CLONING OF THE V-ATPASE SUBUNIT-G IN PLANT - FUNCTIONAL EXPRESSION AND SUBCELLULAR-LOCALIZATION

A 13-kDa tobacco plasma membrane protein was isolated from two-dimensi onal electrophoresis gels. After microsequencing, RT-PCR techniques an d cDNA library screening allowed for the cloning of two cDNAs. These c DNAs encoded for the subunit G of the vacuolar H+-ATPase, the first on e identified in plants. Analysis of mRNA distribution showed a maximum level in the leaves and in the stem of the apical part of the tobacco plant. Heterologous functional complementation of the yeast mutant (D elta vma10::URA3) was achieved with the two cDNAs. After fractionation of microsomal membranes on linear sucrose gradient, Western blots wer e performed using antibodies against recombinant protein and three pea ks were identified: one which comigrated with the tonoplast marker and the others at slightly higher density corresponding to endoplasmic re ticulum and to plasma membrane fractions. (C) 1998 Federation of Europ ean Biochemical Societies.