The Role of GMXCXXC Metal Binding Sites in the Copper-induced Redistribution of the Menkes Protein

Abstract

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. 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 are in the of cellular copper of been and a these are copper proteins essential is demonstrated in the Menkes and The in these were and MNK and MNK and highly membrane proteins of and respectively. to the family of P-type the and the with a of CPX-type or a conserved in the channel and a of metal binding sites used metal binding Menkes TGN, used metal binding Menkes TGN, the copper a MBS MBSs MBSs and the MNK and six metal binding sites The motifs are also in and in proteins the of these motifs in metal the of the N-terminal motifs of both and MNK has been the of These a binding of one copper metal binding for both MNK and and a to and the of the MNK and has been in a mutant in MNK and copper to the high The MBS was to be critical for of MNK and was to be in A further demonstrated that the of a region MBS 6 and MNK is localized to the network (TGN) of MNK from the TGN to the plasma membrane was demonstrated in cells that the MNK this was experiments with cells MNK from a of from the TGN to an as intracellular has also been these is the in the and trafficking of these copper-transporting region that be in the trafficking of MNK is the N-terminal region containing the six of this was to the of the N-terminal MBSs of MNK in copper-induced redistribution. report that not of the MBSs are essential for the of MNK and for a Furthermore, the presence of one MBS a region of amino acids to the channel is necessary and sufficient for the copper-induced trafficking of the Menkes the that that be required for the of MNK from the TGN to the plasma membrane not amino acids of the MNK of the six MBSs in MNK trafficking was and mutagenesis of the MNK MNK mutated in MBS MBS 6 MBSs 1–3 MBSs 4–6 and MBS 5 and MBS 6 were acids the of MNK were deleted to and in this were MBS 5 and MBS 6 to with one MBS of the mutated MNK were and the intracellular of the mutated MNK was the MNK or were for and copper was the region of of the cells with the TGN and with with cells Thus, the mutated were not Furthermore, the not MNK the that even the not changes in the of were exposed to the copper concentrations for were and The shown the of cells cells cells in μm copper for MNK that was mutated in either MBS or MBS 6 was and was the and the plasma membrane were in the of cells mutated or proteins as in copper concentrations not Thus, a to the MNK not the of in cells in their of the of with cells can be resulting from of the To a of the of were from cells that the mutant MNK and These were and that the mutant proteins were of the and to the of MNK of the were A protein of the of was in of and also the of the to the mutated with the Menkes protein MNK was in the This of corresponded well with the of the in the experiments described intracellular of and was of these mutated proteins localized to the TGN in cells cultured in cells were for in μm copper, to the plasma membrane was in the cells and in high copper the of the plasma membrane of these cells and was the 300 μm copper the and the were for and in media containing μm copper, were in the of the for the MNK not to MNK and and remained the TGN in high copper concentrations These demonstrated that the first MBSs were not essential for the of the MBSs to the channel were further the critical for a with MNK mutated in MBS 5 and MBS 6 was cells with this that TGN of the presence of elevated copper, remained the TGN, MNK to the plasma membrane This that MBSs were not sufficient for the of amino acids the MBSs in the N-terminal region a in copper-induced trafficking and both MBS 5 and MBS 6 or one of the were a MNK that lacked MBSs was and mutated MBS 5 and MBS 6 an the MNK that in mutant proteins were located the TGN of when cells were in μm copper for an was the and the plasma membrane These that amino acids of the MNK were not necessary for the copper-induced of the this region not a critical or these that a MBS located a of amino acids from the membrane channel was sufficient for MNK ability of the mutated proteins to confer copper was the of and cells and with the cells and cells MNK in copper concentrations The of copper of and was to that of the cells with an of 6, and the of cells and the was to μm with a of and μm copper, 6 the in copper of cells with cells be a in the of MNK in cells be to these either a MBS or the first MBSs from to the copper-induced intracellular of MNK as shown with and the protein region the first four MBSs is not necessary for one MBS the region MBS 5 to MBS 6 is necessary and sufficient for MNK and the ability of MNK to confer to copper is to its ability to copper-induced demonstrated a of MNK from the TGN to the plasma membrane in a to the was demonstrated in cells that MNK To the MNK response to elevated copper was that the as a in the six MBSs copper, a that the of MNK this used an extensive mutagenesis to define the of the six MBSs in the copper-induced trafficking of of a MBS not the copper-induced of MNK from the TGN to the plasma as demonstrated of MBS and MBS 6 The of that been in Menkes MBS 6 and the first four of the the were either or that are to in a of the MNK These that that the of a MBS are not to a and are that six MBSs are not necessary for MNK