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• W2023416426 abstract "Copper is an essential co-factor for several key metabolic processes. This requirement in humans is underscored by Menkes disease, an X-linked copper deficiency disorder caused by mutations in the copper transporting P-type ATPase, MNK. MNK is located in the trans-Golgi network where it transports copper to secreted cuproenzymes. Increases in copper concentration stimulate the trafficking of MNK to the plasma membrane where it effluxes copper. In this study, a Menkes disease mutation, G1019D, located in the large cytoplasmic loop of MNK, was characterized in transfected cultured cells. In copper-limiting conditions the G1019D mutant protein was retained in the endoplasmic reticulum. However, this mislocalization was corrected by the addition of copper to cells via a process that was dependent upon the copper binding sites at the N-terminal region of MNK. Reduced growth temperature and the chemical chaperone, glycerol, were found to correct the mislocalization of the G1019D mutant, suggesting this mutation interferes with protein folding in the secretory pathway. These findings identify G1019D as the first conditional mutation associated with Menkes disease and demonstrate correction of the mislocalized protein by copper supplementation. Our findings provide a molecular framework for understanding how mutations that affect the proper folding of the MNK transporter in Menkes patients may be responsive to parenteral copper therapy. Copper is an essential co-factor for several key metabolic processes. This requirement in humans is underscored by Menkes disease, an X-linked copper deficiency disorder caused by mutations in the copper transporting P-type ATPase, MNK. MNK is located in the trans-Golgi network where it transports copper to secreted cuproenzymes. Increases in copper concentration stimulate the trafficking of MNK to the plasma membrane where it effluxes copper. In this study, a Menkes disease mutation, G1019D, located in the large cytoplasmic loop of MNK, was characterized in transfected cultured cells. In copper-limiting conditions the G1019D mutant protein was retained in the endoplasmic reticulum. However, this mislocalization was corrected by the addition of copper to cells via a process that was dependent upon the copper binding sites at the N-terminal region of MNK. Reduced growth temperature and the chemical chaperone, glycerol, were found to correct the mislocalization of the G1019D mutant, suggesting this mutation interferes with protein folding in the secretory pathway. These findings identify G1019D as the first conditional mutation associated with Menkes disease and demonstrate correction of the mislocalized protein by copper supplementation. Our findings provide a molecular framework for understanding how mutations that affect the proper folding of the MNK transporter in Menkes patients may be responsive to parenteral copper therapy. Copper is essential for the growth and development of all organisms. Several key metabolic processes are copper-dependent, including oxidative respiration, neurotransmitter synthesis, connective tissue formation, free radical detoxification, iron homeostasis, and pigmentation (1Linder M.C. Wooten L. Cerveza P. Cotton S. Shulze R. Lomeli N. Am. J. Clin. Nutr. 1998; 67: 965S-971SCrossref PubMed Scopus (239) Google Scholar). This copper dependence for humans is most dramatically illustrated in Menkes disease, an X-linked recessive copper deficiency disorder that is generally lethal in early childhood (2Kaler S.G. Am. J. Clin. Nutr. 1998; 67: 1029S-1034SCrossref PubMed Scopus (106) Google Scholar). Menkes disease is caused by mutations in a transmembrane copper transporting P-type ATPase, MNK (or ATP7A), which is expressed in virtually all non-hepatic tissues (3Mercer J.F.B. Livingston J. Hall B.K. Paynter J.A. Begy C. Chandrasekharappa S. Lockhart P. Grimes A. Bhave M. Siemenack D. Glover T.W. Nat. Genet. 1993; 3: 20-25Crossref PubMed Scopus (628) Google Scholar, 4Vulpe C. Levinson B. Whitney S. Packman S. Gitschier J. Nat. Genet. 1993; 3: 7-13Crossref PubMed Scopus (1219) Google Scholar, 5Chelly J. Tumer Z. Tonnerson T. Petterson A. Ishikawa-Brush Y. Tommerup N. Horn N. Monaco A.P. Nat. Genet. 