title: A mutagenic study identifying critical residues for the structure and function of rice manganese transporter OsMTP8.1.
abstract: Rice (Oryza sativa) MTP8.1 (OsMTP8.1) is a tonoplast-localized manganese transporter of the cation diffusion facilitator family. Here we present a structure-function analysis of OsMTP8.1 based on the site-directed and random mutagenesis and complementation assays in manganese hypersensitive yeast, in combination with three-dimensional (3D) structure modeling based on the crystal structure of the Escherichia coli CDF family member, EcYiiP. Two metal-binding sites are conserved in OsMTP8.1 with EcYiiP, one is between transmembrane helices TM2 and TM5, the other is the cytoplasmic C-terminus. In addition to these two metal-binding sites, there may exist other Mn-binding sites such as that at the very end of the CTD. Two residues (R167 and L296) may play an important role for the hinge-like movement of CTDs. Several mutations such as E357A and V374D may affect dimer formation, and S132A may induce a conformational change, resulting in a loss of transport function or modification in metal selectivity. The N-terminus of OsMTP8.1 was not functional for Mn transport activity, and the real function of NTD remains to be investigated in the future. The findings of the present study illustrate the structure-function relationship of OsMTP8.1 in Mn transport activity, which may also be applied to other plant Mn-CDF proteins.
authors.affiliation: Department of Biochemistry &Molecular Biology, College of Life Sciences, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China.
authors.forename: Xi
authors.initials: X
authors.lastname: Chen
authors.affiliation: Department of Biochemistry &Molecular Biology, College of Life Sciences, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China.
authors.forename: Jiyu
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Biochemistry &Molecular Biology, College of Life Sciences, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China.
authors.forename: Lihua
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Biochemistry &Molecular Biology, College of Life Sciences, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China.
authors.forename: Gang
authors.initials: G
authors.lastname: Ma
authors.affiliation: Department of Biochemistry &Molecular Biology, College of Life Sciences, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
mesh_terms: D001665:Binding Sites; D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008345:Manganese / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D012275:Oryza / Q000737:chemistry*; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D000072417:Protein Domains; D011523:Protoplasts / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics; D013329:Structure-Activity Relationship; D015032:Zinc / Q000494:pharmacology
chemical_list: D027682:Cation Transport Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; D008345:Manganese; D015032:Zinc