HETEROLOGOUS EXPRESSION OF PLANT VACUOLAR PYROPHOSPHATASE IN YEAST DEMONSTRATES SUFFICIENCY OF THE SUBSTRATE-BINDING SUBUNIT FOR PROTON TRANSPORT

The membrane bounding the vacuole of plant cells contains two electrogenic proton pumps. These are the vacuolar H+-ATPase (EC 3.6.1.3), an enzyme common to all eukaryotes, and a vacuolar H+-translocating pyrophosphatase (EC 3.6.1.1), which is ubiquitous in plants but otherwise known in only a few phototrophic bacteria. Although the substrate-binding subunit of the vacuolar H+-pyrophosphatase has been identified and purified and cDNAs encoding it have been isolated and characterized, the minimal unit competent in pyrophosphate (PPi)-energized H+ translocation is not known. Here we address this question and show that heterologous expression of the cDNA (AVP) encoding the substrate-binding submit of the vacuolar H+-pyrophosphatase from the vascular plant Arabidopsis thaliana in the yeast Saccharomyces cerevisiae results in the production of vacuolarly localized functional enzyme active in PHi-dependent H+ translocation. Since the heterologously expressed pump is indistinguishable from the native plant enzyme with respect to PH hydrolysis, H+ translocation, activation by potassium, and selective inhibition by calcium and 1,1-diphosphonates, it is concluded that all of the known catalytic functions of the enzyme map to the one subunit encoded by AVP.