mostly bound with albumin via its N-terminal tripeptide (Asp-Ala-His) and then taken up into hepatocytes as a copper-histidine complex. This hepatic uptake is mediated by hCTRl. Subsequently, the cytosolic copper forms a complex with glutathione, metallothionein, or the copper chaperone. The copper chaperones hCOX17 and hCCS deliver copper to cytochrome-c oxidase in the mitochondria and the cytoplasmic copper/zinc superoxide dismutase, respectively. HAH1 distributes copper to ATP7B that resides in the trans-Golgi (GA) or in the post-Golgi compartments (post-GA), likely endosomes, and then copper is transported into these organelles by ATP7B. In the secretory pathway, after ceruloplasmin (CPN) incorporates copper at the Golgi, holoCPN (Cu-CPN) is secreted into blood through secretory vesicles (SV). In the excretory pathway, copper in the trans-Golgi or in the post-Golgi is excreted into bile through lysosomes (Lys). The recycling of ATP7B between the trans-Golgi and the post-Golgi is regulated by intracellular copper concentration. Under the basal condition, ATP7B resides in the trans-Golgi; however, it rapidly moves to the post-Golgi compartments when the level of intracellular copper is elevated.

transporter protein detectable in all human tissues, including intestine and liver (44). Normally, the intestinal cells release copper under regulation of ATP7A, except in patients with Menkes disease in whom intestinal cells entrap copper because of defective ATP7A (9). The released copper is mostly bound with albumin via its N-terminal tripeptide (Asp-Ala-His) in the portal blood, and then taken up into hepatocytes as a copper-histidine complex (9). This hepatic uptake is also mediated by hCTRl. Subsequently, the cytosolic copper forms a complex with glutathione, metallothionein, or copper chaperone (1,5). The copper chaperones hCOX17 and hCCS deliver copper to cytochrome-c oxidase in the mitochondria and the cytoplasmic copper/zinc superoxide dismutase, respectively (4,5). Based on structural and functional similarities to the yeast Atxl, we assume that HAH1 distributes copper to ATP7B (46). Then, copper is transported into the export pathways by ATP7B at the Golgi or post-Golgi compartments. In the export pathways, copper is secreted into blood, following its incorpora-

tion into CPN, and is excreted into bile through lysosomes. The latter process is more important for maintaining the hepatic concentration of copper, because the bile mobilizes three-quarters of the absorbed copper per day (1). A unified model of hepatic copper transport is illustrated in Fig. 2.

0 0

Post a comment