ATP analogue binding to the A subunit induces conformational changes in the E subunit that involves a disulfide bond formation in plant V-ATPase

Vacuolar H+-ATPase (V-ATPase) consists of a catalytic head, a stalk part an d a membrane domain. We indirectly investigated the interaction between the A subunit (catalytic head) and the E subunit (stalk part) using an ATP ana logue, adenosine 5'-[beta,gamma -imino]triphosphate (AMP-PNP), which holds the enzyme in the substrate-binding state. AMP-PNP treatment caused a mobil ity shift of the E subunit with a faster migration in SDS/polyacrylamide ge l electrophoresis without a reductant, while ATP treatment did not. A mobil ity shift of the E subunit has been detected in several plants. As polypept ides with intramolecular disulfide bonds migrate faster than those without disulfide bonds, the mobility shift may be due to the formation of an intra molecular disulfide bond by two cysteine residues conserved among several p lant species. The mobility shift may be involved in the binding of AMP-PNP to the ATP-binding site, which exists in the A and B subunits, as it was in hibited by the addition of ATP. Pretreatment with 2'-3'-O-(4-benzoylbenzoyl )-ATP (Bz-ATP), which modifies the ATP-binding site of the B subunit under UV illumination, did not inhibit the mobility shift of the E subunit caused by AMP-PNP treatment. The response of V-ATPase following the AMP-PNP bindi ng may cause a conformational change in the E subunit into a form that is s usceptible to oxidation of cysteine residues. This is the first demonstrati on of interaction between the A and E subunits in the substrate-binding sta te of a plant V-ATPase.