The Role of GMXCXXC Metal Binding Sites in the Copper-induced Redistribution of the Menkes Protein
1999; Elsevier BV; Volume: 274; Issue: 16 Linguagem: Inglês
10.1074/jbc.274.16.11170
ISSN1083-351X
AutoresDaniel Strausak, Sharon La Fontaine, J. E. Hill, Stephen Firth, Paul J. Lockhart, Julian F. B. Mercer,
Tópico(s)RNA and protein synthesis mechanisms
ResumoThe 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 thetrans-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 μm 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. 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 thetrans-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 μm 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. Copper is an essential element required for enzymes such as cytochrome c oxidase, lysyl oxidase, and dopamine-β-hydroxylase that employ its fundamental redox properties in the respiratory chain, connective tissue biosynthesis, and catecholamine production, respectively. Nevertheless, these biochemically useful redox properties can also lead to serious cellular damage through the formation of highly reactive free radicals. Thus, intracellular copper concentrations must be tightly regulated. Various molecules are involved in the maintenance of cellular copper homeostasis, many of which have been recently identified and characterized (for a review, see Ref. 1Vulpe C.D. Packman S. Annu. Rev. Nutr. 1995; 15: 293-322Crossref PubMed Scopus (241) Google Scholar). Among these are copper efflux proteins whose essential role is demonstrated in the genetically inherited Menkes and Wilson diseases. The genes affected in these diseases were identified by several independent groups (2Mercer J.F. Livingston J. Hall B. Paynter J.A. Begy C. Chandrasekharappa S. Lockhart P. Grimes A. Bhave M. Siemieniak D. Glover T.W. Nat. Genet. 1993; 3: 20-25Crossref PubMed Scopus (624) Google Scholar, 3Chelly J. Tumer Z. Tonnesen T. Petterson A. Ishikawa-Brush Y. Tommerup N. Horn N. Monaco A.P. Nat. Genet. 1993; 3: 14-19Crossref PubMed Scopus (623) Google Scholar, 4Vulpe C. Levinson B. Whitney S. Packman S. Gitschier J. Nat. Genet. 1993; 3: 7-13Crossref PubMed Scopus (1208) Google Scholar, 5Bull P.C. Thomas G.R. Rommens J.M. Forbes J.R. Cox D.W. Nat. Genet. 1993; 5: 327-337Crossref PubMed Scopus (1688) Google Scholar, 6Tanzi R.E. Petrukhin K. Chernov I. Pellequer J.L. Wasco W. Ross B. Romano D.M. Parano E. Pavone L. Brzustowicz L.M. Devoto M. Peppercorn J. Bush A.I. Sternlieb I. Pirastu M. Gusella J.F. Evgrafov O. Penchaszadeh G.K. Honig B. Edelman I.S. Soares M.B. Scheinberg I.H. Gilliam T.C. Nat. Genet. 1993; 5: 338-343Crossref PubMed Scopus (1174) Google Scholar) and designated MNK (ATP7A) and WND(ATP7B). MNK and WND encode highly homologous membrane proteins of 178 and 165 kDa, respectively. They belong to the family of P-type ATPases classically represented by the human Ca2+-ATPases and the ubiquitous Na+/K+-ATPases (7Pedersen P.L. Carafoli E. Trends Biochem. Sci. 1987; 12: 146-150Abstract Full Text PDF Scopus (818) Google Scholar). Together with Cd2+-ATPases, Cu+/2+-ATPases form a subfamily of CPX-type ATPases (X = Cys, His, or Ser) characterized by a conserved Cys-Pro-X motif in the proposed ion transduction channel and a variable number of GMXCXXC putative heavy metal binding sites (MBS) 1The abbreviations used are: MBS, metal binding site; MNK, Menkes protein; TGN, trans-Golgi network; CHO, Chinese hamster ovary; kb, kilobase(s); BME, Eagle's basal media; wt, wild-type; WND, Wilson protein.1The abbreviations used are: MBS, metal binding site; MNK, Menkes protein; TGN, trans-Golgi network; CHO, Chinese hamster ovary; kb, kilobase(s); BME, Eagle's basal media; wt, wild-type; WND, Wilson protein. at the N terminus (8Solioz M. Vulpe C. Trends Biochem. Sci. 1996; 21: 237-241Abstract Full Text PDF PubMed Scopus (415) Google Scholar). Bacterial copper transporters contain a single MBS (9Odermatt A. Suter H. Krapf R. Solioz M. J. Biol. Chem. 1993; 268: 12775-12779Abstract Full Text PDF PubMed Google Scholar), yeast possess two MBSs (10Fu D. Beeler T.J. Dunn T.M. Yeast. 1995; 11: 283-292Crossref PubMed Scopus (142) Google Scholar), nematodes have three MBSs (11Sambongi Y. Wakabayashi T. Yoshimizu T. Omote H. Oka T. Futai M. J. Biochem. (Tokyo). 