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Plant Physiology 99:161-169 (1992)
© 1992 American Society of Plant Biologists

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Membranes and Bioenergetics

Dissociation and Reassembly of the Vacuolar H+-ATPase Complex from Oat Roots 1

John M. Ward, Anke Reinders, Hei-Ti Hsu and Heven Sze

Department of Botany, University of Maryland, College Park, Maryland 20742, U.S. Department of Agriculture, Agricultural Research Service, Florist and Nursery Crops Laboratory, Beltsville, Maryland 20705

Conditions for the dissociation and reassembly of the multi-subunit 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.

1 This work was supported in part by grants from the National Science Foundation DBM 86-14204 and DCB 90-06402 (to H.S.) and the Maryland Agricultural Experiment Station (Contribution No. 8389, Scientific Article No. 6220).




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