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. ***