1997; 121: 1169-1175Crossref PubMed Scopus (50) Google Scholar), and the human MNK and WND have six metal binding sites (8Solioz M. Vulpe C. Trends Biochem. Sci. 1996; 21: 237-241Abstract Full Text PDF PubMed Scopus (415) Google Scholar). The same motifs are also present in cadmium transporters and in mercury-binding proteins (12Silver S. Phung L.T. Annu. Rev. Microbiol. 1996; 50: 753-789Crossref PubMed Scopus (1030) Google Scholar), underscoring the role of these motifs in specifically complexing heavy metal ions.Elucidating the function of the N-terminal GMXCXXC motifs of both WND and MNK has been the subject of several studies (13DiDonato M. Narindrasorasak S. Forbes J.R. Cox D.W. Sarkar B. J. Biol. Chem. 1997; 272: 33279-33282Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 14Lutsenko S. Petrukhin K. Cooper M.J. Gilliam C.T. Kaplan J.H. J. Biol. Chem. 1997; 272: 18939-18944Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 15Payne A.S. Gitlin J.D. J. Biol. Chem. 1998; 273: 3765-3770Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 16Iida M. Terada K. Sambongi Y. Wakabayashi T. Miura N. Koyama K. Futai M. Sugiyama T. FEBS Lett. 1998; 428: 281-285Crossref PubMed Scopus (96) Google Scholar). These studies suggest a binding stoichiometry of one copper atom per metal binding site for both MNK and WND N termini and a capacity to bind Zn, Ni, and Co (13DiDonato M. Narindrasorasak S. Forbes J.R. Cox D.W. Sarkar B. J. Biol. Chem. 1997; 272: 33279-33282Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 14Lutsenko S. Petrukhin K. Cooper M.J. Gilliam C.T. Kaplan J.H. J. Biol. Chem. 1997; 272: 18939-18944Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Thus far, the biochemical function of the MNK and WND N termini has only been studied in a Δccc2 mutant yeast strain in which human MNK and WND fully restored copper delivery to the high affinity iron uptake system (15Payne A.S. Gitlin J.D. J. Biol. Chem. 1998; 273: 3765-3770Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 16Iida M. Terada K. Sambongi Y. Wakabayashi T. Miura N. Koyama K. Futai M. Sugiyama T. FEBS Lett. 1998; 428: 281-285Crossref PubMed Scopus (96) Google Scholar). The third MBS was found to be most critical for catalytic activity of MNK and was suggested to be involved in protein-protein interaction (15Payne A.S. Gitlin J.D. J. Biol. Chem. 1998; 273: 3765-3770Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). A further study demonstrated that the deletion of a region comprising MBS 6 rendered WND inactive (16Iida M. Terada K. Sambongi Y. Wakabayashi T. Miura N. Koyama K. Futai M. Sugiyama T. FEBS Lett. 1998; 428: 281-285Crossref PubMed Scopus (96) Google Scholar).In CHO-K1 cells, human fibroblasts, and HeLa cells, MNK is localized to the trans-Golgi network (TGN) (17Petris M.J. Mercer J.F. Culvenor J.G. Lockhart P. Gleeson P.A. Camakaris J. EMBO J. 1996; 15: 6084-6095Crossref PubMed Scopus (528) Google Scholar, 18Dierick H.A. Adam A.N. Escara-Wilke J.F. Glover T.W. Hum. Mol. Genet. 1997; 6: 409-416Crossref PubMed Scopus (98) Google Scholar, 19Yamaguchi Y. Heiny M.E. Suzuki M. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14030-14035Crossref PubMed Scopus (190) Google Scholar). Copper-dependent redistribution of MNK from the TGN to the plasma membrane was demonstrated in copper-resistant CHO-K1 cells that overexpressed the endogenous hamster MNK homologue (17Petris M.J. Mercer J.F. Culvenor J.G. Lockhart P. Gleeson P.A. Camakaris J. EMBO J. 1996; 15: 6084-6095Crossref PubMed Scopus (528) Google Scholar). Recently, this observation was supported by experiments with stable CHO-K1 cells expressing human MNK from a cDNA construct (20La Fontaine S. Firth S.D. Lockhart P.J. Brooks H. Parton R.G. Camakaris J. Mercer J.F.B. Hum. Mol. Genet. 1998; 7: 1293-1300Crossref PubMed Scopus (78) Google Scholar). Copper-induced movement of WND from the TGN to an as yet unidentified intracellular compartment has also been observed (21Yang X.L. Miura N. Kawarada Y. Terada K. Petrukhin K. Gilliam T.C. Sugiyama T. Biochem. J. 1997; 326: 897-902Crossref PubMed Scopus (86) Google Scholar, 22Payne A.S. Kelly E.J. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10854-10859Crossref PubMed Scopus (185) Google Scholar, 23Lutsenko S. Cooper M.