A Trimeric Quaternary Structure Is Conserved in Bacterial and Human Glutamate Transporters

Abstract

Neuronal and glial glutamate transporters play a central role in the termination of synaptic transmission and in extracellular glutamate homeostasis in the mammalian central nervous system. They are known to be multimers; however, the number of subunits forming a functional transporter is controversial. We studied the subunit stoichiometry of two distantly related glutamate transporters, the human glial glutamate transporter hEAAT2 and a bacterial glutamate transporter from Escherichia coli, ecgltP. Using blue native polyacrylamide gel electrophoresis, analysis of concatenated transporters, and chemical cross-linking, we demonstrated that human and prokaryotic glutamate transporters expressed in Xenopus laevis oocytes or in mammalian cells are assembled as trimers composed of three identical subunits. In an inducible mammalian cell line expressing hEAAT2 the glutamate uptake currents correlate to the amount of trimeric transporters. Overexpression and purification of in in a of trimeric transporters that functional in that an trimeric the native and functional of glutamate transporters. Neuronal and glial glutamate transporters play a central role in the termination of synaptic transmission and in extracellular glutamate homeostasis in the mammalian central nervous system. They are known to be multimers; however, the number of subunits forming a functional transporter is controversial. We studied the subunit stoichiometry of two distantly related glutamate transporters, the human glial glutamate transporter hEAAT2 and a bacterial glutamate transporter from Escherichia coli, ecgltP. Using blue native polyacrylamide gel electrophoresis, analysis of concatenated transporters, and chemical cross-linking, we demonstrated that human and prokaryotic glutamate transporters expressed in Xenopus laevis oocytes or in mammalian cells are assembled as trimers composed of three identical subunits. In an inducible mammalian cell line expressing hEAAT2 the glutamate uptake currents correlate to the amount of trimeric transporters. Overexpression and purification of in in a of trimeric transporters that functional in that an trimeric the native and functional of glutamate transporters. is the in the mammalian central nervous system. the from glial and glutamate transporters glutamate from the synaptic to glutamate and to glutamate mammalian glutamate transporters, blue blue and to to a of the glutamate transporters two They as of and and as is that the and the are of the a a subunit glutamate transporters are known to be and a is to the subunit stoichiometry of known a that glutamate transporters we the subunit stoichiometry of two distantly related glutamate transporters, the human glial glutamate transporter hEAAT2 and the bacterial glutamate transporter from Escherichia from a of that the two transporters as an trimeric of glutamate of in Xenopus and in a or to the hEAAT2 a from and a and the an of to a a a in a the and of and of oocytes as an inducible cell a an hEAAT2 the cells and the and cell the and uptake of of cell and the of Xenopus and currents in oocytes a and currents and and a of a and the currents the to in the and of glutamate currents the uptake uptake currents in mammalian cells an as and a of a to and the extracellular glutamate cells a of an glutamate uptake as the the in the and in the of glutamate a to and of Xenopus and and oocytes the in the or an the oocytes in as the that and in the and cells expressing the transporter a of in and and in and from of cells as to in of or from a in or the the the from the the and and in the of the or in the of and to of as in two of the gel and and and and the to a and a the and of the a to an and in a in in to the a the in and uptake of the in of or the in of and a of hEAAT2 an or a to hEAAT2 and to the transporters a and transporters expressed in Xenopus and currents a We in two to the of the of glutamate and from the In the of the is and the be to the currents in the and of glutamate are in and and hEAAT2 In a hEAAT2 currents that the to currents hEAAT2 a in the of glutamate that glutamate We that the of the the and the of currents of currents in oocytes or and of in Xenopus hEAAT2 and transporters expressed in Xenopus oocytes and expressed in Xenopus oocytes and a and the hEAAT2 and the from the and a analysis the in of the and to that the the and the are are expressed in are from a in the to the in the the of be to the of the of the from the hEAAT2 two and are the and of hEAAT2 in Xenopus oocytes a in a of the to the of of of the two in the as the hEAAT2 and that of the two in of the of the of and a to the of a that of the hEAAT2 be to the hEAAT2 in to from and the to the hEAAT2 analysis demonstrated that of the hEAAT2 the the We that the of hEAAT2 subunits are in a in the the prokaryotic an analysis as in analysis gel and the of the of hEAAT2 transporters expressed in Xenopus oocytes as a of in the of the or the are of the that hEAAT2 transporters as an of in Xenopus of transporters in Xenopus oocytes and mammalian cells of a gel from Xenopus as to of the gel in analysis and are the of the to the of the polyacrylamide the of the that of the are of a gel transporter from the cells the