Molecular properties of metal-uptake systems supported by proton pumps in plant cells

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We obtained the following results concerning Ca^<2+>/H^+ exchanger, Zn^<2+>/H^+ exchanger, metal-binding protein, proton pumps in the vacuolar membranes in this project.(1) Cation/H^+ exchangers: Rice has five isoforms of cation/H^+ exchanger (CAX). CAX1a among five isoforms is expressed in all tissues examined and is highly expressed in companion and endodermis cells, which has high concentration of Ca^<2+>. We estimate that CAX1a functions to reserve Ca^<2+> into vacuoles and is involved in the Ca^<2+> detoxification.(2) Novel metal binding protein (PCaP1): We found a novel metal cation-binding protein, AtPCaP1, in Arabidopsis thaliana. The protein is N-myristoylated and stably associated with the plasma membrane in vitro and in vivo. AtPCaP1 binds to Ca^<2+>, Cu^<2+>, Ca^<2+>/calmodulin complex, and phosphatidylinositol phosphates. From these biochemical properties, we estimate that the protein is involved in signal transduction of environmental stresses, such as metal and pathogen stresses.(3) Vacuolar zinc transporter (MTP1): Arabidopsis MTP1 has been demonstrated to be located to the vacuolar membrane. The knockout mutant of MTP1 was highly sensitive to high concentrations (more than 0.2 mM) of zinc. Thus, MTP1 might be involved in the Zn detoxification by transport of the excess Zn^<2+> ion in the cytoplasm into vacuoles. By heterologous expression of MTP1 in yeast and direct measurement of zinc transport using Zn-65, we demonstrated that MTP1 functions as Zn^<2+>/H^+ exchanger. Furthermore, we found that the cytoplasmic histidine-rich region functions as a buffering pocket of Zn2+ and a sensor of the zinc level at the cytoplasmic surface.(4) Vacuolar proton pump: Vacuolar membrane H^+-pyrophosphatase (H^+-PPase) acidifies vacuoles and maintains the pH gradient across the membrane. We determined the essential residues and functional motifs in the enzyme.