SUMMARY Apreviouslyundescribedautosomaldominantsyndrome hasbeen observedin a largekindred with affected relatives spanning at least five generations. The phenotype is highlyvariableand appears to showcomplete penetrance. Affected persons have a progressively coarse, acromegaloid-like facial appearance and thickening of the lips and intraoral mucosa. Thedifferences are discussed between this syndrome and three rather similar syndromes,pachydermoperiostosis, the Ascher syndrome, and multiple neuroma syndrome. In this paper we describe a large kindred with apreviously unreported syndrome segregating in anautosomal dominantfashion. Despite similarities infacial appearance, the syndrome appears to bedistinct from acromegaly, pachydermoperiostosis,and the Ascher and multiple neuroma syndromes.Thereis no knownassociated health risk. Although some affected persons were concerned about theirappearance, most were unaware of any problemuntil the proband requested corrective surgery. At Received for
Side effects of new medications often do not reveal themselves in the course of initial clinical trials but are discovered when their use becomes a standard of care and, therefore, widespread (eg, cyclooxygenase-2 inhibitors, hormone replacement therapy, thalidomide). This is particularly true for
A new inherited variant of α 1 -antitrypsin (protease inhibitor or Pi) has been found in five individuals of a family of Welsh origin. The new allele is called Pi N , as the α 1 AT product migrates in acid starch gel between the products of the Pi M and Pi P alleles. The individuals carrying the Pi N allele are all of Pi type MN. The new variant has been compared in several electrophoretic systems with other variants migrating in a similar region by acid starch gel electrophoresis (M, P, S, V, W and X). Acid starch gel and crossed antigen-antibody electrophoresis are most suitable for distinguishing the Pi N product. By immunofixation electrophoresis, N has a mobility only slightly different from that of M, however the value of this method can be seen for distinguishing other slow variants which cannot be clearly distinguished on acid starch gels. Twenty-three variants of α 1 AT are now known. Twenty-two of these are electrophoretic variants and one, the null allele (Pi − ), produces no α 1 AT.
We have analyzed the functional effect of site-directed mutations and deletions in the copper-binding domain of ATP7B (the copper transporting P-type ATPase defective in Wilson disease) using a yeast complementation assay. We have shown that the sixth copper-binding motif alone is sufficient, but not essential, for normal ATP7B function. The N-terminal two or three copper-binding motifs alone are not sufficient for ATP7B function. The first two or three N-terminal motifs of the copper-binding domain are not equivalent to, and cannot replace, the C-terminal motifs when placed in the same sequence position with respect to the transmembrane channel. From our data, we propose that the copper-binding motifs closest to the channel are required for the copper-transport function of ATP7B. We propose that cooperative copper binding to the copper-binding domain of ATP7B is not critical for copper transport function, but that cooperative copper binding involving the N-terminal two or three copper-binding motifs may be involved in initiating copper-dependent intracellular trafficking. Our data also suggest a functional difference between the copper-binding domains of ATP7A and ATP7B. We have analyzed the functional effect of site-directed mutations and deletions in the copper-binding domain of ATP7B (the copper transporting P-type ATPase defective in Wilson disease) using a yeast complementation assay. We have shown that the sixth copper-binding motif alone is sufficient, but not essential, for normal ATP7B function. The N-terminal two or three copper-binding motifs alone are not sufficient for ATP7B function. The first two or three N-terminal motifs of the copper-binding domain are not equivalent to, and cannot replace, the C-terminal motifs when placed in the same sequence position with respect to the transmembrane channel. From our data, we propose that the copper-binding motifs closest to the channel are required for the copper-transport function of ATP7B. We propose that cooperative copper binding to