of of trimeric from two and of glutamate uptake from the the line a of the of the amount of the a of is and to an and the number of be from to the number of in transporter is to subunit or of a in the and of a of three in the of of to a of the and an of two of and the the and analysis of the gel in the of a of three to the of and the the hEAAT2 transporter a trimeric as of and that of hEAAT2 in oocytes or of the subunits. is the of the hEAAT2 transporters from the from the of of transporters be the or we an inducible mammalian cell line that hEAAT2 and cell line to of hEAAT2 the to the to the hEAAT2 transporters expressed in mammalian cells and of the cells and in in the amount of the hEAAT2 and glutamate uptake analysis that the hEAAT2 in a trimeric a of in cells and that are a of the of the trimeric hEAAT2 and the glutamate uptake currents the line a of a the the as the amount of trimeric hEAAT2 and of hEAAT2 trimers are that glutamate uptake is hEAAT2 We that a trimeric of the hEAAT2 transporter is a of of in cells the native and functional of the hEAAT2 as in in In a an and an of of to be that of of the analysis that the is the in the as as in the of and of to the of the the and of transporters in Xenopus oocytes of a gel ecgltP. as to of the gel in analysis and are the of the to the of the polyacrylamide the of the analysis of expressed in Xenopus the two to the that are the of the concatenated of is as the trimeric the of concatenated and that hEAAT2 and glutamate transporters are assembled as trimers from a that to the of the in the that the to an and the and to and we a concatenated of the transporters two in a the as a the of the of the In two and to the of a of that and that the as the that the from the to the the and to the and trimeric the two to and are that are and a trimeric that that two from an of two of two subunits or the of three of subunit to the in of the as the assembled from three an of is of a of to a from a the as the is in analysis two the are a the as the and a a to the the that two the in and the of are to in Xenopus oocytes the of to in of hEAAT2 or and to glutamate transporter subunits. and to transporter from Xenopus as as to and to trimers from a and in to and trimers and hEAAT2 transporter subunits to and to trimers trimers hEAAT2 transporters, the trimeric of glutamate transporters. transporters and and hEAAT2 transporters from oocytes and as to and and of and and of a expressed in and a analysis of the demonstrated a an of to the expressed in Xenopus oocytes of the and analysis of the a as a that the of the in the to the from to to the the as that of the is of a and functional of in of from from a of from a the and the of of ecgltP. the of in of the of in of of or analysis to the of the ecgltP. in as a the the and or a the and of the the from the as and hEAAT2 expressed in Xenopus oocytes and mammalian is a and the of to a glutamate uptake the the of glutamate transporters. glutamate uptake the and the or in of transporters Using analysis and of native or and transporters expressed in and trimeric demonstrated that transporters are in native however, the the subunit stoichiometry from as a number of transporter be a transporter that to a number of the In to a trimeric stoichiometry two prokaryotic glutamate transporters and and the that and prokaryotic transporters subunit that is the analysis and chemical of transporters expressed in Xenopus oocytes a trimeric stoichiometry prokaryotic and transporters. of hEAAT2 in an inducible mammalian cell line demonstrated that is of the and in mammalian as as in glutamate and hEAAT2 are that the is the functional of glutamate and concatenated and a we and the trimeric and the we the of and in the glutamate transporter the is the transporters number of glutamate 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central or a central or subunit is the in the mammalian central nervous system. the from glial and glutamate transporters glutamate from the synaptic to glutamate and to glutamate mammalian glutamate transporters, blue blue and to to a of the glutamate transporters two They as of and and as is that the and the are of the a a subunit glutamate transporters are known to be and a is to the subunit stoichiometry of known a that glutamate transporters we the subunit stoichiometry of two distantly related glutamate transporters, the human glial glutamate transporter hEAAT2 and the bacterial glutamate transporter from Escherichia from a of that the two transporters as an trimeric of glutamate transporters. of in Xenopus and in a or to the hEAAT2 a from and a and the an of to a a a in a the and of and of oocytes as an inducible cell a an hEAAT2 the cells and the and cell the and uptake of of cell and the of Xenopus and currents in oocytes a and currents and and a of a and the currents the to in the and of glutamate currents the uptake uptake currents in mammalian cells an as and a of a to and the extracellular glutamate cells a of an glutamate uptake as the the in the and in the of glutamate a to and of Xenopus and and oocytes the in the or an the oocytes in as the that and in the and cells expressing the transporter a of in and and in and from of cells as to in of or from a in or the the the from the the and and in the of the or in the of and to of as