A mutagenic study identifying critical residues for the structure and function of rice manganese transporter OsMTP8.1.

Xi Chen,Gang Ma,Lihua Wang,Jiyu Li,Wei Zhang

doi:10.1038/srep32073

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.

Highlights

Manganese (Mn) is an essential micronutrient for plant growth and development
Transport activity of OsMTP8.1 was assessed by complementation assay using a number of yeast mutants that were deficient in various metal transporters
The results showed that a pmr[1] strain but not other mutants were restored after transformation with OsMTP8.1 (Fig. 1A), thereby suggesting that OsMTP8.1 may be a Mn-specific transporter

Summary

Introduction

Manganese (Mn) is an essential micronutrient for plant growth and development. In addition to being a cofactor in many enzymatic processes involved in glucose metabolism and energy production, it acts as a cofactor of MnSOD in the mitochondrion and as part of the water-splitting complex in PSII in the chloroplast[1,2]. MTPs belong to the cation diffusion facilitator (CDF) transporter family and are important for the maintenance of cation homeostasis in bacteria, yeast, plants, and mammals[14,15,16]. By using site-directed mutagenesis, Kawachi et al studied the structural basis of Zn2+ selectivity and transport activity in AtMTP121. They found that two Zn-binding sites (sites A and C) are conserved in AtMTP1 with EcYiiP. They found that the N-terminus of AtMTP1 is very important for metal selectivity They suggested that the N-terminal domain, the His-rich loop, and the leucine zipper motif in TM6 may contribute to the high Zn2+ selectivity in AtMTP1. The sequence of plant Mn-CDF members are highly similar and differ from EcYiiP and AtMTP1 only in terms of structure-function relationships

Methods

Results

Conclusion