Deletion of a Histidine-rich Loop of AtMTP1, a Vacuolar Zn2+/H+ Antiporter of Arabidopsis thaliana, Stimulates the Transport Activity

Abstract

Arabidopsis thaliana AtMTP1 belongs to the cation diffusion facilitator family and is localized on the vacuolar membrane. We investigated the enzymatic kinetics of AtMTP1 by a heterologous expression system in the yeast Saccharomyces cerevisiae, which lacked genes for vacuolar membrane zinc transporters ZRC1 and COT1. The yeast mutant expressing AtMTP1 heterologously to of zinc of yeast expressing AtMTP1 by the zinc by vacuolar membrane yeast and zinc on a by vacuolar The by of the The for and of AtMTP1 to and We a mutant AtMTP1 lacked the to of a in the of expressing the mutant to of and to The and AtMTP1 a in and a is for zinc We a a of and a of the zinc the in the of the of Arabidopsis thaliana AtMTP1 belongs to the cation diffusion facilitator family and is localized on the vacuolar membrane. We investigated the enzymatic kinetics of AtMTP1 by a heterologous expression system in the yeast Saccharomyces cerevisiae, which lacked genes for vacuolar membrane zinc transporters ZRC1 and COT1. The yeast mutant expressing AtMTP1 heterologously to of zinc of yeast expressing AtMTP1 by the zinc by vacuolar membrane yeast and zinc on a by vacuolar The by of the The for and of AtMTP1 to and We a mutant AtMTP1 lacked the to of a in the of expressing the mutant to of and to The and AtMTP1 a in and a is for zinc We a a of and a of the zinc the in the of the of is a a for and a for of and and zinc in in zinc in The of zinc to the of and to of zinc transporters and zinc zinc transporters and The family in Arabidopsis thaliana of and a thaliana AtMTP1 and localized on the membrane of in the Arabidopsis investigated of to to of the and of AtMTP1 and to a family of the cation diffusion facilitator which in and and to and to zinc the the in thaliana AtMTP1 the to and and a the and is in The in to a zinc the and of zinc transporters zinc of by of the in and by a yeast a mutant of ZRC1 and thaliana vacuolar zinc AtMTP1 a in zinc and a The of by the mutant of AtMTP1 The of cation diffusion facilitator family of a and the AtMTP1 in Saccharomyces to the for and of the on a zinc to zinc in yeast We of zinc and a expression of a mutant which lacked a of the the of We the and the of the of the to and and mutant of yeast by the The ZRC1 and the the The and and and in the yeast in yeast and and by the on of AtMTP1 in thaliana by The AtMTP1 by and The the of yeast expression The a which zinc by the and the expression of AtMTP1 by the of for AtMTP1 of AtMTP1 mutant the to of in by a and in We the and The a mutant The mutant AtMTP1 a of yeast by the for the AtMTP1 to the in yeast and and for and and and The a the by a and The in and and of in in yeast a zinc to the in by the which yeast and and for and of for The and the and The to and and in the to a of zinc in of and the to a of and to a of the to a of The in the for The by of of zinc and and for The and the in of and on zinc of a The by of yeast to of the the to and the of the of The to the of and The zinc is to the of to vacuolar membrane the in of of and and The of for a of to the of the membrane for and by the the a in The of and The the by a a of vacuolar in the of and The of in yeast vacuolar the of of a for and for and by and to membrane the membrane the and of for of to the of AtMTP1 by a and to the of and of AtMTP1 in the of AtMTP1 in AtMTP1 in of the vacuolar membrane yeast expressing AtMTP1 by a zinc on a yeast expressing AtMTP1 and the to the yeast The yeast the in the of to and of a the of AtMTP1 in a mutant of AtMTP1 and and in yeast lacked and genes membrane of yeast AtMTP1 and the The the of AtMTP1 zinc of yeast expressing and mutant yeast expressing AtMTP1 to and to the of zinc of yeast in AtMTP1 in of and of The of the of zinc in yeast a zinc to the the of and to the zinc The in of of yeast a in the in a a of of yeast the yeast for of of zinc in yeast in yeast AtMTP1 by the yeast and yeast for and a AtMTP1 to the zinc and in the in vacuolar the of yeast yeast of zinc in The membrane by and the of membrane for in of the to the by a of the in the of a by to the zinc is a by by yeast vacuolar membrane vacuolar