the copper-binding domain of ATP7B is not critical for copper transport function, but that cooperative copper binding involving the N-terminal two or three copper-binding motifs may be involved in initiating copper-dependent intracellular trafficking. Our data also suggest a functional difference between the copper-binding domains of ATP7A and ATP7B. Heavy metal transporting P-type ATPases (designated CPx-type ATPases) are distinguished from other P-type ATPases, by the presence of a large N-terminal metal-binding domain (1Solioz M. Bittar E.E. Anderson J.P. Advances in Molecular and Cell Biology. JAI Press, London1998: 167-203Google Scholar). This domain contains repeats of a GMXCXXCXXXIE motif evolutionarily conserved in heavy metal-binding proteins. Metal binding to these motifs occurs via the cysteine (C) residues (2Lutsenko 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, 3DiDonato 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, 4Gitschier J. Moffat B. Reilly D. Wood W.I. Fairbrother W.J. Nat. Struct. Biol. 1998; 5: 47-54Crossref PubMed Scopus (211) Google Scholar). The number of motifs varies between proteins. The copper-binding domains of ATP7A (human Menkes disease protein) and ATP7B (human Wilson disease protein) contain six repeats of the copper-binding motif, whereas Ccc2p, the yeast orthologue of ATP7B/ATP7A, has an N-terminal domain containing two motifs (5Vulpe C. Levinson B. Whitney S. Packman S. Gitschier J. Nat. Genet. 1993; 3: 7-13Crossref PubMed Scopus (1208) Google Scholar, 6Mercer J.F.B. Livingstone 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, 7Chelly 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, 8Tanzi R.E. Petrukhin K.E. 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: 344-350Crossref PubMed Scopus (1173) Google Scholar, 9Bull P.C. Thomas G.R. Rommens J.M. Forbes J.R. Cox D.W. Nat. Genet. 1993; 5: 327-337Crossref PubMed Scopus (1686) Google Scholar, 10Yuan D.S. Stearman R. Dancis A. Dunn T. Beeler T. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2632-2636Crossref PubMed Scopus (390) Google Scholar). The copper-binding domains of ATP7A and ATP7B expressed and purified from bacteria have been shown to bind at least six atoms of copper (2Lutsenko 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, 3DiDonato 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). These domains bind copper presented as either Cu(I) or Cu(II). However once bound, copper is found only as Cu(I). Copper binding to the N-terminal domain of ATP7B may be cooperative, based on results from competitive zinc binding experiments (3DiDonato 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).The solution structure of the fourth copper-binding motif of ATP7A has been solved by NMR (4Gitschier J. Moffat B. Reilly D. Wood W.I. Fairbrother W.J. Nat. Struct. Biol. 1998; 5: 47-54Crossref PubMed Scopus (211) Google Scholar). The structure of this motif was solved with silver(I) bound, which was assumed to be similar to the copper bound form. The fourth ATP7A motif folded independently, and the structure closely resembled the overall fold of the MerP mercury-binding protein which has a mercury-binding domain well conserved at the sequence level with the copper-binding motifs found in CPx-type ATPases. The structure of the fourth ATP7A motif likely represents the prototypical fold of copper-binding motifs found in CPx-type ATPases. Each motif may fold individually, and then fold again with respect to the other motifs to form the complete copper-binding domain.There have been several hypotheses presented regarding the role of copper binding to the copper-binding domain in the overall function of CPx-type ATPases. The copper-binding domain has been proposed to remove copper from cytosolic ligands and transiently bind copper prior to transport (5Vulpe C. Levinson B. Whitney S. Packman S. Gitschier J. Nat. Genet. 1993; 3: 7-13Crossref PubMed Scopus (1208) Google Scholar, 11Bull P.C. Cox D.W. Trends Genet. 1994; 10: 246-252Abstract Full Text PDF PubMed Scopus (272) Google Scholar). A recent paper substantiated this hypothesis (12Pufahl R.A. Singer C.P. Peariso K.L. Lin S.J. Schmidt P.J. Fahrni C.J. Culotta V.C. Penner-Hahn J.E. O'Halloran T.V. Science. 