Trafficking of the Menkes copper transporter ATP7A is regulated by clathrin-, AP-2–, AP-1–, and Rab22-dependent steps

The transporter ATP7A mediates systemic copper absorption and provides cuproenzymes in the trans-Golgi network (TGN) with copper. To regulate metal homeostasis, ATP7A constitutively cycles between the TGN and plasma membrane (PM). ATP7A trafficking to the PM is elevated in response to increased copper load and is reversed when copper concentrations are lowered. Molecular mechanisms underlying this trafficking are poorly understood. We assess the role of clathrin, adaptor complexes, lipid rafts, and Rab22a in an attempt to decipher the regulatory proteins involved in ATP7A cycling. While RNA interference (RNAi)–mediated depletion of caveolin 1/2 or flotillin had no effect on ATP7A localization, clathrin heavy chain depletion or expression of AP180 dominant-negative mutant not only disrupted clathrin-regulated pathways, but also blocked PM-to-TGN internalization of ATP7A. Depletion of the μ subunits of either adaptor protein-2 (AP-2) or AP-1 using RNAi further provides evidence that both clathrin adaptors are important for trafficking of ATP7A from the PM to the TGN. Expression of the GTP-locked Rab22a_Q64L mutant caused fragmentation of TGN membrane domains enriched for ATP7A. These appear to be a subdomain of the mammalian TGN, showing only partial overlap with the TGN marker golgin-97. Of importance, ATP7A remained in the Rab22a_Q64L-generated structures after copper treatment and washout, suggesting that forward trafficking out of this compartment was blocked. This study provides evidence that multiple membrane-associated factors, including clathrin, AP-2, AP-1, and Rab22, are regulators of ATP7A trafficking.     

PLEKHA5, PLEKHA6, and PLEKHA7 bind to PDZD11 to target the Menkes ATPase ATP7A to the cell periphery and regulate copper homeostasis

Copper homeostasis is crucial for cellular physiology and development, and its dysregulation leads to disease. The Menkes ATPase ATP7A plays a key role in copper efflux, by trafficking from the Golgi to the plasma membrane upon cell exposure to elevated copper, but the mechanisms that target ATP7A to the cell periphery are poorly understood. PDZD11 interacts with the C-terminus of ATP7A, which contains sequences involved in ATP7A trafficking, but the role of PDZD11 in ATP7A localization is unknown. Here we identify PLEKHA5 and PLEKHA6 as new interactors of PDZD11 that bind to the PDZD11 N-terminus through their WW domains similarly to the junctional protein PLEKHA7. Using CRISPR-KO kidney epithelial cells, we show by immunofluorescence microscopy that WW-PLEKHAs (PLEKHA5, PLEKHA6, PLEKHA7) recruit PDZD11 to distinct plasma membrane localizations and that they are required for the efficient anterograde targeting of ATP7A to the cell periphery in elevated copper conditions. Pull-down experiments show that WW-PLEKHAs promote PDZD11 interaction with the C-terminus of ATP7A. However, WW-PLEKHAs and PDZD11 are not necessary for ATP7A Golgi localization in basal copper, ATP7A copper-induced exit from the Golgi, and ATP7A retrograde trafficking to the Golgi. Finally, measuring bioavailable and total cellular copper, metallothionein-1 expression, and cell viability shows that WW-PLEKHAs and PDZD11 are required for maintaining low intracellular copper levels when cells are exposed to elevated copper. These data indicate that WW-PLEKHAs-PDZD11 complexes regulate the localization and function of ATP7A to promote copper extrusion in elevated copper.     

Cell-Specific Trafficking Suggests a new role for Renal ATP7B in the Intracellular Copper Storage