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6004-6009Crossref PubMed Scopus (125) Google Scholar). Despite these data, little is known about the structural domains involved in the exocytic and endocytic trafficking of these copper-transporting proteins. An obvious candidate region that may be involved in the copper-dependent trafficking of MNK is the N-terminal region containing the six copper-binding GMXCXXC motifs.The objective of this study was to clarify the role of the N-terminal MBSs of MNK in copper-induced redistribution. We report here that not all of the MBSs are essential for the relocalization of MNK and for conferring a copper-resistant phenotype. Furthermore, the presence of only one MBS within a region of ∼170 amino acids adjacent to the proposed transduction channel is necessary and sufficient for the copper-induced exocytic trafficking of the Menkes protein. In addition, the data indicate that protein-protein interactions that may be required for the translocation of MNK from the TGN to the plasma membrane do not involve amino acids 8–485 of the MNK N terminus.RESULTSThe role of the six MBSs in MNK trafficking was investigated using site-directed and deletion mutagenesis of the MNK N terminus. Constructs encoding MNK mutated in MBS 1 alone (MNKm1), MBS 6 alone (MNKm6), MBSs 1–3 (MNKm1-3), MBSs 4–6 (MNKm4-6), and MBS 5 and MBS 6 (MNKm5+6) were generated. Amino acids 8–485 at the N terminus of MNK were deleted to create MNKΔ1-4, and in this construct, mutations were separately introduced within MBS 5 (MNKΔ1-4m5) and MBS 6 (MNKΔ1-4m6) to generate constructs with only one functional MBS (Fig.1).All of the mutated MNK cDNA constructs were initially transiently transfected into CHO-K1 cells, and the intracellular distribution of the mutated MNK molecules was assessed by indirect immunofluorescence using polyclonal antibodies directed against the MNK N or C terminus. Stable cell lines were established for constructs containingMNKm1-3, MNKm4-6 and MNKm1-6. In basal copper concentrations, fluorescent staining was observed within the perinuclear region of all of the transfected cells (Figs.Figure 2, Figure 3, Figure 4, Figure 5, Figure 6; − Cu), consistent with location at the TGN and with previous results obtained with CHO-K1 cells (17Petris M.J. Mercer J.F. Culvenor J.G. Lockhart P. Gleeson P.A. Camakaris J. EMBO J. 1996; 15: 6084-6095Crossref PubMed Scopus (528) Google Scholar, 20La Fontaine S. Firth S.D. Lockhart P.J. Brooks H. Parton R.G. Camakaris J. Mercer J.F.B. Hum. Mol. Genet. 1998; 7: 1293-1300Crossref PubMed Scopus (78) Google Scholar). Thus, the overexpressed, mutated MNKs were not mislocalized under basal conditions. Furthermore, the mutations did not affect MNK epitope recognition by the polyclonal antibody, suggesting that even multiple point mutations within the N terminus did not induce significant antigenic changes in the molecule.Figure 6Copper resistance characteristics of MNK-expressing clones. Cell lines were exposed to the indicated copper concentrations for 7 days. Surviving colonies were then stained and scored. The results shown represent the normalized mean ± S.D. (bars) of three independent colony counts. ● (A–C), CHO-K1; ○ (A–C), CHO-K1 overexpressing wt MNK; ▴ (A), 114 cells (MNKm1-3); ▪ (B), 115 cells (MNKm4-6); ■ (C), 116 cells (MNKm1-6).View Large Image Figure ViewerDownload (PPT)After incubation in 200 μm copper for 3 h, transiently expressed human MNK that was mutated in either MBS 1 (MNKm1) or MBS 6 (MNKm6) was redistributed and was clearly present within the cytoplasm and at the plasma membrane (Fig. 2). There were no obvious differences in the staining pattern of cells expressing mutated or wt proteins as observed after incubation in different copper concentrations (data not shown). Thus, a single GMXCXXC to GMXSXXS mutation within the MNK N terminus did not influence the copper-inducible relocation of MNK.Proteins transiently expressed in cells differ significantly in their levels of expression. Therefore, the interpretation of results obtained with transiently transfected cells can be complicated by artifacts resulting from overexpression of the target protein. To allow a clearer analysis of the effects of mutations, stable cell lines were generated from CHO-K1 cells that expressed the mutant MNK forms MNKm1-3, MNKm4-6, and MNKm1-6. These cell lines were designated 114, 115, and 116, respectively.To confirm that the expressed mutant proteins were of the expected sizes and to determine the level of MNK expression, Western blots on whole cell lysates of the stable cell lines were carried out (Fig.3 A). A protein of the expected size of ∼178 kDa was detected in each of cell lines 114, 115, and 116, also demonstrating the capacity of the anti-MNK antibody to