in two of the gel and and and and the to a and a the and of the a to an and in a in in to the a the in and uptake of the in of or the in of and a of of in Xenopus and in a or to the hEAAT2 a from and a and the an of to a a a in a the and of and of oocytes as an inducible cell a an hEAAT2 the cells and the and cell the and uptake of of cell and the of Xenopus and currents in oocytes a and currents and and a of a and the currents the to in the and of glutamate currents the uptake uptake currents in mammalian cells an as and a 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hEAAT2 transporter a trimeric as of and that of hEAAT2 in oocytes or of the subunits. is the of the hEAAT2 transporters from the from the of of transporters be the or we an inducible mammalian cell line that hEAAT2 and cell line to of hEAAT2 the to the to the hEAAT2 transporters expressed in mammalian cells and of the cells and in in the amount of the hEAAT2 and glutamate uptake analysis that the hEAAT2 in a trimeric a of in cells and that are a of the of the trimeric hEAAT2 and the glutamate uptake currents the line a of a the the as the amount of trimeric hEAAT2 and of hEAAT2 trimers are that glutamate uptake is hEAAT2 We that a trimeric of the hEAAT2 transporter is a of of in cells the native and functional of the hEAAT2 as in in In a an and an of of to be that of of the analysis that the is the in the as as in the of and of to the of the the and of transporters in Xenopus oocytes of a gel ecgltP. as to of the gel in analysis and are the of the to the of the polyacrylamide the of the analysis of expressed in Xenopus the two to the that are the of the concatenated of is as the trimeric the of concatenated and that hEAAT2 and glutamate transporters are assembled as trimers from a that to the of the in the that the to an and the and to and we a concatenated of the transporters two in a the as a the of the of the In two and to the of a of that and that the as the that the from the to the the and to the and trimeric the two to and are that are and a trimeric that that two from an of two of two subunits or the of three of subunit to the in of the as the assembled from three an of is of a of to a from a the as the is in analysis two the are a the as the and a a to the the that two the in and the of are to in Xenopus oocytes the of to in of hEAAT2 or and to glutamate transporter subunits. and to transporter from Xenopus as as to and to trimers from a and in to and trimers and hEAAT2 transporter subunits to and to trimers trimers hEAAT2 transporters, the trimeric of glutamate transporters. transporters and and hEAAT2 transporters from oocytes and as to and and of and and of a expressed in and a analysis of the demonstrated a an of to the expressed in Xenopus oocytes of the and analysis of the a as a that the of the in the to the from to to the the as that of the is of a and functional of in of from from a of from a the and the of of ecgltP. the of in of the of in of of or analysis to the of the ecgltP. in as a the the and or a the and of the the from the as and hEAAT2 expressed in Xenopus oocytes and mammalian is a and the of to a glutamate uptake the the of glutamate transporters. glutamate uptake the and the or in hEAAT2 an or a to hEAAT2 and to the transporters a and transporters expressed in Xenopus and currents a We in two to the of the of glutamate and from the In the of the is and the be to the currents in the and of glutamate are in and and hEAAT2 In a hEAAT2 currents that the to currents hEAAT2 a in the of glutamate that glutamate We that the of the the and the of currents of currents in oocytes or and of in Xenopus hEAAT2 and transporters expressed in Xenopus oocytes and expressed in Xenopus oocytes and a and the hEAAT2 and the from the and a analysis the in of the and to that the the and the are are expressed in are from a in the to the in the the of be to the of the of the from the hEAAT2 two and are the and of hEAAT2 in Xenopus oocytes a in a of the to the of of of the two in the as the hEAAT2 and that of the two in of the of the of and a to the of a that of the hEAAT2 be to the hEAAT2 in to from and the to the hEAAT2 analysis demonstrated that of the hEAAT2 the the We that the of hEAAT2 subunits are in a in the the prokaryotic an analysis hEAAT2 as in analysis gel and the of the of hEAAT2 transporters expressed in Xenopus oocytes as a of in the of the or the are of the that hEAAT2 transporters as an of in Xenopus of is and to an and the number of be from to the number of in transporter is to subunit or of a in the and of a of three in the of of to a of the and an of two of and the the and analysis of the gel in the of a of three to the of and the the hEAAT2 transporter a trimeric as of and that of hEAAT2 in oocytes or of the subunits. is the of the hEAAT2 transporters from the from the of of transporters be the or we an inducible mammalian cell line that hEAAT2 and cell line to of hEAAT2 the to the to the hEAAT2 transporters expressed in mammalian cells and of the cells and in in the amount of the hEAAT2 and glutamate uptake analysis that the hEAAT2 in a trimeric a of in cells and that are a of the of the trimeric hEAAT2 and the glutamate uptake currents the line a of a the the as the amount of trimeric hEAAT2 and of hEAAT2 trimers are that glutamate