Deletion of a histidine-rich loop of AtMTP1, a vacuolar Zn 2+/H+ antiporter of Arabidopsis thaliana, stimulates the transport activity

Miki Kawachi, Yoshihiro Kobae, Tetsuro Mimura, Masayoshi Maeshima

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127 Citations (Scopus)

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 was tolerant to 10 mM ZnCl2. Active transport of zinc into vacuoles of living yeast cells expressing AtMTP1 was confirmed by the fluorescent zinc indicator FuraZin-1. Zinc transport was quantitatively analyzed by using vacuolar membrane vesicles prepared from AtMTP1-expressing yeast cells and radioisotope 65Zn2+. Active zinc uptake depended on a pH gradient generated by endogenous vacuolar H+-ATPase. The activity was inhibited by bafilomycin A1, an inhibitor of the H +-ATPase. The Km for Zn2+ and Vmax of AtMTP1 were determined to be 0.30 μM and 1.22 nmol/min/mg, respectively. We prepared a mutant AtMTP1 that lacked the major part (32 residues from 185 to 216) of a long histidine-rich hydrophilic loop in the central part of AtMTP1. Yeast cells expressing the mutant became hyperresistant to high concentrations of Zn2+ and resistant to Co2+. The Km and Vmax values were increased 2-11-fold. These results indicate that AtMTP1 functions as a Zn2+/H+ antiporter in vacuoles and that a histidine-rich region is not essential for zinc transport. We propose that a histidine-rich loop functions as a buffering pocket of Zn2+ and a sensor of the zinc level at the cytoplasmic surface. This loop may be involved in the maintenance of the level of cytoplasmic Zn2+.

Original languageEnglish
Pages (from-to)8374-8383
Number of pages10
JournalJournal of Biological Chemistry
Volume283
Issue number13
DOIs
Publication statusPublished - 2008 Mar 28

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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