Peripheral and integral subunits of the tonoplast H+-ATPase from oat roots.
The subunit organization of the tonoplast H+-pumping ATPase from oat roots (Avena sativa L. var. Lang) was investigated. Tonoplast vesicles were treated with low ionic strength solutions (0.1 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer or 0.1 mM Na EDTA), carbonate, or a chaotropic reagent (KI), and then centrifuged to give a soluble fraction and a pellet. Treatments with low ionic strength solutions or KI resulted in 70-80% reduction in the membrane-associated ATPase activity, but did not affect the K+-stimulated pyrophosphatase activity. Polypeptides of 72, 60, and 41 kDa were solubilized from tonoplast vesicles by these wash treatments. These polypeptides reacted with polyclonal antibodies against the holoenzyme of tonoplast ATPase (anti-ATPase) and copurified with the tonoplast ATPase activity during gel filtration chromatography (Sepharose CL-6B). Mono-specific antibody against the 72- or 60-kDa polypeptide reacted with the solubilized 72- or 60-kDa polypeptide, respectively. However, the N,N-[14C]dicyclohexylcarbodiimide-binding 16-kDa polypeptide and a 13-kDa polypeptide that also reacted with anti-ATPase and copurified with the tonoplast ATPase activity during gel filtration remained in the pellets after the wash treatments. We conclude that the 72- and 60-kDa polypeptides appear to be peripheral subunits of the tonoplast ATPase and that the 16-kDa polypeptide is probably embedded in the membrane bilayer. Additional subunits of the ATPase complex may include a 41-kDa (peripheral) and a 13-kDa (integral) polypeptide. Based on these results, a working model of the tonoplast ATPase analogous to the F1F0-ATPase is proposed.
D000918:Antibody Specificity; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D046911:Macromolecular Substances; D008958:Models, Molecular; D008970:Molecular Weight; D009994:Osmolar Concentration; D010944:Plants / Q000201:enzymology*; D011193:Potassium Iodide / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000032:analysis*; D012995:Solubility
D046911:Macromolecular Substances; D011193:Potassium Iodide; D004024:Dicyclohexylcarbodiimide; D004492:Edetic Acid; D006180:Proton-Translocating ATPases