Dissociation and Reassembly of the Vacuolar H⁺-ATPase Complex from Oat Roots

• Published in: Plant physiology (Bethesda) 1992-05, Vol.99 (1), p.161-169
• Main Authors: Ward, John M., Reinders, Anke, Hei-Ti Hsu, Sze, Heven
• Format: Article
• Language: English
• Subjects: Adenosine triphosphatases; Anions; Biological and medical sciences; Cell physiology; Dialysis; Enzymes; Fundamental and applied biological sciences. Psychology; Hydrolysis; Membranes and Bioenergetics; Monoclonal antibodies; Oats; Plant physiology and development; Plant roots; Plants; Plasma membrane and permeation; Tonoplast
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.99.1.161

Conditions for the dissociation and reassembly of the multisubunit vacuolar proton-translocating ATPase (H+-ATPase) from oat roots (Avena sativa var Lang) were investigated. The peripheral sector of the vacuolar H+-ATPase is dissociated from the membrane integral sector by chaotropic anions. Membranes treated with 0.5 molar KI lost 90% of membrane-bound ATP hydrolytic activity; however, in the presence of Mg2+ and ATP, only 0.1 molar KI was required for complete inactivation of ATPase and H+-pumping activities. A high-affinity binding site for MgATP (dissociation constant = 34 micromolar) was involved in this destabilization. The relative loss of ATPase activity induced by KI, KNO3, or KCl was accompanied by a corresponding increase in the peripheral subunits in the supernatant, including the nucleotide-binding polypeptides of 70 and 60 kilodaltons. The order of effectiveness of the various ions in reducing ATPase activity was: KSCN > KI > KNO3 > KBr > K-acetate > K2SO4 > KCl. The specificity of nucleotides (ATP > GTP > ITP) in dissociating the ATPase is consistent with the participation of a catalytic site in destabilizing the enzyme complex. Following KI-induced dissociation of the H+-ATPase, the removal of KI and MgATP by dialysis resulted in restoration of activity. During dialysis for 24 hours, ATP hydrolysis activity increased to about 50% of the control. Hydrolysis of ATP was coupled to H+ pumping as seen from the recovery of H+ transport following 6 hours of dialysis. Loss of the 70 and 60 kilodalton subunits from the supernatant as probed by monoclonal antibodies further confirmed that the H+-ATPase complex had reassembled during dialysis. These data demonstrate that removal of KI and MgATP resulted in reassociation of the peripheral sector with the membrane integral sector of the vacuolar H+-ATPase to form a functional H+ pump. The ability to dissociate and reassociate in vitro may have implications for the regulation, biosynthesis, and assembly of the vacuolar H+-ATPase in vivo.