elevated copper, was to to the plasma that the first are not essential for the copper-induced trafficking of MNK to and remained the region, even high copper concentrations and This that the MBSs close to the membrane channel were for the intracellular trafficking of MNK. for this was with the containing MBS 5 and MBS 6 that that one MBS close to the channel was necessary and sufficient for MNK trafficking These experiments demonstrated that 478 amino acids the MNK not a critical sequence and were not required for the trafficking of the hypothesis that the metal binding sites a ability of the mutated MNK to corresponded well with their ability to confer copper resistance. The cells and and that MNK mutated in MBSs 4–6 and MBSs not in copper μm for the and MNK and were to copper This demonstrated that the was the of MNK. MNK mutated in the first MBSs were to copper, that MBSs 1–3 were not essential for both MNK trafficking and copper that MNK the TGN and the plasma membrane in cells cultured in media of MNK an in the of of MNK from the TGN to the plasma membrane or a in the of from the plasma these for its in the TGN been The first is a acid sequence containing as a region for of MNK TGN has been shown to a the region of MNK are a of plasma membrane proteins resulting in of these membrane proteins these one hypothesis to the of MNK the plasma membrane is that copper the of the protein a that the This as a of the binding of copper to MBS 5 or MBS To this is of the of of MNK in the presence and of copper be to copper-induced is that changes the the of copper to the channel and the of a copper the changes that are with the copper of MNK a that is the to an in the of MNK that are for the plasma this the of the MBS to the channel be critical either for the of copper to the channel or for the of copper from copper such as that with the protein region MBS 5 and MBS This is experiments from that in and of are to copper the trafficking of the and that a in a region from the metal binding sites in the in also abolished copper-induced These that in MNK that copper also copper-induced to the plasma of 478 N-terminal amino acids containing four MBSs that as with and a with one MBS or MBSs the be sufficient to of copper in The of MBSs the has one in in in and six in This that MNK and the MBSs of the The MBSs to the of copper in the or copper to the channel in the cells of to further the of the six MBSs in the copper-transporting copper of the mutant MNK be 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 are in the of cellular copper of been and a these are copper proteins essential is demonstrated in the Menkes and The in these were and MNK and MNK and highly membrane proteins of and respectively. to the family of P-type the and the with a of CPX-type or a conserved in the channel and a of metal binding sites used metal binding Menkes TGN, used metal binding Menkes TGN, the copper a MBS MBSs MBSs and the MNK and six metal binding sites The motifs are also in and in proteins the of these motifs in metal the of the N-terminal motifs of both and MNK has been the of These a binding of one copper metal binding for both MNK and and a to and the of the MNK and has been in a mutant in MNK and copper to the high The MBS was to be critical for of MNK and was to be in A further demonstrated that the of a region MBS 6 and MNK is localized to the network (TGN) of MNK from the TGN to the plasma membrane was demonstrated in cells that the MNK this was experiments with cells MNK from a of from the TGN to an as intracellular has also been these is the in the and trafficking of these copper-transporting region that be in the trafficking of MNK is the N-terminal region containing the six The of this was to the of the N-terminal MBSs of MNK in copper-induced redistribution. report that not of the MBSs are essential for the of MNK and for a Furthermore, the presence of one MBS a region of amino acids to the channel is necessary and sufficient for the copper-induced trafficking of the Menkes the that that be required for the of MNK from the TGN to the plasma membrane not amino acids of the MNK of the six MBSs in MNK trafficking was and mutagenesis of the MNK MNK mutated in MBS MBS 6 MBSs 1–3 MBSs 4–6 and MBS 5 and MBS 6 were acids the of MNK were deleted to and in this were MBS 5 and MBS 6 to with one MBS of the mutated MNK were and the intracellular of the mutated MNK was the MNK or were for and copper was the region of of the cells with the TGN and with with cells Thus, the mutated were not Furthermore, the not MNK the that even the not changes in the in μm copper for MNK that was mutated in either MBS or MBS 6 was and was the and the plasma membrane were in the of cells mutated or proteins as in copper concentrations not Thus, a to the MNK not the of in cells in their of the of with cells can be resulting from of the To a of the of were from cells that the mutant MNK and These were and that the mutant proteins were of the and to the of MNK of the were A protein of the of was in of and also the of the to the mutated with the Menkes protein MNK was in the This of corresponded well with the of the in the experiments described intracellular of and was of these mutated proteins localized to the TGN in cells cultured in cells were for in μm copper, to the plasma membrane was in the cells and in high copper the of the plasma membrane of these cells and was the 