Molecular structure and functional mechanism of a novel proton pump H+-pyrophosphatase

在五年的项目期间，我们获得了以下关于H+-焦磷酸酶和金属转运体的结果。(1) H^+-焦磷酸酶（H^+-PPase）：H^+-PPase使植物液泡或酸化体酸化，并维持跨膜的pH梯度。我们的实验发现，拟南芥中H^+-PPase的敲除突变体不能正常生长，这表明H^+-PPase是植物中必不可少的膜运输系统之一。(2) H^+-PPase的生化性质和膜拓扑结构：除了植物的H^+-PPase外，我们纯化并鉴定了coelicolor链霉菌H^+-PPase，因为它及其突变酶可以在大肠杆菌中表达。我们发现S. coelicolor酶具有热稳定性等特性。我们还确定了H^+-PPase的膜拓扑结构和酶的基本基序。我们的结构模型已被接受为各种生物中H^+-PPases的标准。(3) H+-PPase的结晶：制备了大量的Fv片段，并与活性形式的H+-PPase发生特异性反应。我们利用溶解植物液泡膜的Fv片段成功制备了大量的Fv- h +-PPase复合物。现在，我们正在确定酶结晶的最佳条件。(4)阳离子/H^+交换剂：水稻有5种阳离子/H^+交换剂（CAX）同工型。这五种异构体随金属离子选择性和组织特异性基因表达而变化。其中CAXIa在所有被检组织中均有表达，且在伴细胞和内胚层细胞中高表达，具有高浓度的Ca^<2+>。我们估计CAXla的功能是将Ca^<2+>保留到液泡中，并参与Ca^<2+>的解毒。膜的拓扑结构和几个功能基序也被确定。

During the project period for five years, we obtained the following results concerning H+-pyrophosphatase and metal transporters.(1) H^+-pyrophosphatase (H^+-PPase) :H^+-PPase acidifies plant vacuoles or acidocalcisomes and maintains the pH gradient across the membrane. Our experiments revealed that a knockout mutant of H^+-PPase in Arabidopsis could not grow normally, suggesting that H^+-PPase is one of the essential membrane transport systems in plants.(2) Biochemical properties and membrane topology of H^+-PPase :In addition to plant H^+-PPase, we purified and characterized Streptomyces coelicolor H^+-PPase, since it and its mutant enzymes can be expressed in E. col. We revealed that S. coelicolor enzyme has a characteristic properties such as heat stability. We also determined the membrane topology of H^+-PPase and essential motifs in the enzyme. Our structural model has been accepted as the standard of H^+-PPases in various organisms.(3) Crystallization of H^+-PPase :We prepared a large quantity of Fv fragment, which specifically reacts with the active form of H+-PPase. We succeeded in preparation of a large quantity of the Fv-H+-PPase complex using the Fv fragment from the solubilized plant vacuolar membranes. Now, we are determining the best conditions for crystallization of the enzyme.(4) Cation/H^+ exchangers :Rice has five isoforms of cation/H^+ exchanger (CAX). These five isoforms varied with metal ion selectivity and tissue specific gene expression. CAXIa among them 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 CAXla functions to reserve Ca^<2+> into vacuoles and is involved in the Ca^<2+> detoxification. The membrane topology and several functional motifs were also determined.