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

在本项目中，我们获得了关于液泡膜中的Ca^&lt；2&H交换器、锌离子/H交换器、金属结合蛋白、质子泵等方面的研究结果。(1)阳离子/H交换器：水稻有五种阳离子/H交换器(CAX)。五种异构体中的CAX1a在所有组织中都有表达，在伴生细胞和内皮细胞中高表达，内皮细胞中钙离子含量较高。我们估计CAX1a具有将钙储存到液泡中的功能，并参与钙的解毒。(2)新的金属结合蛋白(PCaP1)：我们在拟南芥中发现了一个新的金属阳离子结合蛋白AtPCaP1。该蛋白是N-肉豆蔻酰化的，在体外和体内与质膜稳定结合。AtPCaP1与钙、铜、钙调素复合体和磷脂酰肌醇磷酸盐结合。根据这些生化性质，我们推测该蛋白参与了环境胁迫的信号转导，如金属胁迫和病原体胁迫。(3)空泡锌转运蛋白(MTP1)：拟南芥MTP1已被证明位于液泡膜上。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多余的锌离子运输到液泡中而参与锌的解毒。通过MTP1在酵母中的异源表达和锌离子转运的直接测定，我们证明了MTP1具有锌离子交换功能。此外，我们还发现，细胞质富含组氨酸的区域作为锌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)酸化液泡并维持跨膜的pH梯度。我们确定了该酶的必需残基和功能基序。

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.