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J. Biol. Chem., Vol. 283, Issue 13, 8374-8383, March 28, 2008

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Deletion of a Histidine-rich Loop of AtMTP1, a Vacuolar Zn²⁺/H⁺ Antiporter of Arabidopsis thaliana, Stimulates the Transport Activity^*

Miki Kawachi¹, Yoshihiro Kobae, Tetsuro Mimura, and Masayoshi Maeshima²

From the Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601 and the Department of Biology, Graduate School of Science, Kobe University, Kobe 657-8501, Japan

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 K_m for Zn²⁺ and V_max 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 K_m and V_max 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⁺.

Received for publication, September 12, 2007 , and in revised form, January 15, 2008.

^* This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Sports, Culture, Science, and Technology of Japan, PROBRAIN, RITE, and a grant from Global Research Program of the Ministry of Science and Technology, Korea (to M. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Recipient of Japan Society for the Promotion of Science for Young Scientists Research Fellowship 19-7413.

² To whom correspondence should be addressed. Tel.: 81-52-789-4096; Fax: 81-52-789-4096; E-mail: maeshima{at}agr.nagoya-u.ac.jp.

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