Molecular Characterization of Vacuolar H+-Translocating Inorganic Pyrophosphatase

植物细胞的液泡膜包含两个初级质子泵：一个v型atp酶（EC 3.6.1.3）和一个焦磷酸盐（PPi）激活的质子易位无机焦磷酸酶（V-PPase: EC 3.6.1.1）。这两种酶都介导从细胞质到液泡腔的电致氢易位，以建立酸内pH梯度和膜内正电位。这可以用来激发各种溶质的二次h耦合输运。计划的项目是基于我们最近从拟南芥和Beta vulgaris中克隆编码V- PPase底物结合亚基的cdna，并从前者中分离基因组克隆。将考察两个主要领域。(1)拟南芥克隆的特征研究将完成，并确定它们是否对应于底物结合亚基的异构体，是否对应于含有底物结合亚基序列的V-PPase的其他未识别亚基，或者是否对应于非V-PPase基因。(2)通过对拟南芥和烟叶中V-PPase的表达分析，以及反义和过表达转化子的形成，研究V-PPase在体内的功能。结果将揭示V- PPase的整体亚基组成以及在其他细胞器中可能存在核编码同源物。V-PPase可能参与初级钾离子转运，以及质子转运，以及PPi作为植物系统中关键代谢物的日益认识，暗示了泵在pH停滞，耐厌氧，蔗糖动员，膨胀调节和气孔功能中的作用。在具有不同程度V-PPase表达的转基因植物中，对这些过程中的一个或多个进行扰动，将有助于阐明V-PPase的真正生理功能。质子转位焦磷酸酶（PPase）存在于光养细菌和线粒体的能量偶联膜中。然而，所谓的V-PPase是焦磷酸盐驱动的离子转位酶在“非能量偶联”膜中的第一个例子。这与它独特的起源、离子易位的新类别、以及它在植物中的丰度和普遍性相结合，使得对该系统的全面表征对一般知识很重要。所描述的工作可能有助于对PPi在植物蔗糖代谢中的作用，植物细胞膨胀和营养成分调节的潜在机制，以及植物在有限氧可用性条件下确保细胞pH调节和持续代谢活性的特有策略提供新的见解。＊＊＊

9305281 Rea The vacuolar membrane of plant cells contains two primary proton pumps: a V-type ATPase (EC 3.6.1.3) and a pyrophosphate (PPi) energized proton-translocating inorganic pyrophosphatase (V-PPase: EC 3.6.1.1). Both enzymes mediate electrogenic H-translocation from the cytosol to vacuole lumen to establish an inside-acid pH gradient and inside-positive membrane potential. This may then be used to energize the secondary H-coupled transport of a wide range of solutes. The project planned is based on our recent molecular cloning of cDNAs encoding the substrate-binding subunit of the V- PPase from Arabidopsis thaliana and Beta vulgaris and the isolation of genomic clones from the former organism. Two main areas will be examined. (1) Studies will be finished to characterize Arabidopsis clones and to determine if they correspond to isoforms of the substrate-binding subunit, to additional unidentified subunits of the V-PPase containing sequence identities with the substrate- binding subunit, or to non-V-PPase genes. (2) Experiments will also be performed to study the in vivo function of the V-PPase in Arabidopsis and Nicotiana plumbaginifolia by expression analysis, and the formation of antisense and overexpression transformants. Results will shed light on the overall subunit composition of V- PPase and the possible existence of nuclear-coded homologs in other organelles. The possible involvement of the V-PPase in primary K ion-translocation, as well as proton-translocation, and the increasing recognition of PPi as a key metabolite in plant systems, implicate the pump in pH stasis, resistance to anaerobiosis, sucrose mobilization, turgor regulation and stomatal function. Perturbation of one or more of these processes in transgenic plants with different degrees ofV-PPase expression, will facilitate elucidation of the true physiological function of the V-PPase. %%% Proton-translocating pyrophosphatases (PPase) have been found in energy-coupling membranes of mitochondria and in phototrophic bacteria. The so called V-PPase, however, is the first example of a pyrophosphate-driven ion translocase in a "non-energy coupling" membrane. This in conjunction with its unique origins, membership in a new category of ion-translocases, and its abundance and ubiquity in plants, makes the thorough characterization of the system important for general knowledge. The work described may contribute new insights into the role of PPi in plant sucrose metabolism, the mechanisms underlying the regulation of plant cell turgor and nutrient composition, and in strategies peculiar to plants for ensuring cellular pH regulation and sustained metabolic activity under conditions of limited oxygen availability. ***