yeast expressing of in for and to The by the of the membrane a membrane and of in and and vacuolar membrane yeast for in the of The by the of for of membrane zinc to the the of The of in membrane to and the of the zinc of the AtMTP1 of a The of the on the The zinc of AtMTP1 The the The a of and the of membrane by the kinetics of membrane zinc of vacuolar membrane yeast mutant AtMTP1 the of The membrane in for The and The by the of and for the of the a membrane and of of in the membrane The zinc zinc and of zinc on of zinc in vacuolar membrane yeast expressing The membrane in the for The by the of for of in membrane The zinc of a of by AtMTP1 in yeast vacuolar the of vacuolar mutant AtMTP1 of membrane and the in for The for and zinc of zinc on of zinc in vacuolar membrane AtMTP1 mutant and The membrane in the for The by the and zinc for and of and The of and for the and mutant of membrane the in the of in to on the the to and and to of the by and The in the of We the of the a of is the in the on AtMTP1 the in the of the The of on the AtMTP1 the the AtMTP1 to a of on zinc by membrane yeast of in for The by of and for of in the membrane The zinc in the of is to of the and of AtMTP1 a a a We by heterologous expression of a mutant which lacked a of the in mutant the and mutant AtMTP1 in the vacuolar membrane by The mutant AtMTP1 a the the mutant lacked The of the of in yeast expressing mutant in the of the to The yeast which the AtMTP1 to zinc The expressing a mutant to zinc and a to zinc the the is for zinc to on of in to and yeast the and AtMTP1 the AtMTP1 the yeast expressing the mutant to the is for zinc and for and of of the the enzymatic yeast expressing AtMTP1 of the The and AtMTP1 a in the for and the a of and the for of the and AtMTP1 to and of the and mutant AtMTP1 and of The of and mutant AtMTP1 on the of vacuolar membrane The of the of and the in yeast expressing the mutant to of expressing of the to the in the of the AtMTP1 of a AtMTP1 a in for and in the of which lacked a of the The yeast expressing AtMTP1 to in the the by a of a of to the of zinc by AtMTP1 and the of expression of thaliana vacuolar membrane AtMTP1 in a system for of zinc transporters in AtMTP1 and The vacuolar of AtMTP1 and by expression of in thaliana and of The of zinc zinc by of AtMTP1 and by of AtMTP1 the of yeast vacuolar and The mutant is to zinc and to in the of of and to to zinc by of zinc in yeast and is the for zinc AtMTP1 localized in the and in yeast of AtMTP1 a in the AtMTP1 by a by The heterologously AtMTP1 in yeast to on the vacuolar by a of The of by a the membrane. yeast and AtMTP1 the by a and by a The membrane zinc in a and to a in of the is in of the of is of AtMTP1 a by vacuolar and the of AtMTP1 by the and the and and the The in yeast vacuolar membrane the to of the The and AtMTP1 the by AtMTP1 in yeast to in the yeast The mutant of thaliana to to and AtMTP1 a a to zinc in and in the vacuolar a to and yeast vacuolar the to zinc and vacuolar membrane to a for and thaliana vacuolar membrane a to to zinc of a in the of to the a membrane zinc a to the and of the of zinc the of the a of is for zinc the in the the of zinc the to the in the to for zinc in The in of to of AtMTP1 is in zinc the of the to the The is a for the a a and a The of is to of a and of to and and the to of the the in the of The the is in the the to in the yeast expressing a mutant the of the to the to and and for to the the of to a for and of the mutant AtMTP1 to and to by the of the The yeast expressing a mutant to in the The the of and of and and in and We the of the the of and the mutant AtMTP1 and a of the cation diffusion facilitator family thaliana AtMTP1 The and and of AtMTP1 by The of the AtMTP1 is the is to zinc to is the in the The of the mutant the of the the of zinc and the to of AtMTP1 is of and and a to and the a to of The the in the and the the to the of for of and for of of AtMTP1 lacked the the in the the for and the the a and of to the in the a to the of a and the zinc of the AtMTP1 a in the for The of AtMTP1 to the the the in the and a of zinc in the for zinc the the by the and is to in the and the zinc is the and the to the zinc of AtMTP1 and The of AtMTP1 for in yeast membrane to the zinc ZRC1 and