uptake is hEAAT2 We that a trimeric of the hEAAT2 transporter is a of of in cells the native and functional of the hEAAT2 as in in In a an and an of of to be that of of the analysis that the is the in the as as in the of and of to the of the the and that hEAAT2 and glutamate transporters are assembled as trimers from a that to the of the in the that the to an and the and to and we a concatenated of the transporters two in a the as a the of the of the In two and to the of a of that and that the as the that the from the to the the and to the and trimeric the two to and are that are and a trimeric that that two from an of two of two subunits or the of three of subunit to the in of the as the assembled from three an of is of a of to a from a the as the is in analysis two the are a the as the and a a to the the that two the in and the of are to in Xenopus oocytes the of to in system. of hEAAT2 or and to glutamate transporter subunits. and to transporter from Xenopus as as to and to trimers from a and in to and trimers and hEAAT2 transporter subunits to and to trimers trimers hEAAT2 transporters, the trimeric and of a expressed in and a analysis of the demonstrated a an of to the expressed in Xenopus oocytes of the and analysis of the a as a that the of the in the to the from to to the the as that of the is of a We analysis to the of the ecgltP. in as a the the and or a the and of the the from the as and hEAAT2 expressed in Xenopus oocytes and mammalian is a and the of to a glutamate uptake the the of glutamate transporters. glutamate uptake the and the or in of transporters Using analysis and of native or and transporters expressed in and trimeric demonstrated that transporters are in native however, the the subunit stoichiometry from as a number of transporter be a transporter that to a number of the In to a trimeric stoichiometry two prokaryotic glutamate transporters and and the that and prokaryotic transporters subunit that is the analysis and chemical of transporters expressed in Xenopus oocytes a trimeric stoichiometry prokaryotic and transporters. of hEAAT2 in an inducible mammalian cell line demonstrated that is of the and in mammalian as as in glutamate and hEAAT2 are that the is the functional of glutamate and concatenated and a we and the trimeric and the we the of and in the glutamate transporter the is the transporters number of glutamate transporter subunit the number of subunits the and the and of of and of are a trimeric subunit stoichiometry to prokaryotic and glutamate trimeric of glutamate transporters is in to the in of transporters, the transporter and transporters prokaryotic transporter and a mammalian transporter to is the of transporters and transporters to be or to transporters, transporters a that to be to that of to of and human transporters expressed in Xenopus oocytes and as an that is the transporters and as the is in transporters that as In of as the that be the of transporters be a number of subunits the of transporter and the subunit the transporters a the of three identical subunits. that is to is as the however, the that subunits are in glutamate transporters. and of expressed transporters and In or a be and purification of hEAAT2 and in Xenopus oocytes as as in mammalian trimeric of glutamate transporters is and to be the in and that of glutamate transporters two we the three subunits to the of a central or a central or subunit of transporters Using analysis and of native or and transporters expressed in and trimeric demonstrated that transporters are in native however, the the subunit stoichiometry from as a number of transporter be a transporter that to a number of the In to a trimeric stoichiometry two prokaryotic glutamate transporters and and the that and prokaryotic transporters subunit We that is the analysis and chemical of transporters expressed in Xenopus oocytes a trimeric stoichiometry prokaryotic and transporters. of hEAAT2 in an inducible mammalian cell line demonstrated that is of the and in mammalian as as in glutamate and hEAAT2 are that the is the functional of glutamate and concatenated and a we and the trimeric and the we the of and in the glutamate transporter the is the transporters number of glutamate transporter subunit the number of subunits the and the and of of and of are a trimeric subunit stoichiometry to prokaryotic and glutamate transporters. trimeric of glutamate transporters is in to the in of transporters, the transporter and transporters prokaryotic transporter and a mammalian transporter to is the of transporters and transporters to be or to transporters, transporters a that to be to that of to of and human transporters expressed in Xenopus oocytes and as an that is the transporters and as the is in transporters that as In of as the that be the of transporters be a number of subunits the of transporter and the subunit the transporters a the of three identical subunits. that is to is as the however, the that subunits are in glutamate transporters. and of expressed transporters and In or a be and purification of hEAAT2 and in Xenopus oocytes as as in mammalian trimeric of glutamate transporters is and to be the in and that of glutamate transporters two we the three subunits to the of a central or a central or subunit We the the and of the and in the inducible cell and