recognize the mutated proteins. Compared with the endogenous CHO-K1 Menkes protein homologue, significantly more MNK was expressed in the transfected cell lines. This level of expression corresponded well with the intensity of the fluorescent signal observed in the whole cell fluorescence experiments described below.The intracellular location of MNKm1-3, MNKm4-6, and MNKm1-6 was assessed by confocal immunofluorescence microscopy. All of these mutated proteins localized to the TGN in cells cultured in basal medium (Fig. 3 B). When cells were incubated for 3 h in 80 μm copper, some redistribution to the plasma membrane was noticeable in the wt cells and in cell line 114 (MNKm1-3). In high copper concentrations, the labeling of the plasma membrane of these cells intensified and was the predominant signal at 300 μm copper (Fig. 3 B). When the 114 and the wt cell lines were incubated for 10, 20, 40, 60, and 120 min in media containing 100 μm copper, there were no noticeable differences in the kinetics of the translocation observed for the two MNK forms (data not shown). In contrast to wt MNK and MNKm1-3, MNKm4-6 and MNKm1-6 remained at the TGN after incubation in high copper concentrations (300 μm). These data demonstrated that the first three MBSs were not essential for the copper-dependent redistribution of MNK, whereas the three MBSs closest to the transport channel were critical.To further localize the regions critical for relocalization, a construct with MNK mutated in MBS 5 and MBS 6 was created (MNKm5+6). CHO-K1 cells transiently transfected with this construct showed clear perinuclear staining that indicated TGN localization of MNKm5+6. In the presence of elevated copper, MNKm5+6 remained at the TGN, whereas wt MNK relocated to the plasma membrane (Fig.4). This result showed that MBSs 1–4 were not sufficient for the copper-dependent redistribution of MNK.To determine whether amino acids other than the MBSs in the N-terminal region play a role in copper-induced trafficking and whether both MBS 5 and MBS 6 or just one of the two were essential, a construct encoding MNK that lacked MBSs 1–4 was generated (MNKΔ1-4) and mutated individually at MBS 5 (MNKΔ1-4m5) and MBS 6 (MNKΔ1-4m6). Immunofluorescence microscopy using an antibody directed against the MNK C terminus showed that in basal media, all three mutant proteins were located at the TGN of transiently transfected CHO-K1 cells. However, when cells were incubated in 200 μm copper for 3 h, an elevated, dispersed fluorescence was evident throughout the cytoplasm and at the plasma membrane (Fig.5). These results showed that amino acids 8–485 of the MNK N terminus were not necessary for the copper-induced redistribution of the protein; therefore, this region did not contain a critical copper-sensing or copper-responsive targeting signal. In addition, these data indicated that a single MBS located within a maximum distance of 170 amino acids from the membrane channel was sufficient for MNK trafficking.The ability of the mutated proteins to confer copper resistance was determined by assessing the survival of 114, 115 and 116 cells (expressing MNKm1-3, MNKm4-6, and MNKm1-6, respectively) compared with the parental CHO-K1 cells and CHO-K1 cells expressing wt MNK in increasing copper concentrations (Fig.6). The level of copper resistance of cell lines 115 and 116 was similar to that of the parental CHO-K1 cells with an estimated LD50 of 60–65 μmCuCl2 (Fig. 6, B and C). In contrast, the survival of 114 cells and the wt cell line was still evident up to 105 μm CuCl2, with a LD50 of 75 and 105 μm copper, respectively (Fig. 6 A). However, the difference in copper resistance of 114 cells compared with wt cells may be explained by a ∼50% reduction in the level of MNK expression in 114 cells (Fig. 3 A); therefore, MNKm1-3 may be functionally equivalent to wt MNK.Taken together, these results showed that: (i) point mutations converting either a single MBS or the first three MBSs from GMXCXXC to GMXSXXS have no effect on the copper-induced intracellular redistribution of MNK as shown with MNKm1, MNKm6, and MNKm1-3; (ii) the protein region encompassing the first four MBSs is not necessary for copper-regulated trafficking; (iii) one MBS within the region comprising MBS 5 to MBS 6 is necessary and sufficient for copper-dependent MNK trafficking; and (iv) the ability of MNK to confer resistance to copper is linked to its ability to undergo copper-induced redistribution.DISCUSSIONPetris et al. (17Petris M.J. Mercer J.F. Culvenor J.G. Lockhart P. Gleeson P.A. Camakaris J. EMBO J. 1996; 15: 6084-6095Crossref PubMed Scopus (528) Google Scholar) previously demonstrated a copper-responsive relocalization of MNK from the TGN to the plasma membrane in copper-resistant CHO-K1 cells. Recently, a copper-dependent redistribution to the cell surface was demonstrated in CHO-K1 cells that expressed human MNK (20La Fontaine S. Firth S.D. Lockhart P.J. Brooks H. Parton R.G. Camakaris J. Mercer J.F.B. Hum. Mol. Genet. 