The of AtMTP1 is of to zinc and the zinc in the AtMTP1 mutant The is of the of the of in the the of AtMTP1 a to and zinc in the and a of the zinc in the by the and the the to the the zinc is The to a for of zinc for the zinc belongs to the family and the of a in of and a of The of a zinc is to to AtMTP1 is a of the zinc the of the and of the the to and of the of the the and mutant AtMTP1 the the of the is a a for and a for of and and zinc in in zinc in The of zinc to the of and to of zinc transporters and zinc zinc transporters and The family in Arabidopsis thaliana of and a thaliana AtMTP1 and localized on the membrane of in the Arabidopsis investigated of to to of the and of AtMTP1 and to a family of the cation diffusion facilitator which in and and to and vacuolar vacuolar AtMTP1 to zinc the the in thaliana AtMTP1 the to and and a the and is in The in to a zinc the and of zinc transporters zinc of by of the in and by a yeast a mutant of ZRC1 and thaliana vacuolar zinc AtMTP1 a in zinc and a The of by the mutant of AtMTP1 The of cation diffusion facilitator family of a and the AtMTP1 in Saccharomyces to the for and of the on a zinc to zinc in yeast We of zinc and a expression of a mutant which lacked a of the the of We the and the of the of the to and and mutant of yeast by the The ZRC1 and the the The and and and in the yeast in yeast and and by the on of AtMTP1 in thaliana by The AtMTP1 by and The the of yeast expression The a which zinc by the and the expression of AtMTP1 by the of for AtMTP1 of AtMTP1 mutant the to of in by a and in We the and The a mutant The mutant AtMTP1 a of yeast by the for the AtMTP1 to the in yeast and and for and and and The a the by a and The in and and of in in yeast a zinc to the in by the which yeast and and for and of for The and the and The to and and in the to a of zinc in of and the to a of and to a of the to a of The in the for The by of of zinc and and for The and the in of and on zinc of a The by of yeast to of the the to and the of the of The to the of and The zinc is to the of to vacuolar membrane the in of of and and The of for a of to the of the membrane for and by the the a in The of and The the by a a of vacuolar in the of and The of in yeast vacuolar the of of a for and for and by and to membrane the membrane the and of for of to the of AtMTP1 by a and to the of and and mutant of yeast by the The ZRC1 and the the The and and and in the yeast in yeast and and by the on of AtMTP1 in thaliana by The AtMTP1 by and The the of yeast expression The a which zinc by the and the expression of AtMTP1 by the of for AtMTP1 of AtMTP1 mutant the to of in by a and in We the and The a mutant The mutant AtMTP1 a of yeast by the for the AtMTP1 to the in yeast and and for and and and The a the by a and The in and and of in in yeast a zinc to the in by the which yeast and and for and of for The and the and The to and and in the to a of zinc in of and the to a of and to a of the to a of The in the for The by of of zinc and and for The and the in of and on zinc of a The by of yeast to of the the to and the of the of The to the of and The zinc is to the of to vacuolar membrane the in of of and and The of for a of to the of the membrane for and by the the a in The of and The the by a a of vacuolar in the of and The of in yeast vacuolar the of of a for and for and by and to membrane the membrane the and of for of to the of AtMTP1 by a and to the of and of AtMTP1 in the of AtMTP1 in AtMTP1 in of the vacuolar membrane yeast expressing AtMTP1 by a zinc on a yeast expressing AtMTP1 and the to the yeast The yeast the in the of to and of a the the of zinc in yeast a zinc to the the of and to the zinc The in of of yeast a in the in a a of of yeast the yeast for of of zinc in yeast in yeast AtMTP1 by the yeast and yeast for and a AtMTP1 to the zinc and in the in vacuolar the of yeast yeast of zinc in The membrane by and the of membrane for in of the to the by a of the in the of a by to the zinc is a by by yeast vacuolar membrane vacuolar yeast expressing of in for and to The by the of the membrane a membrane and of in and and vacuolar membrane yeast for in the of The by the of for of membrane zinc to the the of The of in membrane to and the of the zinc of the AtMTP1 of a