1997; 278: 853-856Crossref PubMed Scopus (585) Google Scholar). Atx1p is a yeast copper chaperone protein required upstream of Ccc2p for iron and copper homeostasis in yeast (13Lin S.-J. Pufahl R.A. Dancis A. O'Halloran T.V. Culotta V.C. J. Biol. Chem. 1997; 272: 9215-9220Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar). Yeast two-hybrid analysis demonstrated that Atx1p, which contains one copper-binding motif, was able to directly interact with the putative copper-binding domain of Ccc2p, but no other predicted domain of Ccc2p (12Pufahl R.A. Singer C.P. Peariso K.L. Lin S.J. Schmidt P.J. Fahrni C.J. Culotta V.C. Penner-Hahn J.E. O'Halloran T.V. Science. 1997; 278: 853-856Crossref PubMed Scopus (585) Google Scholar). This interaction was dependent on copper ions and suggested that Atx1p could donate copper to Ccc2p by direct interaction and copper exchange between homologous GMXCXXCXXXIE motifs (12Pufahl R.A. Singer C.P. Peariso K.L. Lin S.J. Schmidt P.J. Fahrni C.J. Culotta V.C. Penner-Hahn J.E. O'Halloran T.V. Science. 1997; 278: 853-856Crossref PubMed Scopus (585) Google Scholar). Since a human orthologue of Atx1p exists (ATOX1: originally designated HAH1) and was able to functionally replace Atx1p (14Klomp 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), likely ATP7B also requires a copper chaperone, probably ATOX1, as its major source of copper for transport. These data support the hypothesis that copper removed from a cytosolic chaperone protein and transiently bound to the ATP7B copper-binding domain is the source of copper for subsequent transport.Another proposed role for the copper-binding domain is that of a copper sensor (3DiDonato 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, 5Vulpe C. Levinson B. Whitney S. Packman S. Gitschier J. Nat. Genet. 1993; 3: 7-13Crossref PubMed Scopus (1208) Google Scholar, 15Petris M.J. Mercer J.F.B. Culvenor J.G. Lockhart P. Gleeson P.A. Camakaris J. EMBO J. 1996; 15: 6084-6095Crossref PubMed Scopus (528) Google Scholar). When mammalian cells were exposed to high concentrations of copper, ATP7A and ATP7B underwent a reversible copper-regulated trafficking event, from the trans-Golgi network to the plasma membrane or a post-Golgi vesicular compartment, respectively (15Petris M.J. Mercer J.F.B. Culvenor J.G. Lockhart P. Gleeson P.A. Camakaris J. EMBO J. 1996; 15: 6084-6095Crossref PubMed Scopus (528) Google Scholar, 16Hung I.H. Suzuki M. Yamaguchi Y. Yuan D.S. Klausner R.D. Gitlin J.D. J. Biol. Chem. 1997; 272: 21461-21466Crossref PubMed Scopus (287) Google Scholar). This trafficking may represent a change in physiologic function of ATP7B from cupro-enzyme biosynthesis in the Golgi apparatus, to a copper efflux function in the plasma membrane or secretory vesicles. The observed trafficking event may be triggered by conformation changes induced by cooperative copper binding to the N-terminal domains of these proteins (3DiDonato 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).The metal binding properties of the ATP7B copper-binding domain are beginning to be understood. However, the functional significance of copper binding to the copper-binding domain of ATP7B is not yet well characterized. In this study we describe the functional consequences of mutations and deletions in the copper-binding domain of ATP7B in an attempt to further understand the role of this domain in the overall copper transport function of ATP7B.The assay used in our study is based on complementation ofccc2 mutant yeast by ATP7B, an assay we have previously used to determine the effect of Wilson disease missense mutations on ATP7B function (17Forbes J.R. Cox D.W. Am. J. Hum. Genet. 1998; 63: 1663-1674Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). In yeast Saccharomyces cerevisiae, the plasma membrane protein Ctr1p transports copper into the cytoplasm, where it is carried by the copper chaperone Atx1p to Ccc2p (13Lin S.-J. Pufahl R.A. Dancis A. O'Halloran T.V. Culotta V.C. J. Biol. Chem. 1997; 272: 9215-9220Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar, 18Dancis A. Yuan D.S. Haile D. Askwith C. Eide D. Moehle C. Kaplan J. Klausner R.D. Cell. 1994; 76: 393-402Abstract Full Text PDF PubMed Scopus (561) Google Scholar). Copper is supplied by Ccc2p, across the membrane of a post-Golgi vesicular compartment (19Yuan D.S. Dancis A. Klausner R.D. J. Biol. Chem. 1997; 272: 25787-25793Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), to the multi-copper containing oxidase Fet3p. Fet3p functions, at the plasma membrane, together with the high-affinity iron transporter Ftr1p to import iron (20Stearman R. Yuan D.S. Yamaguchi-Iwai Y. Klausner R.D. Dancis A. Science. 