Human Cu-ATPases ATP7A and ATP7B maintain copper homeostasis through regulated trafficking between intracellular compartments. Inactivation of these transporters causes Menkes disease and Wilson disease, respectively. In Menkes disease, copper accumulates in kidneys and causes tubular damage, indicating that the renal ATP7B does not compensate for the loss of ATP7A function. We show that this is likely due to a kidney-specific regulation of ATP7B. Unlike ATP7A (or hepatic ATP7B) which traffics from the TGN to export copper, renal ATP7B does not traffic and therefore is unlikely to mediate copper export. The lack of ATP7B trafficking is not on account of the loss of a kinase-mediated phosphorylation or simultaneous presence of ATP7A in renal cells. Rather, the renal ATP7B appears 2–3 kDa smaller than hepatic ATP7B. Recombinant ATP7B expressed in renal cells is similar to hepatic protein in size and trafficking. The analysis of ATP7B mRNA revealed a complex behavior of exon 1 upon amplification, suggesting that it could be inefficiently translated. Recombinant ATP7B lacking exon 1 traffics differently in renal and hepatic cells, but does not fully recapitulate the endogenous phenotype. We discuss factors that may contribute to cell-specific behavior of ATP7B and propose a role for renal ATP7B in intracellular copper storage.     

COX19 mediates the transduction of a mitochondrial redox signal from SCO1 that regulates ATP7A-mediated cellular copper efflux

SCO1 and SCO2 are metallochaperones whose principal function is to add two copper ions to the catalytic core of cytochrome c oxidase (COX). However, affected tissues of SCO1 and SCO2 patients exhibit a combined deficiency in COX activity and total copper content, suggesting additional roles for these proteins in the regulation of cellular copper homeostasis. Here we show that both the redox state of the copper-binding cysteines of SCO1 and the abundance of SCO2 correlate with cellular copper content and that these relationships are perturbed by mutations in SCO1 or SCO2, producing a state of apparent copper overload. The copper deficiency in SCO patient fibroblasts is rescued by knockdown of ATP7A, a trans-Golgi, copper-transporting ATPase that traffics to the plasma membrane during copper overload to promote efflux. To investigate how a signal from SCO1 could be relayed to ATP7A, we examined the abundance and subcellular distribution of several soluble COX assembly factors. We found that COX19 partitions between mitochondria and the cytosol in a copper-dependent manner and that its knockdown partially rescues the copper deficiency in patient cells. These results demonstrate that COX19 is necessary for the transduction of a SCO1-dependent mitochondrial redox signal that regulates ATP7A-mediated cellular copper efflux.     

Glutaredoxin1 protects neuronal cells from copper-induced toxicity

Glutaredoxin1 (GRX1) is a glutathione (GSH)-dependent thiol oxidoreductase. The GRX1/GSH system is important for the protection of proteins from oxidative damage and in the regulation of protein function. Previously we demonstrated that GRX1/GSH regulates the activity of the essential copper-transporting P_1B-Type ATPases (ATP7A, ATP7B) in a copper-responsive manner. It has also been established that GRX1 binds copper with high affinity and regulates the redox chemistry of the metallochaperone ATOX1, which delivers copper to the copper-ATPases. In this study, to further define the role of GRX1 in copper homeostasis, we examined the effects of manipulating GRX1 expression on copper homeostasis and cell survival in mouse embryonic fibroblasts and in human neuroblastoma cells (SH-SY5Y). GRX1 knockout led to cellular copper retention (especially when cultured with elevated copper) and reduced copper tolerance, while in GRX1-overexpressing cells challenged with elevated copper, there was a reduction in both intracellular copper levels and copper-induced reactive oxygen species, coupled with enhanced cell proliferation. These effects are consistent with a role for GRX1 in regulating ATP7A-mediated copper export, and further support a new function for GRX1 in neuronal copper homeostasis and in protection from copper-mediated oxidative injury.     

Activation of ADP-ribosylation factor regulates biogenesis of the ATP7A-containing trans-Golgi network compartment and its Cu-induced trafficking

