Functional analysis of the sheep Wilson disease protein (sATP7B) in CHO cells

doi:10.1078/0171-9335-00165

European Journal of Cell Biology

Volume 80, Issue 5, May 2001, Pages 349-357

https://doi.org/10.1078/0171-9335-00165 Get rights and content

Summary

In this study we investigated the function of the sheep orthologue of ATP7B (sATP7B), the protein affected in the human copper toxicosis disorder Wilson disease. Two forms of sATP7B are found in the sheep, a ‘normal’ form and one with an alternate N terminus, both of which were expressed in CHO-K1 cells. Cells expressing either form of sATP7B were more resistant to copper than the parental CHO-K1 cells. Subcellular localisation studies showed that both forms of sATP7B were similarly located in the trans-Golgi network (TGN). When the extracellular copper concentration was increased, each form of sATP7B redistributed to a punctate, vesicular compartment that extended throughout the cytoplasm. Both forms of sATP7B recycled to the perinuclear location within one hour when the cells were subsequently incubated in basal medium. After treatment of cells with bafilomycin A₁ sATP7B accumulated in cytoplasmic vesicles, implying that ATP7B continuously recycles via the endocytic pathway. These results suggest that both forms of sATP7B are functional copper-transport proteins and that the intracellular location and trafficking of the sheep protein within the cell also appears normal.