1996; 271: 1552-1557Crossref PubMed Scopus (574) Google Scholar). When yeast cells lack Ccc2p, copper is not incorporated into Fet3p, and subsequently the cells lack high-affinity iron uptake leaving them unable to grow on iron-limited medium (10Yuan D.S. Stearman R. Dancis A. Dunn T. Beeler T. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2632-2636Crossref PubMed Scopus (390) Google Scholar, 20Stearman R. Yuan D.S. Yamaguchi-Iwai Y. Klausner R.D. Dancis A. Science. 1996; 271: 1552-1557Crossref PubMed Scopus (574) Google Scholar). We have shown that ATP7B is able to complement the yeast mutant ccc2, delivering copper to Fet3p, thereby restoring the ability ofccc2 mutant yeast cells to grow on iron-limited medium, providing a sensitive assay for the copper transporter function of ATP7B (17Forbes J.R. Cox D.W. Am. J. Hum. Genet. 1998; 63: 1663-1674Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar).Similar yeast complementation assays have recently been used to study the functional effect of mutations in the copper-binding domain of ATP7A (21Payne A.S. Gitlin J.D. J. Biol. Chem. 1998; 273: 3765-3770Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) and the effect of deletions in the copper-binding domain of ATP7B (22Iida M. Terada K. Sambongi Y. Wakabayashi T. Miuna N. Koyama K. Futai M. Sugiyama T. FEBS Lett. 1998; 428: 281-285Crossref PubMed Scopus (96) Google Scholar). Our study represents the most comprehensive analysis to date of the copper-binding domain of ATP7B. Additionally, our data suggest a functional difference between the copper-binding domains of ATP7B, and that reported for ATP7A.RESULTSThe copper-binding domains of the mutant ATP7B expression constructs analyzed for function in this study are shown in Fig.1. All constructs were expressed inccc2 mutant yeast from the single copy integrating vector unless otherwise noted.Series 1 constructs were those in which both cysteine residues in each of the copper-binding motifs were mutated to serine sequentially from the N-terminal to C-terminal end of the copper-binding domain (Fig. 1). These were analyzed for function based on their ability to complement the high affinity iron uptake deficiency phenotype of ccc2mutant yeast. When expressed in yeast, all proteins but Cu1–6C/S were able to complement the ccc2 yeast mutant allowing the cells to grow on iron-limited medium (Fig.2 A). We have shown previously that mutant proteins unable to fully complement ccc2 mutant yeast at single copy expression levels could often complement when overexpressed from a multicopy vector (17Forbes J.R. Cox D.W. Am. J. Hum. Genet. 1998; 63: 1663-1674Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Overexpression from the multicopy vector produced approximately 30-fold more ATP7B protein than from the single copy vector, and saturated the yeast cell membranes with ATP7B protein (data not shown). A mutant protein which failed to complement when expressed in multicopy was considered completely non-functional. Cu1–6C/S also failed to complement when expressed from the multicopy vector indicating that the mutant protein was nonfunctional (data not shown).Figure 2Complementation of ccc2mutant yeast by ATP7B copper-binding domain mutation series 1 constructs (Fig. 1). A, plating assays were performed as described under "Experimental Procedures."B, Fet3p oxidase assays were performed as described under "Experimental Procedures." Holo-Fet3p activity, Fet3p copper loadedin vivo, was detected by homogenizing yeast in buffer containing the copper chelator bathocuproine disulfonate and reducing agent ascorbate to prevent artifactual copper loading of apo-Fet3p during processing. Total-Fet3p activity, holo-Fet3p plus apo-Fet3p activity, was detected by homogenizing yeast in the presence of copper to reconstitute apo-Fet3p in vitro. Western blots were done, using anti-ATP7B.C10 antibody, on 10 μg of solubilized membrane protein prepared for the oxidase assay.View Large Image Figure ViewerDownload (PPT)Fet3p oxidase assays were employed to measure the function