ATP7A (MNK) regulates copper homeostasis by translocating from a compartment localized within the trans-Golgi network to the plasma membrane (PM) in response to increased copper load. The mechanisms that regulate the biogenesis of the MNK compartment and the trafficking of MNK are unclear. Here we show that the architecture of the MNK compartment is linked to the structure of the Golgi ribbon. Depletion of p115 tethering factor, which causes fragmentation of the Golgi ribbon, also disrupts the MNK compartment. In p115-depleted cells, MNK localizes to punctate structures that pattern on Golgi ministacks dispersed throughout the cell. Despite altered localization MNK trafficking still occurs, and MNK relocates from and returns to the fragmented compartment in response to copper. We further show that the biogenesis of the MNK compartment requires activation of ADP-ribosylation factor (Arf)1 GTPase, shown previously to facilitate the biogenesis of the Golgi ribbon. Activation of cellular Arf1 is prevented by 1) expressing an inactive "empty" form of Arf (Arf1/N126I), 2) expressing an inactive form of GBF1 (GBF1/E794K), guanine nucleotide exchange factor for Arf1, or 3) treating cells with brefeldin A, an inhibitor of GBF1 that disrupts MNK into a diffuse pattern. Importantly, preventing Arf activation inhibits copper-responsive trafficking of MNK to the PM. Our findings support a model in which active Arf is essential for the generation of the MNK compartment and for copper-responsive trafficking of MNK from there to the PM. Our findings provide an exciting foundation for identifying Arf1 effectors that facilitate the biogenesis of the MNK compartment and MNK traffic.     

Conservation of copper-transporting P(IB)-type ATPase function

Copper-transporting P(IB)-type ATPases are highly conserved, and while unicellular eukaryotes and invertebrates have only one, a gene duplication has occurred during vertebrate evolution. Copper-induced trafficking of mammalian ATP7A and ATP7B from the trans-Golgi Network towards the plasma membrane is critical for their role in copper homeostasis. In polarized epithelial cells ATP7A and ATP7B traffic towards the basolateral and apical membranes respectively. We examined the localization and function of DmATP7, the single Drosophila melanogaster orthologue, in cultured D. melanogaster and mammalian cells to explore the conservation of P(IB)-type ATPase function. Comparative genomic analysis demonstrated motifs involved in basolateral targeting and retention of ATP7A were conserved in DmATP7, whereas ATP7B targeting motifs were not. DmATP7 expression was able to correct the copper hyper-accumulation phenotype of cultured fibroblasts from a Menkes disease patient expressing a null ATP7A allele. DmATP7 was able to transport copper to the cupro-enzyme tyrosinase and under elevated copper conditions DmATP7 was able to traffic towards the plasma membrane and efflux copper, essentially phenocopying ATP7A. When expressed in polarized Madin-Darby Canine Kidney cells, DmATP7 translocated towards the basolateral membrane when exposed to elevated copper, similar to ATP7A. These results demonstrate DmATP7 is able to functionally compensate for the absence of ATP7A, with important trafficking motifs conserved in these distantly related orthologues.     

Copper-regulated trafficking of the Menkes disease copper ATPase is associated with formation of a phosphorylated catalytic intermediate

The Menkes protein (MNK; ATP7A) is a copper-transporting P-type ATPase that is defective in the copper deficiency disorder, Menkes disease. MNK is localized in the trans-Golgi network and transports copper to enzymes synthesized within secretory compartments. However, in cells exposed to excessive copper, MNK traffics to the plasma membrane where it functions in copper efflux. A conserved feature of all P-type ATPases is the formation of an acyl-phosphate intermediate, which occurs as part of the catalytic cycle during cation transport. In this study we investigated the effect of mutations within conserved catalytic regions of MNK on intracellular localization and trafficking from the trans-Golgi network (TGN). Our findings suggest that mutations that block formation of the phosphorylated catalytic intermediate also prevent copper-induced relocalization of MNK from the TGN. Furthermore, mutations in the phosphatase domain, which resulted in hyperphosphorylation of MNK, caused constitutive trafficking from the TGN to the plasma membrane. A similar effect on trafficking was observed with a phosphatase mutation in the closely related copper ATPase, ATP7B, affected in Wilson disease. These findings suggest that the copper-induced trafficking of the Menkes and Wilson disease copper ATPases is associated with the phosphorylated intermediate that is formed during the catalysis of these pumps. Our findings describe a novel mechanism for regulating the subcellular location of a transport protein involving the recognition of intermediate conformations during catalysis.     