1993; 3: 14-19Crossref PubMed Scopus (628) Google Scholar). Menkes disease is characterized by reduced copper transport from intestinal enterocytes into the bloodstream. This reduced supply of dietary copper is further compounded by reduced copper transport across the blood-brain barrier to the central nervous system. Consequently, Menkes patients exhibit a range of symptoms attributable to deficiencies in copper-dependent metabolism, including connective tissue defects, neurological degeneration, mental retardation, seizures, osteoporosis, and hypopigmentation (2Kaler S.G. Am. J. Clin. Nutr. 1998; 67: 1029S-1034SCrossref PubMed Scopus (106) Google Scholar). Studies using cultured cells suggest that MNK is located in the trans-Golgi network (TGN), 1The abbreviations used are: TGN, trans-Golgi network; WtMNK, wild type MNK; ER, endoplasmic reticulum; BCS, bathocuproine disulfonate; PDI, protein-disulfide isomerase; WND, Wilson disease protein where it transports copper to copper-dependent enzymes synthesized within secretory compartments (6Petris M.J. Mercer J.F.B. Culvenor J.G. Lockhart P. Gleeson P.A. Camakaris J. EMBO J. 1996; 15: 6084-6095Crossref PubMed Scopus (538) Google Scholar, 7Yamaguchi Y. Heiny M.E. Suzuki M. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14030-14035Crossref PubMed Scopus (193) Google Scholar, 8Petris M.J. Strausak D. Mercer J.F.B. Hum. Mol. Genet. 2000; 9: 2845-2851Crossref PubMed Scopus (187) Google Scholar). Elevated copper concentrations stimulate the trafficking of MNK to the plasma membrane where it is involved in copper efflux (6Petris M.J. Mercer J.F.B. Culvenor J.G. Lockhart P. Gleeson P.A. Camakaris J. EMBO J. 1996; 15: 6084-6095Crossref PubMed Scopus (538) Google Scholar, 9Camakaris J. Petris M. Bailey L. Shen P. Lockhart P. Glover T.W. Barcroft C.L. Patton J. Mercer J.F.B. Hum. Mol. Genet. 1995; 4: 2117-2123Crossref PubMed Scopus (157) Google Scholar). The copper efflux function of MNK is likely to be essential for supplying the blood with dietary copper, as well as the central nervous system. Menkes patients are treated by parenteral copper injections, which in the most successful cases reduces neurological defects and prolongs life expectancy (2Kaler S.G. Am. J. Clin. Nutr. 1998; 67: 1029S-1034SCrossref PubMed Scopus (106) Google Scholar). However, responses to treatment are variable and likely to depend on the age at which treatment commences, and the extent to which mutations impair copper transport or trafficking functions of the Menkes protein. Indeed, the more positive outcomes are thought to involve patients whose mutations permit the expression of some functional MNK protein (10Kaler S.G. Das S. Levinson B. Goldstein D.S. Holmes C.S. Patronas N.J. Packman S. Gahl W.A. Biochem. Mol. Med. 1996; 57: 37-46Crossref PubMed Scopus (51) Google Scholar). Here we characterize the effect of a Menkes disease missense mutation, G1019D, on the intracellular location and trafficking of MNK. The G1019D mutation was found to impair normal secretion and maturation of MNK in the early secretory pathway. Surprisingly, these defects were exacerbated by copper depletion of the growth media and corrected by copper supplementation. This copper-dependent rescue of mislocalized G1019D mutant protein occurred in a dose-dependent and metal-specific manner and was dependent on the N-terminal copper binding sites. Conditions such as reduced temperature or glycerol supplementation, which are known to correct misfolding of other membrane proteins, also prevented mislocalization of the G1019D mutant protein. Our study reports the first conditional Menkes disease mutation that affects secretion and maturation of the transporter and demonstrates correction of this defect by copper supplementation. These observations provide new insights into understanding how parenteral copper treatment of Menkes patients, whose mutations affect protein folding, may yield positive outcomes. All chemicals were purchased from Sigma. The immortalized Menkes fibroblast cell line, Me32a, was used to generate cell lines expressing wild type MNK (WtMNK) and the G1019D mutant. Me32a cells lack detectable MNK protein due to a frameshift mutation, as described previously (11La Fontaine S. Firth S.D. Camakaris J. Engelzou A. Theophilos M.B. Petris M.J. Lockhart P. Greenough M. Brooks H. Reddel R.R. Mercer J.F.B. J. Biol. Chem. 1998; 273: 31375-31380Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Cells were cultured in minimal essential medium (Invitrogen) supplemented with 10% fetal bovine serum, penicillin/streptomycin in a 5% CO2, 37 °C incubator. In some experiments, copper chloride or the copper chelator, bathocuproine disulfonic acid was added to the culture medium, as indicated in the figure legends. In some experiments various concentrations of tunicamycin or 10% glycerol were added to the growth media for 16 h. The Transformer mutagenesis kit (Clontech) was used to engineer the G1019D mutation into an EcoRV subclone of the MNK cDNA and verified by DNA sequencing. This mutant EcoRV fragment was then cloned into the MNK cDNA expression plasmid (12Petris M.J. Mercer J.F.B. Hum. Mol. Genet. 