of Series 1 ATP7B copper-binding domain mutants. Fet3p receives its copper from Ccc2p within a vesicular compartment and therefore serves as a marker protein for copper transport across the vesicular membrane (19Yuan D.S. Dancis A. Klausner R.D. J. Biol. Chem. 1997; 272: 25787-25793Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). In wild-type yeast and in yeast expressing normal ATP7B, there is little difference between holo- and total-Fet3p activity indicating little or no excess apo-Fet3p production. If high affinity iron uptake is reduced or absent, Fet3p expression is induced in an attempt to compensate (28Askwith C. Eide D. Van Ho A. Bernard P.S. Li L. Davis-Kaplan S. Sipe D.M. Kaplan J. Cell. 1994; 76: 403-410Abstract Full Text PDF PubMed Scopus (582) Google Scholar), therefore a high ratio of total-Fet3p to holo-Fet3p activity indicates an ATP7B mutant protein with absent or reduced function (17Forbes J.R. Cox D.W. Am. J. Hum. Genet. 1998; 63: 1663-1674Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). The Fet3p assay results for the first series of ATP7B mutant proteins showed, with the exception of Cu1–6C/S, little or no difference between holo- and total-Fet3p activity indicating that the first series of copper-binding domain mutant ATP7B proteins analyzed have activity comparable to normal ATP7B (Fig. 2 B). Only the sixth motif was necessary for ATP7B function. Cu1–6C/S protein was nonfunctional judged by its inability to generate detectable holo-Fet3p activity, and the high level of total-Fet3p activity.We have shown previously, by measuring growth curves, thatccc2 mutant yeast expressing ATP7B grows at a rate equal to the wild-type strain in iron-limited medium (17Forbes J.R. Cox D.W. Am. J. Hum. Genet. 1998; 63: 1663-1674Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). We were able to quantitate differences in the ability of mutant ATP7B proteins to complement ccc2 mutant yeast, which was useful as a relative measure of ATP7B function. Growth rates, in iron-limited medium, of yeast expressing ATP7B and Cu1–5C/S were calculated from the linear exponential phase of growth curves done in triplicate (TableI). The ccc2 yeast strain expressing the Cu1–5C/S grew at a rate identical to normal ATP7B. These results confirm that only the sixth copper-binding motif was required for normal transport activity of ATP7B.Table IGrowth rates of ccc2 mutant yeast expressing ATP7B copper-binding domain mutant proteinsStrainGrowth rateΔLog (OD600)/hr%ATP7Bccc20.017 ± 0.00413.5ATP7B0.126 ± 0.000100Cul-5C/S0.128 ± 0.001101.5Cul-5Del0.120 ± 0.00195.2Growth curves in iron-limited medium were generated over 24-h period. Optical density at 600 nm of triplicate cultures was measured at times 0, 3, 6, 12, and 24 h. Growth rates were calculated from the linear exponential growth phase of the cultures using the 3-, 6-, and 12-h time points. Open table in a new tab To determine if copper-binding motifs at the N-terminal end of the copper-binding domain were sufficient for function, Series 2 copper-binding domain mutants were made (Fig. 1). Cu4–6C/S and Cu3–6C/S were made in which the cysteines of copper-binding motifs four to six and three to six were mutated to serine, respectively. These proteins, when expressed, did not restore the ability ofccc2 mutant yeast to grow on iron-limited medium, and generated no detectable holo-Fet3p activity, but had a high total-Fet3p activity indicating they were non-functional (Fig.3). Additionally, expression of these constructs from the multicopy vector failed to complementccc2 mutant yeast (data not shown). However, Cu3–5C/S was able to complement ccc2 mutant yeast and deliver copper to Fet3p as well as normal ATP7B, as indicated by similar levels of holo- and total-Fet3p oxidase activity (Fig. 3). These data demonstrate that two or three N-terminal motifs alone are not sufficient for ATP7B function, and reinforce the functional importance of the sixth copper-binding motif.Figure 3Complementation of ccc2mutant yeast by ATP7B copper-binding domain mutation series 2 constructs (Fig. 1). A, plating assays were performed as described under "Experimental Procedures." B, Fet3p oxidase assays were performed as described under "Experimental Procedures." Holo-Fet3p activity, Fet3p copper loaded in vivo, was detected by homogenizing