The of the on the The zinc of AtMTP1 The the The a of and the of membrane by the kinetics of membrane zinc of vacuolar membrane yeast mutant AtMTP1 the of The membrane in for The and The by the of and for the of the a membrane and of of in the membrane The zinc zinc and of zinc on of zinc in vacuolar membrane yeast expressing The membrane in the for The by the of for of in membrane The zinc of a of by AtMTP1 in yeast vacuolar the of vacuolar mutant AtMTP1 of membrane and the in for The for and zinc of zinc on of zinc in vacuolar membrane AtMTP1 mutant and The membrane in the for The by the and zinc for and of and The of and for the and mutant of membrane the in the of in to on the the to and and to of the by and The in the of We the of the a of is the in the on AtMTP1 the in the of the The of on the AtMTP1 the the AtMTP1 to a of on zinc by membrane yeast of in for The by of and for of in the membrane The zinc in the of is to of the and of AtMTP1 a a a We by heterologous expression of a mutant which lacked a of the in mutant the and mutant AtMTP1 in the vacuolar membrane by The mutant AtMTP1 a the the mutant lacked The of the of in yeast expressing mutant in the of the to The yeast which the AtMTP1 to zinc The expressing a mutant to zinc and a to zinc the the is for zinc to on of in to and yeast the and AtMTP1 the AtMTP1 the yeast expressing the mutant to the is for zinc and for and of of the the enzymatic yeast expressing AtMTP1 of the The and AtMTP1 a in the for and the a of and the for of the and AtMTP1 to and of the and mutant AtMTP1 and of The of and mutant AtMTP1 on the of vacuolar membrane The of the of and the in yeast expressing the mutant to of expressing of the to the in the of the AtMTP1 of a AtMTP1 a in for and in the of which lacked a of the The yeast expressing AtMTP1 to in the the by a of a of AtMTP1 in the of AtMTP1 in AtMTP1 in of the vacuolar membrane yeast expressing AtMTP1 by a zinc on a yeast expressing AtMTP1 and the to the yeast The yeast the in the of to and of a the the of zinc in yeast a zinc to the the of and to the zinc The in of of yeast a in the in a a of of yeast the yeast for of of yeast yeast of zinc in The membrane by and the of membrane for in of the to the by a of the in the of a by to the zinc is a by The the of The of in membrane to and the of the zinc of the AtMTP1 of a The of the on the The zinc of AtMTP1 The the The a of and the of membrane by the The of membrane the in the of in to on the the to and and to of the by and The in the of We the of the a of is the in the on AtMTP1 the in the of the The of on the AtMTP1 the the AtMTP1 to a of AtMTP1 a a a We by heterologous expression of a mutant which lacked a of the in mutant the and mutant AtMTP1 in the vacuolar membrane by The mutant AtMTP1 a the the mutant lacked The of the of in yeast expressing The mutant in the of the to The yeast which the AtMTP1 to zinc The expressing a mutant to zinc and a to zinc the the is for zinc to on of in to and yeast the and AtMTP1 the AtMTP1 the yeast expressing the mutant to the is for zinc and for and of of the the enzymatic yeast expressing AtMTP1 of the The and AtMTP1 a in the for and the a of and the for of the and AtMTP1 to and of the and mutant AtMTP1 and of The of and mutant AtMTP1 on the of vacuolar membrane The of the of and the in yeast expressing the mutant to of expressing of the to the in the of the AtMTP1 of a AtMTP1 a in for and in the of We which lacked a of the The yeast expressing AtMTP1 to in the the by a of a of to the of zinc by AtMTP1 and the of expression of thaliana vacuolar membrane AtMTP1 in a system for of zinc transporters in AtMTP1 and The vacuolar of AtMTP1 and by expression of in thaliana and of The of zinc zinc by of AtMTP1 and by of AtMTP1 the of yeast vacuolar and The mutant is to zinc and to in the of of and to to zinc by of zinc in yeast and is the for zinc AtMTP1 localized in the and in yeast of AtMTP1 a in the AtMTP1 by a by The heterologously AtMTP1 in yeast to on the vacuolar by a of The of by a the membrane. yeast and AtMTP1 the by a and by a The membrane zinc in a and to a in of the is in of the of is of AtMTP1 a by vacuolar and the of AtMTP1 by the and the and and the The in yeast vacuolar membrane the to of the The and