Copper-dependent interaction of dynactin subunit p62 with the N terminus of ATP7B but not ATP7A

The P-type ATPase affected in Wilson disease, ATP7B, is a key liver protein required to regulate and maintain copper homeostasis. When hepatocytes are exposed to elevated copper levels, ATP7B traffics from the trans-Golgi network toward the biliary canalicular membrane to excrete excess copper into bile. The N-terminal region of ATP7B contains six metal-binding sites (MBS), each with the copper-binding motif MXCXXC. These sites are required for the activity and copper-regulated intracellular redistribution of ATP7B. Two proteins are known to interact with the ATP7B N-terminal region: the copper chaperone ATOX1 that delivers copper to ATP7B, and COMMD1 (MURR1) that is potentially involved in vesicular copper sequestration. To identify additional proteins that interact with ATP7B and hence are involved in copper homeostasis, a yeast two-hybrid approach was employed to screen a human liver cDNA library. The dynactin subunit p62 (dynactin 4; DCTN4) was identified as an interacting partner, and this interaction was confirmed by co-immunoprecipitation from mammalian cells. The dynactin complex binds cargo, such as vesicles and organelles, to cytoplasmic dynein for retrograde microtubule-mediated trafficking and could feasibly be involved in the copper-regulated trafficking of ATP7B. The ATP7B/p62 interaction required copper, the metal-binding CXXC motifs, and the region between MBS 4 and MBS 6 of ATP7B. The p62 subunit did not interact with the related copper ATPase, ATP7A. We propose that the ATP7B interaction with p62 is a key component of the copper-induced trafficking pathway that delivers ATP7B to subapical vesicles of hepatocytes for the removal of excess copper into bile.     

EFA6A,an Exchange Factor for Arf6, Regulates NGF-Dependent TrkA Recycling From Early Endosomes and Neurite Outgrowth in PC12 Cells

Endosomal trafficking of TrkA is a critical process for nerve growth factor (NGF)-dependent neuronal cell survival and differentiation. The small GTPase ADP-ribosylation factor 6 (Arf6) is implicated in NGF-dependent processes in PC12 cells through endosomal trafficking and actin cytoskeleton reorganization. However, the regulatory mechanism for Arf6 in NGF signaling is largely unknown. In this study, we demonstrated that EFA6A, an Arf6-specific guanine nucleotide exchange factor, was abundantly expressed in PC12 cells and that knockdown of EFA6A significantly inhibited NGF-dependent Arf6 activation, TrkA recycling from early endosomes to the cell surface, prolonged ERK1/2 phosphorylation, and neurite outgrowth. We also demonstrated that EFA6A forms a protein complex with TrkA through its N-terminal region, thereby enhancing its catalytic activity for Arf6. Similarly, we demonstrated that EFA6A forms a protein complex with TrkA in cultured dorsal root ganglion (DRG) neurons. Furthermore, cultured DRG neurons from EFA6A knockout mice exhibited disturbed NGF-dependent TrkA trafficking compared with wild-type neurons. These findings provide the first evidence for EFA6A as a key regulator of NGF-dependent TrkA trafficking and signaling.     

Clusterin (apolipoprotein J), a molecular chaperone that facilitates degradation of the copper-ATPases ATP7A and ATP7B

The copper-transporting P(1B)-type ATPases (Cu-ATPases) ATP7A and ATP7B are key regulators of physiological copper levels. They function to maintain intracellular copper homeostasis by delivering copper to secretory compartments and by trafficking toward the cell periphery to export excess copper. Mutations in the genes encoding ATP7A and ATP7B lead to copper deficiency and toxicity disorders, Menkes and Wilson diseases, respectively. This report describes the interaction between the Cu-ATPases and clusterin and demonstrates a chaperone-like role for clusterin in facilitating their degradation. Clusterin interacted with both ATP7A and ATP7B in mammalian cells. This interaction increased under conditions of oxidative stress and with mutations in ATP7B that led to its misfolding and mislocalization. A Wilson disease patient mutation (G85V) led to enhanced ATP7B turnover, which was further exacerbated when cells overexpressed clusterin. We demonstrated that clusterin-facilitated degradation of mutant ATP7B is likely to involve the lysosomal pathway. The knockdown and overexpression of clusterin increased and decreased, respectively, the Cu-ATPase-mediated copper export capacity of cells. These results highlight a new role for intracellular clusterin in mediating Cu-ATPase quality control and hence in the normal maintenance of copper homeostasis, and in promoting cell survival in the context of disease. Based on our findings, it is possible that variations in clusterin expression and function could contribute to the variable clinical expression of Menkes and Wilson diseases.     