1999; 8: 2107-2175Crossref PubMed Scopus (139) Google Scholar) by replacing the corresponding wild type fragment. The G1019D mutant fragment was also cloned into the previously generated construct in which all six Cys-X-X-Cys copper binding sites in the N-terminal region were mutated to Ser-X-X-Ser (13Strausak D., La Fontaine S. Hill J. Firth S.D. Lockhart P.J. Mercer J.F.B. J. Biol. Chem. 1999; 274: 11170-11177Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Plasmid constructs bearing the wild type MNK or the G1019D mutant cDNAs were transfected into Me32a cells using LipofectAMINE 2000 (Invitrogen), and G418 selection was used to isolate stably expressing cell lines, as previously described (14Petris M.J. Camakaris J. Greenough M., La Fontaine S. Mercer J.F.B. Hum. Mol. Genet. 1998; 7: 2063-2071Crossref PubMed Scopus (136) Google Scholar). In the experiments shown below in Figs. 6 and 7, Me32a cells were transiently transfected with the indicated plasmids using LipofectAMINE 2000.Figure 7The six N-terminal copper binding sites are essential for the copper-dependent glycosylation of G1019D. Me32a cells were transiently transfected with expression plasmids containing cDNAs for WtMNK, G1019D, mMBS1–6, and mMBS1–6/G1019D and cultured for 48 h in media containing 500 μm BCS (+ BCS) or media supplemented with 100 μm copper (+Cu). Cell lysates were separated using SDS-PAGE, and immunoblot analysis was performed using anti-MNK antibodies as described under “Experimental Procedures.” Tubulin protein levels were detected in parallel to indicate protein loading (lower panel). Note the presence of a single smaller non-glycosylated product for G1019D (lane 2) and mMBS1–6/G1019D (lane 4) in BCS-treated cells, and correction of this defect in copper-treated cells for G1019D (lane 6), but not the mMBS1–6/G1019D protein (lane 8).View Large Image Figure ViewerDownload (PPT) Immunofluorescence microscopy was performed as previously described (14Petris M.J. Camakaris J. Greenough M., La Fontaine S. Mercer J.F.B. Hum. Mol. Genet. 1998; 7: 2063-2071Crossref PubMed Scopus (136) Google Scholar), using affinity-purified MNK antibodies or anti-PDI antibodies (Stressgen Biotech), and detected using Alexa594 anti-sheep or Alexa488 anti-mouse secondary antibodies (Molecular Probes). Protein lysates were prepared as previously described and fractionated using either 6% SDS-PAGE for MNK immunoblots or 8% SDS-PAGE for all other immunoblots (8Petris M.J. Strausak D. Mercer J.F.B. Hum. Mol. Genet. 2000; 9: 2845-2851Crossref PubMed Scopus (187) Google Scholar). Immunoblotting experiments were performed using either anti-MNK antibodies or anti-tubulin antibodies (Sigma), as indicated in the figure legends using an enhanced chemiluminescence detection kit (Roche Molecular Biochemicals). The G1019D mutation was previously identified in a patient with classic Menkes disease (15Tümer Z. Lund C. Tolshave J. Vural B. Tonnesen T. Horn N. Am. J. Hum. Genet. 1997; 60: 63-71PubMed Google Scholar). Glycine 1019 is located in the largest cytoplasmic loop of the ATPase, near the aspartic acid 1044 that is the conserved phosphorylation site of all P-type ATPases (Fig.1 A). Although there has been no specific role assigned to G1019, the non-conservative replacement of a small neutral glycine with a large charged residue such as aspartic acid, was predicted to reduce copper transport activity (15Tümer Z. Lund C. Tolshave J. Vural B. Tonnesen T. Horn N. Am. J. Hum. Genet. 