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 (deltavma10::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.
Rouquié
D
D
Biochimie et Physiologie Moléculaire des Plantes, INRA/ENSA-M/CNRS URA 2133, Montpellier, France.
Tournaire-Roux
C
C
Szponarski
W
W
Rossignol
M
M
Doumas
P
P
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D010947:Plants, Toxic; D006180:Proton-Translocating ATPases / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D013347:Subcellular Fractions / Q000201:enzymology; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics; D025262:Vacuolar Proton-Translocating ATPases
D018076:DNA, Complementary; D010940:Plant Proteins; D012333:RNA, Messenger; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases