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 electrog enic proton pumps. These are the vacuolar H+-ATPase (EC 3.6.1.3), an e nzyme common to all eukaryotes, and a vacuolar H+-translocating pyroph osphatase (EC 3.6.1.1), which is ubiquitous in plants but otherwise kn own in only a few phototrophic bacteria. Although the substrate-bindin g subunit of the vacuolar H+-pyrophosphatase has been identified and p urified and cDNAs encoding it have been isolated and characterized, th e minimal unit competent in pyrophosphate (PPi)-energized H+ transloca tion is not known. Here we address this question and show that heterol ogous expression of the cDNA (AVP) encoding the substrate-binding subm it of the vacuolar H+-pyrophosphatase from the vascular plant Arabidop sis thaliana in the yeast Saccharomyces cerevisiae results in the prod uction of vacuolarly localized functional enzyme active in PHi-depende nt H+ translocation. Since the heterologously expressed pump is indist inguishable 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 kno wn catalytic functions of the enzyme map to the one subunit encoded by AVP.