1998; 7: 1293-1300Crossref PubMed Scopus (78) Google Scholar). To explain the MNK response to elevated copper levels, it was postulated that the N terminus functioned as a copper-sensing domain in which the six MBSs became progressively occupied by copper, causing a conformational change that triggered the redistribution of MNK (17Petris M.J. Mercer J.F. Culvenor J.G. Lockhart P. Gleeson P.A. Camakaris J. EMBO J. 1996; 15: 6084-6095Crossref PubMed Scopus (528) Google Scholar). In this study, we have used an extensive mutagenesis strategy to define the role of the six MBSs in the copper-induced trafficking of MNK.The mutation of a single MBS did not inhibit the copper-induced relocalization of MNK from the TGN to the plasma membrane, as demonstrated by mutations of MBS 1 and MBS 6 (Fig. 2). The majority of point mutations that have been identified in Menkes patients occur within exons 7–10, which encode MBS 6 and the first four transmembrane helices. All of the point mutations identified within the N terminus were either nonsense mutations or insertion/deletion mutations that are predicted to result in a truncation of the MNK gene product (29Tumer Z. Lund C. Tolshave J. Vural B. Tonnesen T. Horn N. Am. J. Hum. Genet. 1997; 60: 63-71PubMed Google Scholar). These observations suggest that point mutations that disrupt the function of a single MBS are not likely to cause a disease phenotype and are supported by our findings that six functional MBSs are not necessary for MNK trafficking.In elevated copper, MNKm1-3 was able to redistribute to the plasma membrane, demonstrating that the first three GMXCXXC repeats are not essential for the copper-induced trafficking of MNK (Fig. 3 B). In contrast to MNKm1-3, MNKm1-6, MNKm4-6, and MNKm5+6 remained predominantly within the perinuclear region, even under very high copper concentrations (300 μm) (Figs. 3 B and 4). This result suggested that the MBSs close to the membrane channel were more important for the intracellular trafficking of MNK. Further support for this conclusion was obtained with the deletion constructs containing only MBS 5 and MBS 6 that showed that only one functional MBS close to the channel was necessary and sufficient for MNK trafficking (Fig. 5). These experiments demonstrated that 478 amino acids within the MNK N terminus did not contain a critical targeting sequence and were not required for the copper-dependent exocytic trafficking of MNK, effectively negating the hypothesis that the multiple metal binding sites constitute a relocalization domain.The ability of the mutated MNK molecules to traffic corresponded well with their ability to confer copper resistance. The parental CHO-K1 cells and cell lines 115 and 116 that overexpressed MNK mutated in MBSs 4–6 and MBSs 1–6, respectively, did not survive in copper levels greater than 75 μm for 7 days. In contrast, the cell lines overexpressing MNKm1-3 and wt MNK (114 and wt cells, respectively) were significantly more resistant to copper (Fig. 6,A–C). This experiment demonstrated that the copper-resistant phenotype was caused by the overexpression of MNK. Therefore, MNK molecules mutated in the first three MBSs were able to effectively efflux copper, indicating that MBSs 1–3 were not essential for both MNK trafficking and copper transport.Earlier data suggested that MNK constitutively recycled between the TGN and the plasma membrane in cells cultured in low-copper media (17Petris M.J. Mercer J.F. Culvenor J.G. Lockhart P. Gleeson P.A. Camakaris J. EMBO J. 1996; 15: 6084-6095Crossref PubMed Scopus (528) Google Scholar). Copper-induced relocalization of MNK could involve an increase in the rate of exocytosis of MNK from the TGN to the plasma membrane or a reduction in the rate of endocytosis from the plasma membrane. No published data distinguish between these two possibilities. However, two regions important for its basal location in the TGN have been identified. The first is a 38-amino acid sequence containing transmembrane domain 3, characterized as a candidate region responsible for Golgi retention of MNK (30Francis M.J. Jones E.E. Levy E.R. Ponnambalam S. Chelly J. Monaco A.P. Hum. Mol. Genet. 