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Cellular multitasking: The dual role of human Cu-ATPases in cofactor delivery and intracellular copper balance
2008, Archives of Biochemistry and Biophysics
Citation Excerpt :
In addition, alternative splicing of exon 1, which encodes the N-terminal extension in ATP7B, further increases diversity of this region. In sheep (Ovis), two forms of ATP7B generated by alternate splicing of exon 1 have been identified [54,55]. The exon 1 of a shorter form, sATP7B, encodes an 18-amino-acid sequence, while in the longer form lATP7B, it is replaced by a 79-amino-acid sequence [55].
The human copper-transporting ATPases (Cu-ATPases) are essential for dietary copper uptake, normal development and function of the CNS, and regulation of copper homeostasis in the body. In a cell, Cu-ATPases maintain the intracellular concentration of copper by transporting copper into intracellular exocytic vesicles. In addition, these P-type ATPases mediate delivery of copper to copper-dependent enzymes in the secretory pathway and in specialized cell compartments such as secretory granules or melanosomes. The multiple functions of human Cu-ATPase necessitate complex regulation of these transporters that is mediated through the presence of regulatory domains in their structure, posttranslational modification and intracellular trafficking, as well as interactions with the copper chaperone Atox1 and other regulatory molecules. In this review, we summarize the current information on the function and regulatory mechanisms acting on human Cu-ATPases ATP7A and ATP7B. Brief comparison with the Cu-ATPase orthologs from other species is included.
ATP7B Copper-Regulated Traffic and Association With the Tight Junctions: Copper Excretion Into the Bile
2008, Gastroenterology
Citation Excerpt :
Can 10 cells forming long-branched BC were incubated with either BCS or CuCl2.13 Immunohistochemical staining showed that copper induced the vectorial translocation of ATP7B from the TGN2 (Supplementary Figure 1; see supplemental material online at www.gastrojournal.org) to the bile canalicular membrane (Figure 2, compare panels A, B). Furthermore, upon addition of 50 μmol/L CuCl2, release of ATP7B from the Golgi was rapid, and within 10 minutes, the transporter was partly relocated to large vesicles scattered throughout the cytoplasm (Supplementary Figure 2; see supplemental material online at www.gastrojournal.org).
Background & Aims: The copper transporter ATP7B plays a central role in the elimination of excess copper by the liver into the bile, yet the site of its action remains controversial. The studies reported here examine the correspondence between the site of ATP7B action and distribution and the pathways of copper disposal by the liver. Methods: Microscopy and cell fractionation studies of polarized Can 10 cells forming long-branched bile canaliculi have been used to study the cellular distribution of ATP7B. Copper excretion into the bile was studied in perfused rat liver. Results: Copper excess provokes a massive download of the ATP7B retained in the trans-Golgi network into the bile canalicular membrane. Furthermore, a stable ATP7B pool is localized to the tight junctions that seal the bile canaliculi. The profile of Cu⁶⁴ excretion into the bile by isolated rat livers perfused under one-pass conditions provides evidence of copper excretion by 2 separate mechanisms, transcytosis across the hepatocyte and paracellular transport throughout the tight junctions. Conclusions: Whereas the ATP7B retained in the trans-Golgi-network is massively translocated to the bile canalicular membrane in response to increased copper levels, a pool of ATP7B associated with the tight junctions remains stable. In situ studies indicate that copper is excreted into the bile by 2 separate pathways. The results are discussed in the frame of the normal and impeded excretion of copper into the bile.
Trafficking of the copper-ATPases, ATP7A and ATP7B: Role in copper homeostasis
2007, Archives of Biochemistry and Biophysics
Citation Excerpt :
The 79 amino acid N-terminal region represents a novel sequence with no similarity to other database sequences. Both forms are functional as they both confer copper resistance to CHO-K1 cells, undergo copper-induced trafficking in these cells [57], and could correct the copper transport defect of Menkes patient fibroblast cells [58]. Many of the sites of ATP7A function are epithelial in origin, for example, the intestinal enterocytes, the mammary gland epithelium, placental trophoblasts and the proximal tubule cells of the kidney.
Copper is essential for human health and copper imbalance is a key factor in the aetiology and pathology of several neurodegenerative diseases. The copper-transporting P-type ATPases, ATP7A and ATP7B are key molecules required for the regulation and maintenance of mammalian copper homeostasis. Their absence or malfunction leads to the genetically inherited disorders, Menkes and Wilson diseases, respectively. These proteins have a dual role in cells, namely to provide copper to essential cuproenzymes and to mediate the excretion of excess intracellular copper. A unique feature of ATP7A and ATP7B that is integral to these functions is their ability to sense and respond to intracellular copper levels, the latter manifested through their copper-regulated trafficking from the transGolgi network to the appropriate cellular membrane domain (basolateral or apical, respectively) to eliminate excess copper from the cell. Research over the last decade has yielded significant insight into the enzymatic properties and cell biology of the copper-ATPases. With recent advances in elucidating their localization and trafficking in human and animal tissues in response to physiological stimuli, we are progressing rapidly towards an integrated understanding of their physiological significance at the level of the whole animal. This knowledge in turn is helping to clarify the biochemical and cellular basis not only for the phenotypes conferred by individual Menkes and Wilson disease patient mutations, but also for the clinical variability of phenotypes associated with each of these diseases. Importantly, this information is also providing a rational basis for the applicability and appropriateness of certain diagnostic markers and therapeutic regimes. This overview will provide an update on the current state of our understanding of the localization and trafficking properties of the copper-ATPases in cells and tissues, the molecular signals and posttranslational interactions that govern their trafficking activities, and the cellular basis for the clinical phenotypes associated with disease-causing mutations.
Biochemical basis of regulation of human copper-transporting ATPases
2007, Archives of Biochemistry and Biophysics
Citation Excerpt :
Interestingly, the sheep orthologue of ATP7B (sATP7B) has two hepatic forms: one “human-like” and another with the alternate N-terminus, which represents longer extension. In non-polarized CHO-K1 cells both forms of sATP7B are similarly located in the TGN and both redistribute to a vesicular compartment in response to elevated copper [73]. How different N-terminal extensions modulate localization and trafficking of sATP7B in polarized cells has not yet been determined.
Copper is essential for cell metabolism as a cofactor of key metabolic enzymes. The biosynthetic incorporation of copper into secreted and plasma membrane-bound proteins requires activity of the copper-transporting ATPases (Cu-ATPases) ATP7A and ATP7B. The Cu-ATPases also export excess copper from the cell and thus critically contribute to the homeostatic control of copper. The trafficking of Cu-ATPases from the trans-Golgi network to endocytic vesicles in response to various signals allows for the balance between the biosynthetic and copper exporting functions of these transporters. Although significant progress has been made towards understanding the biochemical characteristics of human Cu-ATPase, the mechanisms that control their function and intracellular localization remain poorly understood. In this review, we summarize current information on structural features and functional properties of ATP7A and ATP7B. We also describe sequence motifs unique for each Cu-ATPase and speculate about their role in regulating ATP7A and ATP7B activity and trafficking.
ATP7B mediates vesicular sequestration of copper: Insight into biliary copper excretion
2006, Gastroenterology
Background & Aims: The Wilson protein (ATP7B) regulates levels of systemic copper by excreting excess copper into bile. It is not clear whether ATP7B translocates excess intrahepatic copper directly across the canalicular membrane or sequesters this copper into exocytic vesicles, which subsequently fuse with canalicular membrane to expel their contents into bile. The aim of this study was to clarify the mechanism underlying ATP7B-mediated copper detoxification by investigating endogenous ATP7B localization in the HepG2 hepatoma cell line and its ability to mediate vesicular sequestration of excess intracellular copper. Methods: Immunofluorescence microscopy was used to investigate the effect of copper concentration on the localization of endogenous ATP7B in HepG2 cells. Copper accumulation studies to determine whether ATP7B can mediate vesicular sequestration of excess intracellular copper were performed using Chinese hamster ovary cells that exogenously expressed wild-type and mutant ATP7B proteins. Results: In HepG2 cells, elevated copper levels stimulated trafficking of ATP7B to pericanalicular vesicles and not to the canalicular membrane as previously reported. Mutation of an endocytic retrieval signal in ATP7B caused the protein to constitutively localize to vesicles and not to the plasma membrane, suggesting that a vesicular compartment(s) is the final trafficking destination for ATP7B. Expression of wild-type and mutant ATP7B caused Chinese hamster ovary cells to accumulate copper in vesicles, which subsequently undergo exocytosis, releasing copper across the plasma membrane. Conclusions: This report provides compelling evidence that the primary mechanism of biliary copper excretion involves ATP7B-mediated vesicular sequestration of copper rather than direct copper translocation across the canalicular membrane.
Correction of the copper transport defect of Menkes patient fibroblasts by expression of two forms of the sheep Wilson ATPase
2002, Biochimica et Biophysica Acta - Molecular Basis of Disease
The Wilson disease (WD) protein (ATP7B) is a copper-transporting P-type ATPase that is responsible for the efflux of hepatic copper into the bile, a process that is essential for copper homeostasis in mammals. Compared with other mammals, sheep have a variant copper phenotype and do not efficiently excrete copper via the bile, often resulting in excessive copper accumulation in the liver. To investigate the function of sheep ATP7B and its potential role in the copper-accumulation phenotype, cDNAs encoding the two forms of ovine ATP7B were transfected into immortalised fibroblast cell lines derived from a Menkes disease patient and a normal control. Both forms of ATP7B were able to correct the copper-retention phenotype of the Menkes cell line, demonstrating each to be functional copper-transporting molecules and suggesting that the accumulation of copper in the sheep liver is not due to a defect in the copper transport function of either form of sATP7B.