AtMTP1 the by AtMTP1 in yeast to in the yeast The mutant of thaliana to to and AtMTP1 a a to zinc in and in the vacuolar a to and yeast vacuolar the to zinc and vacuolar membrane to a for and thaliana vacuolar membrane a to to zinc of a in the of to the a membrane zinc a to the and of the of zinc the of the a of is for zinc the in the the of zinc the to the in the to for zinc in The in of to of AtMTP1 is in zinc the of the to the The is a for the a a and a The of is to of a and of to and and the to of the the in the of The the is in the the to in the yeast expressing a mutant the of the to the to and and for to the the of to a for and of the mutant AtMTP1 to and to by the of the The yeast expressing a mutant to in the The the of and of and and in and We the of the the of and the mutant AtMTP1 and a the is to zinc to is the in the The of the mutant the of the the of zinc and the to of AtMTP1 is of and and a to and the a to of The the in the and the the to the of for of and for of of AtMTP1 lacked the the in the the for and the the a and of to the in the a to the of a and the zinc of the AtMTP1 a in the for The of AtMTP1 to the the the in the and a of zinc in the for zinc the the by the and is to in the and the zinc is the and the to the zinc of AtMTP1 and The of AtMTP1 for in yeast membrane to the zinc ZRC1 and The of AtMTP1 is of to zinc and the zinc in the AtMTP1 mutant The is of the of the of in the the of AtMTP1 a to and zinc in the and a of the zinc in the by the and the the to the the zinc is The to a for of zinc for the zinc belongs to the family and the of a in of and a of The of a zinc is to to AtMTP1 is a of the zinc the of the and of the the to and of the of the the and mutant AtMTP1 the the of the The of to the of zinc by AtMTP1 and the of expression of thaliana vacuolar membrane AtMTP1 in a system for of zinc transporters in AtMTP1 and The vacuolar of AtMTP1 and by expression of in thaliana and of The of zinc zinc by of AtMTP1 and by of AtMTP1 the of yeast vacuolar and The mutant is to zinc and to in the of of and to to zinc by of zinc in yeast and is the for zinc AtMTP1 localized in the and in yeast of AtMTP1 a in the AtMTP1 by a by The heterologously AtMTP1 in yeast to on the vacuolar by a of The of by a the membrane. yeast and AtMTP1 the by a and by a The membrane zinc in a and to a in of the is in of the of is of AtMTP1 a by vacuolar and We the of AtMTP1 by the and the and and the The in yeast vacuolar membrane the to of the The and AtMTP1 the by AtMTP1 in yeast to in the yeast The mutant of thaliana to to and AtMTP1 a a to zinc in and in the vacuolar a to and yeast vacuolar the to zinc and vacuolar membrane to a for and thaliana vacuolar membrane a to to zinc of a in the of to the a membrane zinc a to the and of the of zinc the of the a of is for zinc the in the the of zinc the to the in the to for zinc in The in of to of AtMTP1 is in zinc We the of the to the The is a for the a a and a The of is to of a and of to and and the to of the the in the of The the is in the the to in the yeast expressing a mutant the of the to the to and and for to the the of to a for and of the mutant AtMTP1 to and to by the of the The yeast expressing a mutant to in the The the of and of and and in and We the of the the of and the mutant AtMTP1 and a The the is to zinc to is the in the The of the mutant the of the the of zinc and the to of AtMTP1 is of and and a to and the a to of The the in the and the the to the of for of and for of of AtMTP1 lacked the the in the the for and the the a and of to the in the a to the of a and the zinc The of the AtMTP1 a in the for The of AtMTP1 to the the the in the and a of zinc in the for zinc the the by the and is to in the and the zinc is the and the to the zinc of AtMTP1 and The of AtMTP1 for in yeast membrane to the zinc ZRC1 and The of AtMTP1 is of to zinc and the zinc in the AtMTP1 mutant The is of the of the of in the the of AtMTP1 a to and zinc in the and a of the zinc in the by the and the the to the the zinc is The to a for of zinc for the zinc belongs to the family and the of a in of and a of The of a zinc is to to AtMTP1 is a of the zinc the of the and of the the to and of the of the the and mutant AtMTP1 the the of the We to for and for of the