Deletion of a histidine-rich loop of AtMTP1, a vacuolar Zn(2+)/H(+) antiporter of Arabidopsis thaliana, stimulates the transport activity.
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 ZnCl(2). 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 (65)Zn(2+). Active zinc uptake depended on a pH gradient generated by endogenous vacuolar H(+)-ATPase. The activity was inhibited by bafilomycin A(1), an inhibitor of the H(+)-ATPase. The K(m) for Zn(2+) and V(max) of AtMTP1 were determined to be 0.30 microm 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 Zn(2+) and resistant to Co(2+). The K(m) and V(max) values were increased 2-11-fold. These results indicate that AtMTP1 functions as a Zn(2+)/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 Zn(2+) and a sensor of the zinc level at the cytoplasmic surface. This loop may be involved in the maintenance of the level of cytoplasmic Zn(2+).
Kawachi
Miki
M
Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan.
Kobae
Yoshihiro
Y
Mimura
Tetsuro
T
Maeshima
Masayoshi
M
D000595:Amino Acid Sequence; D017920:Antiporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017360:Arabidopsis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014617:Vacuoles / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
D017920:Antiporters; D029681:Arabidopsis Proteins; D027682:Cation Transport Proteins; D006639:Histidine; D015032:Zinc; C500829:ZAT protein, Arabidopsis