Molecular Events Initiating Exit of a Copper-transporting ATPase ATP7B from the Trans-Golgi Network

The copper-transporting ATPase ATP7B has a dual intracellular localization: the trans-Golgi network (TGN) and cytosolic vesicles. Changes in copper levels, kinase-mediated phosphorylation, and mutations associated with Wilson disease alter the steady-state distribution of ATP7B between these compartments. To identify a primary molecular event that triggers ATP7B exit from the TGN, we characterized the folding, activity, and trafficking of the ATP7B variants with mutations within the regulatory N-terminal domain (N-ATP7B). We found that structural changes disrupting the inter-domain contacts facilitate ATP7B exit from the TGN. Mutating Ser-340/341 in the N-ATP7B individually or together to Ala, Gly, Thr, or Asp produced active protein and shifted the steady-state localization of ATP7B to vesicles, independently of copper levels. The Ser340/341G mutant had a lower kinase-mediated phosphorylation under basal conditions and no copper-dependent phosphorylation. Thus, negative charges introduced by copper-dependent phosphorylation are not obligatory for ATP7B trafficking from the TGN. The Ser340/341A mutation did not alter the overall fold of N-ATP7B, but significantly decreased interactions with the nucleotide-binding domain, mimicking consequences of copper binding to N-ATP7B. We propose that structural changes that specifically alter the inter-domain contacts initiate exit of ATP7B from the TGN, whereas increased phosphorylation may be needed to maintain an open interface between the domains.     

Critical roles for the COOH terminus of the Cu-ATPase ATP7B in protein stability, trans-Golgi network retention, copper sensing, and retrograde trafficking

ATP7A and ATP7B are copper-transporting P-type ATPases that are essential to eukaryotic copper homeostasis and must traffic between intracellular compartments to carry out their functions. Previously, we identified a nine-amino acid sequence (F37-E45) in the NH(2) terminus of ATP7B that is required to retain the protein in the Golgi when copper levels are low and target it apically in polarized hepatic cells when copper levels rise. To understand further the mechanisms regulating the intracellular dynamics of ATP7B, using multiple functional assays, we characterized the protein phenotypes of 10 engineered and Wilson disease-associated mutations in the ATP7B COOH terminus in polarized hepatic cells and fibroblasts. We also examined the behavior of a chimera between ATP7B and ATP7A. Our results clearly demonstrate the importance of the COOH terminus of ATP7B in the protein's copper-responsive apical trafficking. L1373 at the end of transmembrane domain 8 is required for protein stability and Golgi retention in low copper, the trileucine motif (L1454-L1456) is required for retrograde trafficking, and the COOH terminus of ATP7B exhibits a higher sensitivity to copper than does ATP7A. Importantly, our results demonstrating that four Wilson disease-associated missense mutations behaved in a wild-type manner in all our assays, together with current information in the literature, raise the possibility that several may not be disease-causing mutations.     

Comparative features of copper ATPases ATP7A and ATP7B heterologously expressed in COS-1 cells

ATP7A and ATP7B are P-type ATPases required for copper homeostasis and involved in the etiology of Menkes and Wilson diseases. We used heterologous expression of ATP7A or ATP7B in COS-1 cells infected with adenovirus vectors to characterize differential features pertinent to each protein expressed in the same mammalian cell type, rather than to extrinsic factors related to different cells sustaining expression. Electrophoretic analysis of the expressed protein, before and after purification, prior or subsequent to treatment with endoglycosidase, and evidenced by protein or glycoprotein staining as well as Western blotting, indicates that the ATP7A protein is glycosylated while ATP7B is not. This is consistent with the prevalence of glycosylation motifs in the ATP7A sequence, and not in ATP7B. ATP7A and ATP7B undergo copper-dependent phosphorylation by utilization of ATP, forming equal levels of an "alkali labile" phosphoenzyme intermediate that undergoes similar catalytic (P-type ATPase) turnover in both enzymes. In addition, incubation with ATP yields an "alkali stable" phosphoprotein fraction, attributed to phosphorylation of serines. Alkali stable phosphorylation occurs at lower levels in ATP7A, consistent with a different distribution of serines in the amino acid sequence. Immunostaining of COS-1 cells sustaining heterologous expression shows initial association of both ATP7A and ATP7B with Golgi and the trans-Golgi network. However, in the presence of added copper, ATP7A undergoes prevalent association with the plasma membrane while ATP7B exhibits intense trafficking with cytosolic vesicles. Glycosylation of ATP7A and phosphorylation of ATP7B apparently contribute to their different trafficking and membrane association when expressed in the same cell type.     