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Functional analysis of the sheep Wilson disease protein (sATP7B) in CHO cells

Summary

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Gastroenterology

J. Biol. Chem.

FEBS Lett.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

TGN38 is maintained in the trans-Golgi network by a tyrosine-containing motif in the cytoplasmic domain

EMBO. J.

Null mutation of the murine ATP7B (Wilson disease) gene results in intracellular copper accumulation and late-onset hepatic nodular transformation

Hum. Mol. Genet.

The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene

Nature Genet.

Gene amplification of the Menkes (MNK; ATP7A) P-type ATPase gene of CHO cells is associated with copper resistance and enhanced copper efflux

Hum. Mol. Genet.

Retrieval of TGN proteins from the cell surface requires endosomal acidification

EMBO J.

Adenosine triphosphate-dependent copper transport in isolated rat liver plasma membranes

J. Clin. Invest.

Copper-dependent trafficking of Wilson disease mutant ATP7B proteins

Hum. Mol. Genet.

Identification of a di-leucine motif within the C terminus domain of the Menkes disease protein that mediates endocytosis from the plasma membrane

J. Cell Sci.

Antibodies, A Laboratory Manual

Animal models of human disease: Wilson's disease

Comp. Path. Bull.

Experimental chronic copper toxicity in sheep. Histological and histochemical changes during the development of lesion s in the liver

Res. Vet. Sci.