Abstract
Vacuoles are multifunctional organelles in the mature plant cells and occupy 80~90% of total cell volume. They are important in the homeostasis of the plant cells, which involves the regulation of cell turgor, cytoplasmic pH, and storage of metabolites.Plant vacuolar vesicles contain two parallel proton pumps, namely vacuolar H(+)-pumping adenosine-triphosphatase (V-ATPase; EC 3.6.1.3), which is widely distributed in most eukaryotes, and vacuolar H(+)-pumping inorganic pyrophosphatase (V-PPase; EC 3.6.1.1), which exists primarily in higher plants and several bacteria. V-PPase catalyzes PPi hydrolysis and the electrogenic H(+)-translocation from the cytosol to the vacuolar lumen to generate an inside-acidic pH gradient (□pH) and an interior-positive membrane potential (□□) for the transport of ions and secondary metabolites.Vacuolar H(+)-PPase was purified from etiolated hypocotyls of mung bean seedlings (Vigna radiata L.) by solubilization with sodium deoxycholate and lysophosphatidylcholine, gel filtration as well as ion exchange chromatography.In this study, the treatment of carboxypeptidase Y (CPY) markedly suppressed H+ translocation and purified vacuolar H(+)-PPase activity instead of membrane-bound enzymatic activity in a concentration-dependent manner. It was indicated that the purified vacuolar H(+)-PPase is more sensitive to CPY owing to the removal of the membrane. In addition, CPY obviously decoupled the association of proton translocation and enzymatic activity, suggesting that the C-terminus of the enzyme either participates directly in proton translocation or plays a role in the maintenance of the structure in the proton channel.Furthermore, the incubation with CPY relieved the stimulation of KCl, implying that the C-terminus is most likely the candidate for the region involved in the K(+)-binding and/or probably the K(+)-regulation of the enzyme. On the other hand, the protection studies revealed that PPi is crucial for most protection of vacuolar H(+)-PPase against C-terminal digestion by CPY.As a result, we speculate that the C-terminus of vacuolar H(+)-PPase is not directly involved in the catalytic activity, but provides the site for the cation-binding and regulation as well as supports the structure of a proton channel. Moreover, PPi binding site in catalytic domain may exert long-distance interaction with C-terminus of vacuolar H(+)-PPase, resulting in a tougher conformation against proteolytic attack by CPY.