300 μm copper the and the were for and in media containing μm copper, were in the of the for the MNK not to MNK and and remained the TGN in high copper concentrations These demonstrated that the first MBSs were not essential for the of the MBSs to the channel were further the critical for a with MNK mutated in MBS 5 and MBS 6 was cells with this that TGN of the presence of elevated copper, remained the TGN, MNK to the plasma membrane This that MBSs were not sufficient for the of amino acids the MBSs in the N-terminal region a in copper-induced trafficking and both MBS 5 and MBS 6 or one of the were a MNK that lacked MBSs was and mutated MBS 5 and MBS 6 an the MNK that in mutant proteins were located the TGN of when cells were in μm copper for an was the and the plasma membrane These that amino acids of the MNK were not necessary for the copper-induced of the this region not a critical or these that a MBS located a of amino acids from the membrane channel was sufficient for MNK ability of the mutated proteins to confer copper was the of and cells and with the cells and cells MNK in copper concentrations The of copper of and was to that of the cells with an of 6, and the of cells and the was to μm with a of and μm copper, 6 the in copper of cells with cells be a in the of MNK in cells be to these either a MBS or the first MBSs from to the copper-induced intracellular of MNK as shown with and the protein region the first four MBSs is not necessary for one MBS the region MBS 5 to MBS 6 is necessary and sufficient for MNK and the ability of MNK to confer to copper is to its ability to copper-induced redistribution. The of the six MBSs in MNK trafficking was and mutagenesis of the MNK MNK mutated in MBS MBS 6 MBSs 1–3 MBSs 4–6 and MBS 5 and MBS 6 were acids the of MNK were deleted to and in this were MBS 5 and MBS 6 to with one MBS of the mutated MNK were and the intracellular of the mutated MNK was the MNK or were for and copper was the region of of the cells with the TGN and with with cells Thus, the mutated were not Furthermore, the not MNK the that even the not changes in the in μm copper for MNK that was mutated in either MBS or MBS 6 was and was the and the plasma membrane were in the of cells mutated or proteins as in copper concentrations not Thus, a to the MNK not the of MNK. in cells in their of the of with cells can be resulting from of the To a of the of were from cells that the mutant MNK and These were and respectively. To that the mutant proteins were of the and to the of MNK of the were A protein of the of was in of and also the of the to the mutated with the Menkes protein MNK was in the This of corresponded well with the of the in the experiments described The intracellular of and was of these mutated proteins localized to the TGN in cells cultured in cells were for in μm copper, to the plasma membrane was in the cells and in high copper the of the plasma membrane of these cells and was the 300 μm copper the and the were for and in media containing μm copper, were in the of the for the MNK not to MNK and and remained the TGN in high copper concentrations These demonstrated that the first MBSs were not essential for the of the MBSs to the channel were To further the critical for a with MNK mutated in MBS 5 and MBS 6 was cells with this that TGN of the presence of elevated copper, remained the TGN, MNK to the plasma membrane This that MBSs were not sufficient for the of MNK. To amino acids the MBSs in the N-terminal region a in copper-induced trafficking and both MBS 5 and MBS 6 or one of the were a MNK that lacked MBSs was and mutated MBS 5 and MBS 6 an the MNK that in mutant proteins were located the TGN of when cells were in μm copper for an was the and the plasma membrane These that amino acids of the MNK were not necessary for the copper-induced of the this region not a critical or these that a MBS located a of amino acids from the membrane channel was sufficient for MNK The ability of the mutated proteins to confer copper was the of and cells and with the cells and cells MNK in copper concentrations The of copper of and was to that of the cells with an of 6, and the of cells and the was to μm with a of and μm copper, 6 the in copper of cells with cells be a in the of MNK in cells be to MNK. these either a MBS or the first MBSs from to the copper-induced intracellular of MNK as shown with and the protein region the first four MBSs is not necessary for one MBS the region MBS 5 to MBS 6 is necessary and sufficient for MNK and the ability of MNK to confer to copper is to its ability to copper-induced redistribution. demonstrated a of MNK from the TGN to the plasma membrane in a to the was demonstrated in cells that MNK To the MNK response to elevated copper was that the as a in the six MBSs copper, a that the of MNK this used an extensive mutagenesis to define the of the six MBSs in the copper-induced trafficking of of a MBS not the copper-induced of MNK from the TGN to the plasma as demonstrated of MBS and MBS 6 The of that been in Menkes MBS 6 and the first four of the the were either or that are to in a of the MNK These that that the of a MBS are not to a and are that six MBSs are not necessary for MNK elevated copper, was to to the plasma that the first are not essential for the copper-induced trafficking of MNK to and remained the region, even high copper concentrations and This that the MBSs close to