Subunit C of the vacuolar H+-ATPase of Hordeum vulgare

The molecular cloning of the first subunit C of the plant vacuolar NC-ATPas e is reported, Tonoplast vesicles were purified from barley leaves by sucro se gradient centrifugation, and the tonoplast polypeptides were separated b y two-dimensional (2-D) gel electrophoresis, Using an anti-ATPase holoenzym e antibody, a polypeptide was recognized in the molecular mass range of 40 kDa with an isoelectric point of about 6.0, and tentatively identified as s ubunit C, The polypeptide spot was excised from about 50 2-D gels and subje cted to endo Lys C proteolysis, Two proteolytic peptides were sequenced and the amino acid sequences were used to design degenerated oligonucleotides, followed by PCR amplification with cDNA template and screening elf a cDNA library synthesized from Hordeum vulgare poly A mRNA of epidermis strips. T he full length clone of 1.5 kbp contains an open reading frame of 1062 bp e ncoding a polypeptide of 354 amino acids with a molecular mass of 39 982 Da and an isoelectric point of 6.04, Amino acid identity with sequences of SU C from animals and fungi is in the range of 36.7 to 38.5%, Expression of th e cloned gene was demonstrated by Northern blotting and RT-PCR, (C) 1999 Fe deration of European Biochemical Societies.