Subunit E of the vacuolar H(+)-ATPase of Hordeum vulgare L.: cDNA cloning, expression and immunological analysis

Abstract

A tonoplast protein of 31 kDa apparent molecular mass (TpP 31) was isolated from two-dimensional gels. Amino acid sequences were determined from LysC endoproteinase-peptide fragments. Using degenerate oligonucleotides, a corresponding cDNA clone of 1034 bp was isolated from a barley leaf cDNA library. It encodes for subunit E of the vacuolar H(+)-ATPase, the first one identified in plants so far. The open reading frame extends over 681 bp, encoding a gene product of 227 amino acids and a calculated molecular weight of 26,228 g mol-1. Northern and Western blot analysis indicates constitutive expression of subunit E in all plant organs with only small effects of salt stress. Localization of TpP 31 at the tonoplast was confirmed in fractions of purified vacuolar membrane obtained by free-flow electrophoresis. Immunoprecipitation of newly synthesized 35S-labelled membrane proteins with anti-TpP 31 gave two additional bands with apparent molecular masses of about 53 and 62 kDa. Gel filtration after mild solubilization showed co-purification of TpP 31 with the 55 kDa subunit of the H(+)-ATPase. Both results provide evidence beyond the sequence homology that TpP 31 is a structural component of the vacuolar H(+)-ATPase.