1997; 60: 63-71PubMed Google Scholar). Indeed, a previous study of the recombinant G1019D MNK protein in the yeast,Saccharomyces cerevisiae, demonstrated that this mutation severely reduced but did not eliminate copper transport, as determined by a complementation growth assay (16Payne A.S. Gitlin J.D. J. Biol. Chem. 1998; 273: 3765-3770Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). In this study, we investigated the consequences of the G1019D mutation on the cell biology of the MNK copper transporter. In vitro mutagenesis was used to generate the G1019D mutation in a plasmid bearing the MNK cDNA. Both wild type MNK (WtMNK) and the G1019D mutant protein were stably expressed in the immortalized fibroblast cell line, Me32a. This cell line was derived from a Menkes patient and does not express endogenous MNK protein (11La Fontaine S. Firth S.D. Camakaris J. Engelzou A. Theophilos M.B. Petris M.J. Lockhart P. Greenough M. Brooks H. Reddel R.R. Mercer J.F.B. J. Biol. Chem. 1998; 273: 31375-31380Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Three cell lines expressing the G1019D protein were isolated by selection in G418 and used for immunofluorescence microscopy and immunoblotting experiments. Immunofluorescence microscopy showed that the WtMNK protein was located in the perinuclear region of cells (Fig. 1 B), consistent with its location in the TGN (6Petris M.J. Mercer J.F.B. Culvenor J.G. Lockhart P. Gleeson P.A. Camakaris J. EMBO J. 1996; 15: 6084-6095Crossref PubMed Scopus (538) Google Scholar, 7Yamaguchi Y. Heiny M.E. Suzuki M. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14030-14035Crossref PubMed Scopus (193) Google Scholar). There was no signal detected in Me32a cells stably transfected with the empty vector (data not shown). Although the G1019D protein appeared concentrated in the perinuclear region (Fig.1 B, arrowhead), there was also staining associated with reticular structures extending into cytoplasmic regions. This same distribution was observed in three independent G1019D cell lines (data not shown). This distribution suggested partial location of G1019D protein in both the perinuclear Golgi complex and the endoplasmic reticulum (ER). Immunoblotting analysis detected the expected 180-kDa MNK protein in cells expressing the WtMNK protein (Fig. 1 B). However, the G1019D protein was resolved as two bands that corresponded to the wild type size, and a protein with an apparent molecular mass of 175 kDa (Fig. 1 B). MNK has been previously shown to be a glycosylated protein (7Yamaguchi Y. Heiny M.E. Suzuki M. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14030-14035Crossref PubMed Scopus (193) Google Scholar). The existence of this lower G1019D band, together with the immunofluorescence data suggesting partial localization in the ER, indicated that the G1019D mutation may impair the glycosylation of MNK resulting in this faster migrating species. To explore this further, we treated cells with the glycosylation inhibitor, tunicamycin. This resulted in the generation of the faster migrating non-glycosylated form of the WtMNK protein, which was similar to the lower form of the G1019D protein in untreated cells. Significantly, the G1019D protein was more sensitive to tunicamycin treatment compared with wild type MNK and caused G1019D to migrate as a single lower band. These data suggest that the double band of G1019D protein in untreated cells arises from incomplete or defective glycosylation and were consistent with the immunofluorescence data suggesting partial retention of the G1019D protein in the ER. We characterized the G1019D mutant protein further by exploring whether varying the copper availability affected the localization of the protein. To mimic the copper deficiency that occurs in various tissues of Menkes disease patients, we limited the copper availability by culturing the cell lines in the presence of the copper chelator, bathocuproine disulfonate (BCS). Copper depletion had no apparent effect on the WtMNK protein, which appeared perinuclear in both untreated (Fig. 2 A) and BCS-treated cells (Fig. 2 C). As seen earlier in Fig. 1, in untreated G1019D-expressing cells, G1019D protein was observed in the perinuclear region as well as a diffuse reticular distribution (Fig.2 B). Surprisingly, BCS treatment caused a striking change in the location of the G1019D protein to a predominant reticular distribution, with a notable absence of perinuclear signal (Fig.2 D). This BCS effect on the location of G1019D was due to copper depletion, because it was not observed when BCS was added in combination with copper (Fig. 2 F). Indeed, the addition of BCS and copper had the opposite effect by shifting the G1019D protein to a predominantly perinuclear location (Fig. 2 F). This localization was indistinguishable from the location of the WtMNK protein, which remained perinuclear in cells treated either with BCS alone or BCS plus copper (Fig. 2, C and E). We further increased the media copper levels to an excess concentration of 100 μm Cu, which is known to bring