Molecular Characterization of Vacuolar H+-Translocating Inorganic Pyrophosphatase

9305281 Rea植物细胞的液泡膜含有两个主要的质子泵：V型ATP酶（EC 3.6.1.3）和焦磷酸（PPi）激发的质子转运无机焦磷酸酶（V-PPase：EC 3.6.1.1）。 这两种酶介导产电H-易位从胞质液到液泡腔，以建立内部酸性pH梯度和内部正膜电位。 然后，这可以用于激励广泛范围的溶质的次级H耦合运输。 计划的项目是基于我们最近从拟南芥和甜菜中克隆编码V-PPase底物结合亚基的cDNA，以及从前一种生物体中分离基因组克隆。 将审查两个主要领域。 (1)将完成表征拟南芥克隆的研究，并确定它们是否对应于底物结合亚基的同种型、含有与底物结合亚基的序列同一性的V-PPase的其他未鉴定亚基或非V-PPase基因。 (2)还将进行实验以通过表达分析以及反义和过表达转化体的形成来研究V-PPase在拟南芥和烟草中的体内功能。 结果将阐明V-PPase的整体亚基组成和其他细胞器中可能存在的核编码同源物。 V-PPase可能参与初级钾离子转运和质子转运，以及PPi作为植物系统中的关键代谢物的越来越多的认识，涉及泵的pH停滞，耐缺氧，蔗糖动员，膨压调节和气孔功能。 在V-PPase表达程度不同的转基因植物中，这些过程中的一个或多个的扰动将有助于阐明V-PPase的真正生理功能。 质子转移焦磷酸酶（PPase）存在于线粒体的能量耦合膜和光合细菌中。 然而，所谓的V-PPase是“非能量耦合”膜中焦磷酸盐驱动的离子移位酶的第一个例子。 这与其独特的起源，在一个新的类别的离子移位酶的成员，其在植物中的丰富性和普遍性，使得系统的全面表征的一般知识是重要的。 所描述的工作可能有助于新的见解PPi在植物蔗糖代谢中的作用，植物细胞膨压和营养成分的调节机制，并在植物特有的策略，以确保细胞的pH值调节和持续的代谢活动的条件下，有限的氧气供应。 ***

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. ***