ISOLATION AND CHARACTERIZATION OF CDNAS ENCODING THE VACUOLAR H-PYROPHOSPHATASE OF BETA-VULGARIS()

The H+-pyrophosphatase (V-PPase) of plant vacuolar membranes catalyzes the electrogenic translocation of H+ from the cytosol to vacuole lume n and, in parallel with the vacuolar H+-ATPase located in the same mem brane, establishes the inside-acid, inside-positive H+-electrochemical potential difference responsible for energizing the H+-coupled transp ort of solutes into the vacuole. The results of previous investigation s suggest that the gene encoding the substrate-binding subunit of the V-PPase is present in a single copy in the genome of Arabidopsis thali ana (V. Sarafian, Y. Kim, R.J. Poole, P.A. Rea [1992] Proc Natl Acad S ci USA 89: 1775-1779), but it is not known whether the situation in Ar abidopsis is typical of most vascular plants. With the objective of as sessing the general applicability of this finding and acquiring sequen ce data for structure-function analyses of the enzyme from Beta vulgar is, we have sought to isolate cDNAs encoding the V-PPase from this org anism by screening a Beta cDNA library constructed in lambda ZAP with the Arabidopsis cDNA insert (AVP) encoding the V-PPase. The results of these investigations demonstrate that at least two genes encode the V -PPase in Beta. Restriction and sequence analyses of the cDNAs from Be ta reveal two classes, designated BVP1 and BVP2. BVP1 and BVP2 encode closely related but distinct polypeptides with computed masses of 80,5 50 and 80,000 D, respectively, exhibiting 88% identity with each other and 89% identity with the corresponding polypeptide from Arabidopsis. The nucleotide sequences of BVP1 and BVP2, on the other hand, are 70% identical within their coding regions but less than 28 and 53% identi cal within their respective 5' and 3' noncoding regions. Southern anal yses of Beta genomic DNA confirm that two genes encode the V-PPase, an d northern analyses of polyadenylated RNA isolated from a range of tis sue types and probed with RNAs transcribed from the 3' noncoding seque nces of BVP1 or BVP2 indicate that both genes are expressed in the int act plant. On the basis of these findings and the recent demonstration of the sufficiency of the substrate-binding polypeptide, alone, for a ll of the known catalytic functions of the V-PPase (E.J. Kim, R.-C. Zh en, P.A. Rea [1994] Proc Natl Acad Sci USA [91: 6128-6132]), the two c DNA species isolated from Beta are concluded to encode variants, possi bly isoforms, of the enzyme.