about copper-induced trafficking of MNK to the plasma membrane, as shown for the WtMNK protein (Fig. 2 G). Notably, the G1019D mutant protein also relocalized to the plasma membrane in copper-treated cells (Fig.2 H), suggesting the G1019D mutation did not inhibit copper-induced trafficking from the TGN. The effect of copper depletion on the G1019D protein was further explored by testing whether the BCS-induced changes in subcellular location were also accompanied by changes in its electrophoretic mobility. In untreated cells, the G1019D protein appeared as the double band (Fig. 3 A, lane 1), however, in BCS-treated cells, the lower band was the predominant form of G1019D and there was a marked diminution of the normal sized upper band (Fig. 3 A, lane 2). Significantly, the combined addition of BCS and copper to G1019D-expressing cells reversed the BCS effect by causing a marked increase in levels of the upper band with a concomitant decrease in levels of the lower band (Fig. 3 A, lane 3). None of the treatments affected the mobility of the WtMNK protein (Fig. 3 A, lanes 4–6). These observations, together with the immunofluorescence data in Fig. 2, suggested that copper limitation reduces secretion and glycosylation of the G1019D mutant protein in early secretory compartments, which can be rescued by copper supplementation. We then investigated the concentrations of copper required to increase the levels of the higher molecular weight G1019D product. The shift toward the higher form of the G1019D protein was observed when G1019D-expressing cells were cultured in 5 μm copper (Fig. 3 B, lane 2) and was saturable with 30 μm copper (Fig. 3 B, lane 4). To test metal specificity, we investigated whether exposure of cells to high levels of zinc, silver, or cadmium could enrich levels of the higher form of the G1019D protein (Fig. 3 B, lanes 6–8). The metal concentrations tested were ∼50% of the levels that retarded cell growth (data not shown). None of these heavy metals changed the ratio of upper and lower bands from those observed in untreated G1019D-expressing cells. These data suggest that the copper-dependent maturation of the G1019D protein is dose-dependent and -specific for this metal ion. In Fig. 2 D, a reticular distribution reminiscent of the ER was observed for the G1019D protein when cells were depleted of copper by BCS treatment. However, to more rigorously test this localization, we tested if the BCS-induced distribution of the G1019D protein co-localized with an ER marker, protein disulfide isomerase (PDI). Fig.4 shows that the reticular localization of the G1019D protein in BCS-treated cells (Fig. 4 D) co-localized with PDI (Fig. 4 E), as indicated by the extensive yellow labeling in the merged image (Fig.4 F). There was no apparent co-localization of the WtMNK and PDI in BCS-treated cells (Fig. 4, A–C). These data indicated that the G1019D mutant was retained in the ER under in copper-limiting conditions. Some disease-causing mutations in other membrane proteins have been shown to cause retention in the ER due to protein misfolding, and some of these misfolded proteins can be corrected by reducing the culturing temperature or by treating cells with the chemical chaperone glycerol (17Halaban R. Svedine S. Cheng E. Smicun Y. Aron R. Hebert D.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5889-5894Crossref PubMed Scopus (155) Google Scholar, 18Payne A.S. Kelly E.J. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10854-10859Crossref PubMed Scopus (188) Google Scholar, 19Sato S. Ward C.L. Krouse M.E. Wine J.J. Kopito R.R. J. Biol. Chem. 1996; 271: 635-638Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar, 20Cheng S.H. Gregory R.J. Marshall J. Paul S. Souza D.W. White G.A. O'Riordan C.R. Smith A.E. Cell. 1990; 63: 827-834Abstract Full Text PDF PubMed Scopus (1423) Google Scholar, 21Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 7243-7248Abstract Full Text PDF PubMed Google Scholar). Therefore, we tested whether glycerol or reduced temperature could prevent the BCS-induced retention of the G1019D protein in the ER (Fig. 5 B). Glycerol could indeed suppress the BCS-induced retention of G1019D protein in the ER, as evident by strong perinuclear staining of G1019D when BCS and 10% glycerol was added to the culture media (Fig. 5 D). The BCS-induced reticular distribution of G1019D was also found to be temperature-sensitive, because it was suppressed by growing cells with BCS at a reduced temperature of 30 °C (Fig. 