Reversible redox control of plant vacuolar H+-ATPase activity is related to disulfide bridge formation in subunit E as well as subunit A.
The plant vacuolar proton pump can be subjected to reversible redox regulation in vitro. The redox-dependent activity change involves disulfide bridge formation not only in Vatp A, as reported for bovine V-ATPase, but also in the stalk subunit Vatp E. Microsomal membranes isolated from barley leaves were analysed for their activity of bafilomycin-sensitive ATP hydrolysis and proton pumping using quinacrine fluorescence quenching in vesicle preparations. ATP hydrolysis and proton pumping activity were inhibited by H2O2. H2O2-deactivated ATPase was reactivated by cysteine and glutathione. The glutathione concentration needed for half maximal reactivation was 1 mmol l-1. The activity loss was accompanied by shifts in electrophoretic mobility of Vatp A and E which were reversed upon reductive reactivation. The redox-dependent shift was also seen with recombinant Vatp E, and was absent following site-directed mutagenesis of either of the two cys residues conserved throughout all plant Vatp E sequences. V-ATPase was also inhibited by oxidized thioredoxin. These results support the hypothesis that tuning of vacuolar ATPase activity can be mediated by redox control depending on the metabolic requirements.
Tavakoli
N
N
Lehrstuhl für Stoffwechselphysiologie und Biochemie der Pflanzen, W5, Universität Bielefeld, 33501 Bielefeld, Germany.
Kluge
C
C
Golldack
D
D
Mimura
T
T
Dietz
K J
KJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione / Q000378:metabolism; D001467:Hordeum / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006868:Hydrolysis / Q000187:drug effects; D066298:In Vitro Techniques; D019476:Insect Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011485:Protein Binding; D021122:Protein Subunits; D011796:Quinacrine / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014617:Vacuoles / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism
D004220:Disulfides; D019476:Insect Proteins; D021122:Protein Subunits; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; C443115:V-ATPase subunit E, Manduca sexta; D025262:Vacuolar Proton-Translocating ATPases; D005978:Glutathione; D011796:Quinacrine; D003545:Cysteine