Involvement of protein kinase D in expression and trafficking of ATP7B (copper ATPase)

ATP7B is a P-type ATPase involved in copper transport and homeostasis. In experiments with microsomes isolated from COS-1 cells or HepG2 hepatocytes sustaining ATP7B heterologous expression, we found that ATP7B utilization of ATP includes autophosphorylation of an aspartyl residue serving as ATPase catalytic intermediate as well as phosphorylation of serine residues by protein kinase D (PKD). The latter was abolished by specific PKD inhibition with CID755673. The presence of PKD protein in the microsomal fraction was demonstrated by Western blotting. PKD is a serine/threonine kinase that associates with the trans-Golgi network, regulating fission of transport carriers destined to the cell surface. Parallel studies on cultured cells showed that nascent WT ATP7B transits to the Golgi complex where it undergoes serine phosphorylation by PKD. Misfolded ATP7B protein (especially if subjected to deletions) underwent proteasome-mediated degradation, which provides effective quality control. Inhibition of proteasome-mediated degradation with MG132 yielded additional, but nonfunctional protein. On the other hand, serine phosphorylation protected WT ATP7B from degradation. Protection was enhanced by PKD activation with phorbol esters and limited by PKD inhibition with CID75673. As a final step, phosphorylated ATP7B was transferred from the Golgi complex to cytosolic trafficking vesicles. Phosphorylation and trafficking were completely prevented by mutations of critical copper binding sites, demonstrating copper dependence of both PKD-assisted phosphorylation and trafficking. ATP7B trafficking was markedly reduced by the Ser-478/481/1121/1453 to Ala mutation. We conclude that PKD plays a key role in copper-dependent serine phosphorylation, permitting high levels of ATP7B protein expression and trafficking.     

Evidence for a role for the putative <Emphasis Type="Italic">Drosophila</Emphasis> hGRX1 orthologue in copper homeostasis

Glutaredoxins are a family of small molecular weight proteins that have a central role in cellular redox regulation. Human GRX1 (hGRX1) has also been shown to play an integral role in copper homeostasis by regulating the redox activity of the metalated sites of copper chaperones such as ATOX1 and SOD1, and the copper efflux proteins ATP7A and ATP7B. To further elucidate the role of hGRX1 in copper homeostasis, we examined the impact of RNA interference-mediated knockdown of CG6852, a putative Drosophila orthologue of hGRX1. CG6852 shares ~41 % amino acid identity with hGRX1 and key functional domains including the metal-binding CXXC motif are conserved between the two proteins. Knockdown of CG6852 in the adult midline caused a thoracic cleft and reduced scutellum, phenotypes that were exacerbated by additional knockdown of copper uptake transporters Ctr1A and Ctr1B. Knockdown of CG6852 in the adult eye enhanced a copper-deficiency phenotype caused by Ctr1A knockdown while ubiquitous knockdown of CG6852 resulted a mild systemic copper deficiency. Therefore we conclude that CG6852 is a putative orthologue of hGRX1 and may play an important role in Drosophila copper homeostasis.     

SMAP2, a novel ARF GTPase-activating protein, interacts with clathrin and clathrin assembly protein and functions on the AP-1-positive early endosome/trans-Golgi network