5 F). None of these treatments altered the perinuclear location of WtMNK (Fig. 5,A, C, and E). These data further support the notion that the G1019D mutation causes temperature-sensitive misfolding of the MNK protein in the endoplasmic reticulum, which can be pharmacologically corrected using copper or glycerol. Our studies indicate that increasing copper availability allows the G1019D protein to migrate through the ER and correctly localize to the TGN. One possible manner by which this occurs is via copper binding to the N-terminal copper binding sites of the mutant G1019D protein, which may stabilize the protein and permit glycosylation and secretion to post-ER compartments. To test this hypothesis, we engineered a G1019D mutant in which all six Cys-X-X-Cys copper binding sites were mutated to Ser-X-X-Ser; refered to asmutant Metal BindingSites, mMBS1–6/G1019D. Me32a cells were transiently transfected with plasmids for WtMNK, G1019D, mMBS1–6 (mutant metal binding sites alone), and mMBS1–6/G1019D and then cultured in either basal medium or elevated copper. The localization of these proteins was then investigated by immunofluorescence microscopy. As expected, WtMNK was perinuclear in untreated cells (Fig.6 A) and dispersed to the plasma membrane in elevated copper conditions (Fig. 6 B). As shown earlier, the G1019D protein was both perinuclear and reticular in untreated cells (Fig. 6 C) and dispersed to the plasma membrane by elevated copper (Fig. 6 D). The mutation of the copper binding sites alone did not affect the normal perinuclear location in untreated cells (Fig. 6 E), however, these sites were essential for copper-induced trafficking to the plasma membrane under elevated copper conditions (Fig. 6 F), as shown in previous studies (13Strausak D., La Fontaine S. Hill J. Firth S.D. Lockhart P.J. Mercer J.F.B. J. Biol. Chem. 1999; 274: 11170-11177Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Significantly, when all six copper binding sites were mutated in combination with the G1019D mutation, the resulting mMBS1–6/G1019D mutant was completely located in a reticular distribution (Fig. 6 G), and this resembled the ER retention of G1019D in copper-deficient conditions shown earlier (Fig.2 F). It was notable that increasing the copper concentration of the media failed to change the reticular distribution of the mMBS1–6/G1019D mutant (Fig. 6 H). These data suggest that the secretion of the G1019D mutant protein to post-ER compartments requires the N-terminal copper binding sites. We then determined whether the copper binding sites were required for the enrichment of the higher molecular weight G1019D protein in cells exposed to elevated copper. Me32a cells were transiently transfected with expression plasmids encoding WtMNK, G1019D, mMBS1–6, or mMBS1–6/G1019D mutants and then exposed for 48 h to either BCS or elevated copper. In BCS-treated cells, both G1019D and mMBS1–6/G1019D proteins were resolved as single bands that were smaller than both the WtMNK and mMBS1–6 proteins (Fig. 7). Significantly, elevated copper treatment of cells rescued the G1019D mutant protein by enriching expression of the correctly sized protein (Fig. 7, lane 6), however, copper treatment did not change the apparent size of the smaller mMBS1–6/G1019D protein (Fig. 7,lane 8). These data, together with the immunofluorescence findings in Fig. 6, suggest that copper binding to the N-terminal copper binding sites is required for proper secretion and glycosylation of the G1019D mutant protein. The G1019D mutant protein was previously shown to have reduced copper transport activity in a complementation assay in the yeast,S. cerevisiae (16Payne A.S. Gitlin J.D. J. Biol. Chem. 1998; 273: 3765-3770Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Here we expand on these previous studies by using a mammalian cell model to explore the effect of this mutation on the cell biology of MNK. The recombinant G1019D protein in cultured cells was partially localized in the ER and the TGN, and this was associated with the existence of two electrophoretic mobilities for the protein. Inhibition of glycosylation using tunicamycin resulted in the appearance of only a single lower G1019D band. This suggested that the faster mobility of the lower G1019D band was the result of defective glycosylation, rather than truncation, degradation, or premature termination of the G1019D protein. Our findings suggest that the mislocalization of the G1019D protein probably contributed to