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Cloning of the V‐ATPase subunit G in plant: functional expression and sub‐cellular localization 1
Author(s) -
Rouquié David,
Tournaire-Roux Colette,
Szponarski Wojciech,
Rossignol Michel,
Doumas Patrick
Publication year - 1998
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(98)01252-6
Subject(s) - endoplasmic reticulum , microbiology and biotechnology , biology , complementary dna , complementation , protein subunit , heterologous expression , cloning (programming) , expression cloning , recombinant dna , blot , biochemistry , cdna library , mutant , gene , computer science , programming language
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 (Δvma10::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.