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

在本项目中，我们得到了以下关于Ca^<2+>/H^+交换剂、Zn^<2+>/H^+交换剂、金属结合蛋白、液泡膜质子泵的研究结果。(1)阳离子/H^+交换剂：水稻中有5种阳离子/H^+交换剂（CAX）同工型。CAX1a在5种亚型中均有表达，且在伴生细胞和内胚层细胞中高表达，具有高浓度的Ca^<2+>。我们估计CAX1a的功能是将Ca^<2+>保留到液泡中，并参与Ca^<2+>的解毒。(2)新型金属结合蛋白（PCaP1）：在拟南芥中发现了一种新型金属阳离子结合蛋白AtPCaP1。该蛋白被n -肉豆芽酰基化，并在体内和体外稳定地与质膜结合。AtPCaP1结合Ca^<2+>, Cu^<2+>, Ca^<2+>/钙调蛋白复合物和磷脂酰肌醇磷酸。根据这些生化特性，我们估计该蛋白参与了环境胁迫的信号转导，如金属和病原体胁迫。(3)液泡锌转运蛋白（Vacuolar zinc transporter, MTP1）：已证实拟南芥的MTP1位于液泡膜上。MTP1基因敲除突变体对高浓度锌（大于0.2 mM）高度敏感。因此，MTP1可能通过将细胞质中过量的Zn^<2+ b>离子转运到液泡中参与Zn解毒。通过在酵母中异源表达MTP1并使用Zn-65直接测量锌的运输，我们证明了MTP1作为Zn^<2+>/H^+交换剂的功能。此外，我们发现细胞质富含组氨酸的区域作为Zn2+的缓冲袋和细胞质表面锌水平的传感器。(4)液泡质子泵：液泡膜H^+-焦磷酸酶（H^+-PPase）使液泡酸化并维持膜上的pH梯度。我们确定了酶的基本残基和功能基序。

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.