We recently reported that SMAP1, a GTPase-activating protein (GAP) for Arf6, directly interacts with clathrin and regulates the clathrin-dependent endocytosis of transferrin receptors from the plasma membrane. Here, we identified a SMAP1 homologue that we named SMAP2. Like SMAP1, SMAP2 exhibits GAP activity and interacts with clathrin heavy chain (CHC). Furthermore, we show that SMAP2 interacts with the clathrin assembly protein CALM. Unlike SMAP1, however, SMAP2 appears to be a regulator of Arf1 in vivo, because cells transfected with a GAP-negative SMAP2 mutant were resistant to brefeldin A. SMAP2 colocalized with the adaptor proteins for clathrin AP-1 and EpsinR on the early endosomes/trans-Golgi-network (TGN). Moreover, overexpression of SMAP2 delayed the accumulation of TGN38/46 molecule on the TGN. This suggests that SMAP2 functions in the retrograde, early endosome-to-TGN pathway in a clathrin- and AP-1-dependent manner. Thus, the SMAP gene family constitutes an important ArfGAP subfamily, with each SMAP member exerting both common and distinct functions in vesicle trafficking.     

The role of GMXCXXC metal binding sites in the copper-induced redistribution of the Menkes protein

The Menkes protein (MNK or ATP7A) is a transmembrane, copper-transporting CPX-type ATPase, a subgroup of the extensive family of P-type ATPases. A striking feature of the protein is the presence of six metal binding sites (MBSs) in the N-terminal region with the highly conserved consensus sequence GMXCXXC. MNK is normally located in the trans-Golgi network (TGN) but has been shown to relocalize to the plasma membrane when cells are cultured in media containing high concentrations of copper. The experiments described in this report test the hypothesis that the six MBSs are required for this copper-induced trafficking of MNK. Site-directed mutagenesis was used to convert both cysteine residues in the conserved MBS motifs to serines. Mutation of MBS 1, MBS 6, and MBSs 1-3 resulted in a molecule that appeared to relocalize normally with copper, but when MBSs 4-6 or MBSs 1-6 were mutated, MNK remained in the TGN, even when cells were exposed to 300 microM copper. Furthermore, the ability of the MNK variants to relocalize corresponded well with their ability to confer copper resistance. To further define the critical motifs, MBS 5 and MBS 6 were mutated, and these changes abolished the response to copper. The region from amino acid 8 to amino acid 485 was deleted, resulting in mutant MNK that lacked 478 amino acids from the N-terminal region, including the first four MBSs. This truncated molecule responded normally to copper. Moreover, when either one of the remaining MBS 5 and MBS 6 was mutated to GMXSXXS, the resulting proteins were localized to the TGN in low copper and relocalized in response to elevated copper. These experiments demonstrated that the deleted N-terminal region from amino acid 8 to amino acid 485 was not essential for copper-induced trafficking and that one MBS close to the membrane channel of MNK was necessary and sufficient for the copper-induced redistribution.     

Roles of copper chaperone for superoxide dismutase 1 and metallothionein in copper homeostasis

Copper chaperone for SOD1 (CCS) specifically delivers copper (Cu) to copper, zinc superoxide dismutase (SOD1) in cytoplasm of mammalian cells. In the present study, small interfering RNA (siRNA) targeting CCS was introduced into metallothionein-knockout mouse fibroblasts (MT-KO cells) and their wild type cells (MT-WT cells) to reveal the interactive role of CCS with other Cu-regulating proteins, in particular, MT. CCS knockdown significantly decreased Ctr1, a Cu influx transporter, mRNA expression. On the other hand, Atp7a, a Cu efflux transporter, mRNA expression was increased 3.0 and 2.5 times higher than those of the control in MT-WT and MT-KO cells. These responses of Cu-regulating genes to the CCS knockdown reflected the presence of excess Cu in the cells. To evaluate the Atp7a function in the Cu-replete cells, siRNA of Atp7a and the other Cu transporter, Atp7b were introduced into MT-WT and MT-KO cells. The Atp7a knockdown significantly increased the intracellular Cu concentration, whereas the Atp7b knockdown had no affect. Although two MT isoforms were induced by the CCS knockdown in MT-WT cells, the expression and activity of SOD1 were maintained in both MT-WT and MT-KO cells even when CCS protein expression was reduced to 0.30-0.35 of control. This suggests that the amount of CCS protein exceeds that required to supply Cu to SOD1 in the cells. Further, the CCS knockdown induces Cu accumulation in cells, however, the Cu accumulation is ameliorated by the MT induction, the decrease of Ctr1 expression and the increase of Atp7a expression to maintain Cu homeostasis.