Menkes disease pathology by limiting the transport of dietary copper from enterocytes into circulation and, subsequently, the transport of copper into the appropriate secretory compartments for cuproenzymes. An important finding of our study was that the extent of the mislocalization of the G1019D protein was dependent on copper levels in the cell growth media. Extensive mislocalization in the ER was observed when copper availability was limited by chelation using BCS, and this was associated with the presence of only the lower molecular weight protein. Adding copper in combination with the BCS restored normal perinuclear localization of the G1019D mutant and increased the amount of the normal sized product. This copper-dependent correction was dose-dependent and copper-specific. Collectively, these data suggest that copper limitation impaired glycosylation and secretion of the G1019D mutant protein in early secretory compartments and that these defects could be rescued by copper supplementation. The copper-dependent correction of the G1019D mutant required the N-terminal copper binding sites, because the G1019D mutant in which these sites were mutated was retained in the ER and failed to become glycosylated when exposed to elevated copper. Because mutation of the six copper binding sites alone did not result in ER mislocalization or impaired glycosylation of MNK, these sites are not critical for secretion through the ER per se. Our data suggest that, in the context of a destabilized protein such as G1019D, the binding of copper to these sites may serve to promote a conformation that is able to bypass the ER quality control mechanisms. To date the N-terminal copper binding sites of MNK have been shown to have roles in the acquisition of copper under copper limiting conditions (16Payne A.S. Gitlin J.D. J. Biol. Chem. 1998; 273: 3765-3770Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 22Voskoboinik I. Mar J. Strausak D. Camakaris J. J. Biol. Chem. 2001; 276: 28620-28627Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) and in the copper-regulated trafficking of MNK from the TGN (13Strausak D., La Fontaine S. Hill J. Firth S.D. Lockhart P.J. Mercer J.F.B. J. Biol. Chem. 1999; 274: 11170-11177Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 23Goodyer I.D. Jones E.E. Monaco A.P. Francis M.J. Hum. Mol. Genet. 1999; 8: 1473-1478Crossref PubMed Scopus (60) Google Scholar). Our findings provide evidence that these sites are capable of copper binding early in the biosynthesis of MNK and that this process can be essential for secretion and maturation of mutant MNK proteins, such as G1019D. The export of newly synthesized proteins from the ER to the Golgi is regulated by a quality control mechanism that ensures that only properly folded and assembled proteins exit the ER and misfolded proteins are retained and ultimately degraded. Defective processing of the cystic fibrosis transmembrane regulator protein was originally recognized as an important cause of cystic fibrosis (20Cheng S.H. Gregory R.J. Marshall J. Paul S. Souza D.W. White G.A. O'Riordan C.R. Smith A.E. Cell. 1990; 63: 827-834Abstract Full Text PDF PubMed Scopus (1423) Google Scholar) and has been documented in several proteins involved in other congenital diseases (17Halaban R. Svedine S. Cheng E. Smicun Y. Aron R. Hebert D.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5889-5894Crossref PubMed Scopus (155) Google Scholar, 24Partridge C.J. Beech D.J. Sivaprasadarao A. J. Biol. Chem. 2001; 276: 35947-35952Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The most notable of these is Wilson disease, which is caused by mutations in a copper ATPase, WND, that shows a high degree of sequence similarity to MNK and is also is localized in the TGN (25Hung I.H. Suzuki M. Yamaguchi Y. Yuan D.S. Klausner R.D. Gitlin J.D. J. Biol. Chem. 1997; 272: 21461-21466Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). Wilson disease is caused by a toxic accumulation of hepatic copper due to defective biliary excretion of copper via the WND ATPase. A common Wilson disease mutation, H1069Q, causes retention of the WND protein in the ER (18Payne A.S. Kelly E.J. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10854-10859Crossref PubMed Scopus (188) Google Scholar), and ER retention has been subsequently identified for several other Wilson disease mutations (26Forbes J.R. Cox D.W. Hum. Mol. Genet. 