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

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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.