yeast in buffer containing the copper chelator bathocuproine disulfonate and reducing agent ascorbate to prevent adventitious copper loading of apo-Fet3p during processing. Total-Fet3p activity, holo-Fet3p plus apo-Fet3p activity, was detected by homogenizing yeast in the presence of copper to reconstitute apo-Fet3p in vitro. Western blots were done, using anti-ATP7B.C10 antibody, on 10 μg of solubilized membrane protein prepared for the oxidase assay.View Large Image Figure ViewerDownload (PPT)To determine if the sixth copper-binding motif was essential for ATP7B function, a construct was made (Cu6C/S) in which the cysteine residues within the sixth motif only were mutated to serine (Fig. 1). This protein was able to complement ccc2 mutant yeast, and deliver copper to Fet3p as well as normal ATP7B judged by the similar levels of holo- and total-Fet3p oxidase activity (Fig. 3). These results indicate that the sixth copper-binding motif is sufficient but not essential for ATP7B function.The rodent orthologues of ATP7B all are lacking the fourth copper-binding motif (29Wu J. Forbes J.R. Shiene Chen H. Cox D.W. Nat. Genet. 1994; 7: 541-545Crossref PubMed Scopus (347) Google Scholar, 30Theophilos M.B. Cox D.W. Mercer J.F. Am. J. Hum. Genet. 1996; 5: 1619-1624Google Scholar). However, the spacing and sequence properties between the third and fifth motif are conserved leading to the conclusion that the copper binding property of the fourth motif is not essential for function. As expected, Cu4C/S complementedccc2 mutant yeast cells, allowing normal growth on iron-limited medium, and delivered copper to Fet3p as well as normal ATP7B (ratio of holo- and total-Fet3p oxidase activity similar to ATP7B; Fig. 3).A series of copper-binding domain deletion constructs were analyzed for function. Cu4–6del and Cu3–6del were made so that the third or second copper-binding motifs, respectively, were in the same position relative to the beginning of the first membrane spanning segment as was the sixth copper-binding motif. Neither of these constructs could restore the ability of ccc2 mutant yeast to grow on iron-limited medium (Fig. 4 A). Expression of Cu4–6del or Cu3–6del proteins generated no detectable holo-Fet3p activity, but had a high level of total-Fet3p activity indicating they were nonfunctional (Fig. 4 B). These data indicate that the N-terminal motifs of the copper-binding domain are not equivalent to, and cannot replace, the C-terminal motifs. Deletion of the third to fifth copper-binding motifs (Cu3–5del) resulted in a protein unable to complement ccc2 mutant yeast (Fig. 4 A). Cu3–5del protein was nonfunctional, generating no detectable holo-Fet3p activity, and a high level of total-Fet3p activity (Fig.4 B). None of these deletion constructs could complementccc2 mutant yeast when overexpressed from the multicopy vector, further supporting that they were nonfunctional (data not shown).Figure 4Complementation of ccc2mutant yeast by ATP7B copper-binding domain deletion series constructs (Fig. 1). A, plating assays were performed as described under "Experimental Procedures." B, Fet3p oxidase assays were performed as described under "Experimental Procedures." Holo-Fet3p activity, Fet3p copper loaded in vivo, was detected by homogenizing yeast in buffer containing the copper chelator bathocuproine disulfonate and reducing agent ascorbate to prevent artifactual copper loading of apo-Fet3p during processing. Total-Fet3p activity, holo-Fet3p plus apo-Fet3p activity, was detected by homogenizing yeast in the presence of copper to reconstitute apo-Fet3p in vitro. Western blots were done, using anti-ATP7B.C10 antibody, on 10 μg of solubilized membrane protein prepared for the oxidase assay.View Large Image Figure ViewerDownload (PPT)Cu1–5del was able to complement ccc2 mutant yeast (Fig. 4). The mutant protein delivered copper to Fet3p, and restored the ability of the mutant yeast strain to grow on iron-limited medium. Growth curve analysis revealed that Cu1–5del protein allowed ccc2 mutant yeast to grow at a rate 95% of normal ATP7B expressing ccc2mutant yeast (Table I). Deletion of the entire copper-binding domain (Cudel) resulted in a protein unable to complement ccc2mutant yeast (Fig. 4) even when overexpressed from a multicopy vector (data not shown). Expression of Cudel