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 35 S‐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.