the membrane channel were for the intracellular trafficking of MNK. for this was with the containing MBS 5 and MBS 6 that that one MBS close to the channel was necessary and sufficient for MNK trafficking These experiments demonstrated that 478 amino acids the MNK not a critical sequence and were not required for the trafficking of the hypothesis that the metal binding sites a ability of the mutated MNK to corresponded well with their ability to confer copper resistance. The cells and and that MNK mutated in MBSs 4–6 and MBSs not in copper μm for the and MNK and were to copper This demonstrated that the was the of MNK. MNK mutated in the first MBSs were to copper, that MBSs 1–3 were not essential for both MNK trafficking and copper that MNK the TGN and the plasma membrane in cells cultured in media of MNK an in the of of MNK from the TGN to the plasma membrane or a in the of from the plasma these for its in the TGN been The first is a acid sequence containing as a region for of MNK TGN has been shown to a the region of MNK are a of plasma membrane proteins resulting in of these membrane proteins these one hypothesis to the of MNK the plasma membrane is that copper the of the protein a that the This as a of the binding of copper to MBS 5 or MBS To this is of the of of MNK in the presence and of copper be to copper-induced is that changes the the of copper to the channel and the of a copper the changes that are with the copper of MNK a that is the to an in the of MNK that are for the plasma this the of the MBS to the channel be critical either for the of copper to the channel or for the of copper from copper such as that with the protein region MBS 5 and MBS This is experiments from that in and of are to copper the trafficking of the and that a in a region from the metal binding sites in the in also abolished copper-induced These that in MNK that copper also copper-induced to the plasma of 478 N-terminal amino acids containing four MBSs that as with and a with one MBS or MBSs the be sufficient to of copper in The of MBSs the has one in in in and six in This that MNK and the MBSs of the The MBSs to the of copper in the or copper to the channel in the cells of to further the of the six MBSs in the copper-transporting copper of the mutant MNK be demonstrated a of MNK from the TGN to the plasma membrane in a to the was demonstrated in cells that MNK To the MNK response to elevated copper was that the as a in the six MBSs copper, a that the of MNK this used an extensive mutagenesis to define the of the six MBSs in the copper-induced trafficking of MNK. The of a MBS not the copper-induced of MNK from the TGN to the plasma as demonstrated of MBS and MBS 6 The of that been in Menkes MBS 6 and the first four of the the were either or that are to in a of the MNK These that that the of a MBS are not to a and are that six MBSs are not necessary for MNK elevated copper, was to to the plasma that the first are not essential for the copper-induced trafficking of MNK to and remained the region, even high copper concentrations and This that the MBSs close to the membrane channel were for the intracellular trafficking of MNK. for this was with the containing MBS 5 and MBS 6 that that one MBS close to the channel was necessary and sufficient for MNK trafficking These experiments demonstrated that 478 amino acids the MNK not a critical sequence and were not required for the trafficking of the hypothesis that the metal binding sites a The ability of the mutated MNK to corresponded well with their ability to confer copper resistance. The cells and and that MNK mutated in MBSs 4–6 and MBSs not in copper μm for the and MNK and were to copper This demonstrated that the was the of MNK. MNK mutated in the first MBSs were to copper, that MBSs 1–3 were not essential for both MNK trafficking and copper that MNK the TGN and the plasma membrane in cells cultured in media of MNK an in the of of MNK from the TGN to the plasma membrane or a in the of from the plasma these for its in the TGN been The first is a acid sequence containing as a region for of MNK TGN has been shown to a the region of MNK are a of plasma membrane proteins resulting in of these membrane proteins these one hypothesis to the of MNK the plasma membrane is that copper the of the protein a that the This as a of the binding of copper to MBS 5 or MBS To this is of the of of MNK in the presence and of copper be to copper-induced is that changes the the of copper to the channel and the of a copper the changes that are with the copper of MNK a that is the to an in the of MNK that are for the plasma this the of the MBS to the channel be critical either for the of copper to the channel or for the of copper from copper such as that with the protein region MBS 5 and MBS This is experiments from that in and of are to copper the trafficking of the and that a in a region from the metal binding sites in the in also abolished copper-induced These that in MNK that copper also copper-induced to the plasma The of 478 N-terminal amino acids containing four MBSs that as with and a with one MBS or MBSs the be sufficient to of copper in The of MBSs the has one in in in and six in This that MNK and the MBSs of the The MBSs to the of copper in the or copper to the channel in the cells of to further the of the six MBSs in the copper-transporting copper of the mutant MNK be for for with the and for critical of the