generated no detectable holo-Fet3p activity, but had a high level of total-Fet3p activity indicating the protein was nonfunctional.Judged by Western blot analysis, several mutant proteins appeared to have a higher steady-state protein level with respect to normal ATP7B (Figs. 2 B, 3 B, and 4 B). This result was reproduced in multiple protein preparations (membrane and total cell extracts) and Western blotting experiments. All of these mutant constructs were confirmed to be correctly integrated in single copy by Southern blot analysis of genomic DNA isolated from these strains probed with ATP7B cDNA (data not shown). In most cases, the mutants exhibiting higher protein levels do not complement ccc2mutant yeast. Constructs Cu1–3C/S, Cu1–4C/S, and Cu3–5C/S do fully complement ccc2 mutant yeast and the proteins appeared to be more abundant relative to normal ATP7B. However, since Cu1–5C/S, which was detected at a level equal to normal ATP7B was able to fully complement ccc2 mutant yeast, we believe that the relatively high steady-state protein level of these constructs was not significant to our results. We are currently investigating the cause for the increased protein levels observed.DISCUSSIONWe have used the yeast complementation assay developed in our laboratory to study the functional consequences of mutation and deletions in the copper-binding domain of ATP7B. Fet3p serves as a marker enzyme for the putative copper-transport function of Ccc2p across an as yet unidentified post-Golgi membrane (19Yuan D.S. Dancis A. Klausner R.D. J. Biol. Chem. 1997; 272: 25787-25793Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). ATP7B is able to replace Ccc2p in yeast demonstrating that it too is a putative copper transporter. ATP-dependent copper uptake has been demonstrated in basolateral membranes of rat and human liver (31Dijkstra M. In t'Veld G. van den Berg G.J. Muller M. Kuipers F. Vonk R.J. J. Clin. Invest. 1995; 95: 412-416Crossref PubMed Google Scholar, 32Dijkstra M. van den Berg G.S. Wolters H. In't Veld G. Sloof M.J. Heymans H.S. Kuipers F. Vonk R.J. J. Hepatol. 1996; 25: 37-42Abstract Full Text PDF PubMed Scopus (15) Google Scholar), and in Golgi membranes from rat hepatocytes (33Bingham M.J. Ong T.J. Ingledew W.J. McArdle H.J. Am. J. Physiol. 1996; 271: G741-G746PubMed Google Scholar). However, these activities have not been specifically assigned to Atp7b. Recent reports have shown that expression of ATP7B in Menkes patient fibroblast cell lines was able to reduce copper accumulation in these cells (34La Fontaine S. Firth S.D. Camakaris J. Englezou A. Theophilos M.B. Petris M.J. Howie M. Lockhart P.J. 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, 35Payne A.S. Kelly E.J. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10854-10859Crossref PubMed Scopus (185) Google Scholar). In light of these observations, together with the impaired hepatic copper efflux found in patients with Wilson disease in whichATP7B is mutated (36Danks D.M. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Disease. McGraw-Hill, New York1995: 2211-2235Google Scholar), ATP7B appears to be copper transporter. However, copper transport by ATP7B remains to be directly demonstrated as has been done for ATP7A (37Voskoboinik I. Brooks H. Smith S. Shen P. Camakaris J. FEBS Lett. 1998; 435: 178-182Crossref PubMed Scopus (62) Google Scholar).The copper-binding domain of ATP7B was required absolutely for its copper transport function. Mutation or deletion of all six copper-binding motifs resulted in a protein unable to complementccc2 mutant yeast or deliver copper to Fet3p (Figs. 2 and4). These data indicate that binding of copper to this domain is a requirement for transport across membranes by ATP7B. Copper transiently bound to the copper-binding domain, perhaps delivered by a chaperone protein, is likely the source of copper transferred to the transmembrane domain for subsequent transport.Our data indicate that the copper-binding motifs of the copper-binding domain nearer to the transmembrane domain of ATP7B were more important for the copper transporting activity of ATP7B than were the more N-terminal ones. Only the sixth motif was necessary, but not essential, for normal function of the protein (Figs. 2 and 4 and Table I). Mutation or deletion of the first five motifs resulted in