2000; 9: 1927-1935Crossref PubMed Scopus (138) Google Scholar). In several diseases involving misfolded proteins, glycerol treatment or reduced temperature facilitates the proper folding and secretion of the affected mutant protein in cultured cells. The Wilson disease H1069Q mutation is corrected by reduced temperature, and, similarly, we found both reduced temperature and glycerol could suppress mislocalization of the G1019D protein under copper-depleted conditions. It is possible that the large cytoplasmic loop of MNK and WND copper ATPases, which contains the respective G1019 and H1069 amino acids, is a region important in the folding and secretion of these transporters. It is notable that this cytoplasmic loop of WND has been recently shown to interact with the N-terminal region in a copper-dependent manner in vitro (27Tsivkovskii R. MacArthur B.C. Lutsenko S. J. Biol. Chem. 2001; 276: 2234-2242Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Our data showing a dependence of the N-terminal copper bindings for the secretion of the G1019D mutant highlights the possibility that such an interaction in WND and MNK proteins may influence the processing and secretion of these copper transporters in vivo. It is notable that certain misfolded proteins, such as the vassopressin receptor, P-glycoprotein, and apolipoprotein A, which are retained in the ER, can be rescued by exposing cells to their respective ligands (28Wang J. White A.L. J. Biol. Chem. 1999; 274: 12883-12889Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 29Morello J.P. Salahpour A. Laperriere A. Bernier V. Arthus M.F. Lonergan M. Petaja-Repo U. Angers S. Morin D. Bichet D.G. Bouvier M. J. Clin. Invest. 2000; 105: 887-895Crossref PubMed Scopus (477) Google Scholar, 30Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 709-712Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). It is thought that the binding of the ligands to these proteins early in the secretory pathway confers a more stable conformation of the mutant proteins, allowing evasion of the ER quality control mechanisms. Moreover, in some diseases, such as nephrogenic diabetes insipidus, which involves mutations in the vassopressin receptor, the same ligand with protein-stabilizing ability is used to treat patients (29Morello J.P. Salahpour A. Laperriere A. Bernier V. Arthus M.F. Lonergan M. Petaja-Repo U. Angers S. Morin D. Bichet D.G. Bouvier M. J. Clin. Invest. 2000; 105: 887-895Crossref PubMed Scopus (477) Google Scholar, 31Serradeil-Le Gal C. Cardiovasc. Drug Rev. 2001; 19: 201-214PubMed Google Scholar). The demonstration that copper could correct the mislocalization of the Menkes G1019D mutant is clearly analogous to the ligand-dependent rescue of these other mutant membrane proteins. Moreover, our findings offer novel insights at the molecular level to explain how current treatments for Menkes disease, which involve parenteral administration of copper, may yield positive outcomes in certain individuals whose mutations affect the processing and secretion of MNK. Although we have no specific information on the copper status of the G1019D Menkes patient, there is typically a marked decrease in circulating serum copper levels due to reduced intestinal absorption. A more severe copper deficiency in the brain occurs due to the predicted role of MNK in the transport of copper across the blood-brain barrier. Our findings allow us to speculate that reduced circulating copper levels in the G1019D Menkes patient may have impaired the maturation of the G1019D protein in various tissues, especially in the brain. Moreover, our finding that copper could suppress this mislocalization in cultured cells suggests in principle that a course of treatment involving copper injections may have promoted normal localization of the G1019D protein and sufficient copper transport activity for a positive clinical response. Clearly, future studies with transgenic mice and cultured cells that express various Menkes disease alleles, such as G1019D, will be a useful strategy for understanding the affects of copper treatments on the function of mutant MNK proteins at the cellular and physiological levels. We thank Drs. David Eide and Elizabeth Rogers for critical evaluation of the manuscript and Dr. Julian Mercer for MNK antibodies and the mutant metal binding site plasmid." @default.
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• W2023416426 title "A Conditional Mutation Affecting Localization of the Menkes Disease Copper ATPase" @default.
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