Functional analysis of the rice vacuolar zinc transporter OsMTP1

Abstract

Heavy metal homeostasis is maintained in plant cells by specialized transporters which compartmentalize or efflux metal ions, maintaining cytosolic concentrations within a narrow range. OsMTP1 is a member of the cation diffusion facilitator (CDF)/metal tolerance protein (MTP) family of metal cation transporters in Oryza sativa, which is closely related to Arabidopsis thaliana MTP1. Functional complementation of the Arabidopsis T-DNA insertion mutant mtp1-1 demonstrates that OsMTP1 transports Zn in planta and localizes at the tonoplast. When heterologously expressed in the yeast mutant zrc1 cot1, OsMTP1 complemented its Zn hypersensitivity and was also localized to the vacuole. OsMTP1 alleviated, to some extent, the Co sensitivity of this mutant, rescued the Fe hypersensitivity of the ccc1 mutant at low Fe concentrations, and restored growth of the Cd-hypersensitive mutant ycf1 at low Cd concentrations. These results suggest that OsMTP1 transports Zn but also Co, Fe, and Cd, possibly with lower affinity. Site-directed mutagenesis studies revealed two substitutions in OsMTP1 that alter the transport function of this protein. OsMTP1 harbouring a substitution of Leu82 to a phenylalanine can still transport low levels of Zn, with an enhanced affinity for Fe and Co, and a gain of function for Mn. A substitution of His90 with an aspartic acid completely abolishes Zn transport but improves Fe transport in OsMTP1. These amino acid residues are important in determining substrate specificity and may be a starting point for refining transporter activity in possible biotechnological applications, such as biofortification and phytoremediation.

The PP-motif in luminal loop 2 of ZnT transporters plays a pivotal role in TNAP activation

“…For example, extensive analyses have been conducted in MTP1 in plants and Zrc1 in yeast, and they have identified a number of amino acid residues that are essential for zinc transport activity and for determining metal substrate specificity [23-26]. In vertebrate ZnTs, a few similar studies have been performed, and they confirmed the importance of the HDHD core motif in zinc transport activity and metal substrate specificity [18-22].…”

Got to hide your Zn away: Molecular control of Zn accumulation and biotechnological applications

“…Rice OsMTP1 transports Zn, Co, Fe, Cd and Ni ( Fig. 1) [51,52] and barley HvMTP1 transports Zn and Co [53]. While AtMTP3 transports also Co, AtMTP1 and most of its dicot orthologues seem to be Zn-specific [45].…”

“…Additional residues between transmembrane domains 2 and 3 are also involved in Zn specificity, as single residue mutations were able to confer Co transport activity to AtMTP1 [53]. Similarly, point mutations in OsMTP1 resulted in altered metal selectivity, enhancing Fe and Co transport and adding Mn transport capabilities while lowering or even abolishing Zn transport [51]. Understanding how MTP proteins select each metal and which amino acid residues contribute to selectivity is a key requirement to develop effective biofortification applications.…”

“…The use of transporters to increase Zn concentrations in edible parts of plants, while avoiding toxic elements such as Cd, can utilize recent studies on metal selectivity and structure of transporters, combining crystallographic information, molecular modeling and testing eventual predictions and hypotheses in biological systems, such as yeast and Xenopus oocytes [51,53,76,186]. All of these new approaches should greatly improve our knowledge on Zn homeostasis, and indicate the shortcuts to manipulate plant Zn concentrations for better human nutrition and to design efficient phytoremediation strategies.…”

Papel de COPT1, COPT2, MTP2 y ZIP6 en el transporte y la homeostasis de metales en Medicago truncatula

“…Sin embargo, aparte de transportar Zn 2+ (AtMTP1, AtMTP3, AtMTP5 y AtMTP12; MtMTP1) (Arrivault et al, 2006;Chen et al, 2009;Fujiwara et al, 2015;Kobae et al, 2004), también transportan Co 2+ (AtMTP3) (Arrivault et al, 2006), Fe 2+ y Cd 2+ (OsMTP1) (Menguer et al, 2013). Los MTP del siguiente clado, se han designado mediante ensayos bioinformáticos como transportadores de hierro y zinc (Montanini et al, 2007 , como AtMTP8 (Pedas et al, 2008), OsMTP8.1 (Chen et al, 2013) o AtMTP11 (Delhaize et al, 2007 …”