title: ATP analogue binding to the A subunit induces conformational changes in the E subunit that involves a disulfide bond formation in plant V-ATPase.
abstract: Vacuolar H+-ATPase (V-ATPase) consists of a catalytic head, a stalk part and a membrane domain. We indirectly investigated the interaction between the A subunit (catalytic head) and the E subunit (stalk part) using an ATP analogue, adenosine 5'-[beta,gamma-imino]triphosphate (AMP-PNP), which holds the enzyme in the substrate-binding state. AMP-PNP treatment caused a mobility shift of the E subunit with a faster migration in SDS/polyacrylamide gel electrophoresis without a reductant, while ATP treatment did not. A mobility shift of the E subunit has been detected in several plants. As polypeptides with intramolecular disulfide bonds migrate faster than those without disulfide bonds, the mobility shift may be due to the formation of an intramolecular disulfide bond by two cysteine residues conserved among several plant 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 inhibited 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 binding may cause a conformational change in the E subunit into a form that is susceptible to oxidation of cysteine residues. This is the first demonstration of interaction between the A and E subunits in the substrate-binding state of a plant V-ATPase.
authors.affiliation: Cryobiosystem Research Center, Iwate University, Iwate, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Kawamura
authors.forename: K
authors.initials: K
authors.lastname: Arakawa
authors.forename: M
authors.initials: M
authors.lastname: Maeshima
authors.forename: S
authors.initials: S
authors.lastname: Yoshida
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology*; D000345:Affinity Labels / Q000494:pharmacology; D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000737:chemistry*; D010944:Plants / Q000201:enzymology; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D006180:Proton-Translocating ATPases / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014466:Ultraviolet Rays; D025262:Vacuolar Proton-Translocating ATPases
chemical_list: D000345:Affinity Labels; D004220:Disulfides; D010940:Plant Proteins; D013439:Sulfhydryl Reagents; D000266:Adenylyl Imidodiphosphate; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; D005033:Ethylmaleimide