1998; 7: 1245-1252Crossref PubMed Scopus (80) Google Scholar). In addition, TGN localization has recently been shown to depend on a di-leucine motif, 1487LL1488, within the C-terminal region of MNK (26Petris M.J. Camakaris J. Greenough M. La Fontaine S. Mercer J.F.B. Hum. Mol. Genet. 1998; 7: 2063-2071Crossref PubMed Scopus (134) Google Scholar). Di-leucines are a class of endocytic targeting signals directing plasma membrane proteins into clathrin-coated vesicles, resulting in constitutive internalization of these membrane proteins (31Trowbridge I.S. Collawn J.F. Hopkins C.R. Annu. Rev. Cell Biol. 1993; 9: 129-161Crossref PubMed Scopus (700) Google Scholar). Based on these observations, one hypothesis to explain the accumulation of MNK at the plasma membrane is that copper retards the internalization of the protein by causing a conformational change that obscures the C-terminal di-leucine motif. This conformational change may occur as a consequence of the binding of copper to MBS 5 or MBS 6. To clarify whether this mechanism is operative, some measurements of the rate of internalization of MNK in the presence and absence of copper will be required.An alternative model to explain copper-induced relocalization is that conformational changes induced by copper-binding at the N terminus may activate the delivery of copper to the channel and induce the translocation of a copper ion across the cell membrane. Further conformational changes that are coincident with the copper pumping mechanism of MNK may reveal a motif that is recognized by the vesicular sorting machinery, leading to an increase in the number of MNK molecules that are loaded onto vesicles destined for the plasma membrane. In this mechanism, the proximity of the MBS to the transduction channel may be critical either for the direct delivery of copper to the channel or for the acceptance of copper from copper chaperones such as HAH1 (32Klomp L.W.J. Lin S.J. Yuan D.S. Klausner R.D. Culotta V.C. Gitlin J.D. J. Biol. Chem. 1997; 272: 9221-9226Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar) that specifically interact with the protein region comprising MBS 5 and MBS 6. This model is supported by several recent experiments from our laboratory that showed different mutations in structurally and functionally distinct regions of MNK, which are predicted to disrupt copper transport activity, inhibited the copper-dependent trafficking of the protein. 2S. La Fontaine, L. Ambrosini, and D. Strausak, unpublished observations. In addition, Payneet al. (22Payne A.S. Kelly E.J. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10854-10859Crossref PubMed Scopus (185) Google Scholar) have reported that a mutation in a region distinct from the metal binding sites in the closely related Cu-ATPases affected in Wilson disease also abolished copper-induced relocalization (22Payne A.S. Kelly E.J. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10854-10859Crossref PubMed Scopus (185) Google Scholar). These observations suggest that mutations in MNK that disrupt copper transport may also prevent copper-induced relocalization to the plasma membrane.The redundancy of 478 N-terminal amino acids containing four MBSs implies that as with bacteria and yeast, a Cu-ATPase with only one MBS or two MBSs at the N terminus may be sufficient to maintain some degree of copper homeostasis in mammalian cells. The number of MBSs at the N terminus has increased during evolution: one in bacteria, two in yeast, three in nematodes, and six in mammals. This observation suggests that MNK and WND gained the additional MBSs simply by amplification of the repeated elements. The additional MBSs may have evolved to increase the efficiency of copper scavenging in the cytoplasm or copper ion delivery to the transduction channel in the cells of higher organisms. However, to further clarify the role of the six MBSs in the human copper-transporting ATPases, additional studies involving copper transport activity measurements of the mutant MNK forms will be required. Copper is an essential element required for enzymes such as cyto
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