title: Amino acid screening based on structural modeling identifies critical residues for the function, ion selectivity and structure of Arabidopsis MTP1.
abstract: Arabidopsis thaliana MTP1 is a vacuolar membrane Zn(2+)/H(+) antiporter of the cation diffusion facilitator family. Here we present a structure-function analysis of AtMTP1-mediated transport and its remarkable Zn(2+) selectivity by functional complementation tests of more than 50 mutant variants in metal-sensitive yeast strains. This was combined with homology modeling of AtMTP1 based on the crystal structure of the Escherichia coli broad-specificity divalent cation transporter YiiP. The Zn(2+)-binding sites of EcYiiP in the cytoplasmic C-terminus, and the pore formed by transmembrane helices TM2 and TM5, are conserved in AtMTP1. Although absent in EcYiiP, Cys31 and Cys36 in the extended N-terminal cytosolic domain of AtMTP1 are necessary for complementation of a Zn-sensitive yeast strain. On the cytosolic side of the active Zn(2+)-binding site inside the transmembrane pore, Ala substitution of either Asn258 in TM5 or Ser101 in TM2 non-selectively enhanced the metal tolerance conferred by AtMTP1. Modeling predicts that these residues obstruct the movement of cytosolic Zn(2+) into the intra-membrane Zn(2+)-binding site of AtMTP1. A conformational change in the immediately preceding His-rich cytosolic loop may displace Asn258 and permit Zn(2+) entry into the pore. This would allow dynamic coupling of Zn(2+) transport to the His-rich loop, thus acting as selectivity filter or sensor of cytoplasmic Zn(2+) levels. Individual mutations at diverse sites within AtMTP1 conferred Co and Cd tolerance in yeast, and included deletions in N-terminal and His-rich intra-molecular cytosolic domains, and mutations of single residues flanking the transmembrane pore or participating in intra- or inter-molecular domain interactions, all of which are not conserved in the non-selective EcYiiP.
authors.affiliation: Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Japan.
authors.forename: Miki
authors.initials: M
authors.lastname: Kawachi
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Kobae
authors.forename: Sayaka
authors.initials: S
authors.lastname: Kogawa
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Mimura
authors.forename: Ute
authors.initials: U
authors.lastname: Krämer
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Maeshima
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics*; D017360:Arabidopsis; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D002104:Cadmium / Q000378:metabolism; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003035:Cobalt / Q000378:metabolism; D015151:Immunoblotting; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014617:Vacuoles; D015032:Zinc / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D029681:Arabidopsis Proteins; D002104:Cadmium; D027682:Cation Transport Proteins; D003035:Cobalt; D015032:Zinc; C500829:ZAT protein, Arabidopsis