Cloning of the V-ATPase subunit G in plant:: functional expression and sub-cellular localization

A 13-kDa tobacco plasma membrane protein was isolated from two-dimensional electrophoresis gels. After microsequencing, RT-PCR techniques and cDNA library screening allowed for the cloning of two cDNAs. These cDNAs encoded for the subunit G of the vacuolar H+-ATPase, the first one 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 (Delta vma10::URA3) was achieved with the two cDNAs. After fractionation of microsomal membranes on linear sucrose gradient, Western blots were performed using antibodies against recombinant protein and three peaks were identified: one which comigrated with the tonoplast marker and the others at slightly higher density corresponding to endoplasmic reticulum and to plasma membrane fractions. (C) 1998 Federation of European Biochemical Societies.