title: Molecular dissection of the C-terminal regulatory domain of the plant plasma membrane H+-ATPase AHA2: mapping of residues that when altered give rise to an activated enzyme.
abstract: The plasma membrane H+-ATPase is a proton pump belonging to the P-type ATPase superfamily and is important for nutrient acquisition in plants. The H+-ATPase is controlled by an autoinhibitory C-terminal regulatory domain and is activated by 14-3-3 proteins which bind to this part of the enzyme. Alanine-scanning mutagenesis through 87 consecutive amino acid residues was used to evaluate the role of the C-terminus in autoinhibition of the plasma membrane H+-ATPase AHA2 from Arabidopsis thaliana. Mutant enzymes were expressed in a strain of Saccharomyces cerevisiae with a defective endogenous H+-ATPase. The enzymes were characterized by their ability to promote growth in acidic conditions and to promote H+ extrusion from intact cells, both of which are measures of plasma membrane H+-ATPase activity, and were also characterized with respect to kinetic properties such as affinity for H+ and ATP. Residues that when altered lead to increased pump activity group together in two regions of the C-terminus. One region stretches from K863 to L885 and includes two residues (Q879 and R880) that are conserved between plant and fungal H+-ATPases. The other region, incorporating S904 to L919, is situated in an extension of the C-terminus unique to plant H+-ATPases. Alteration of residues in both regions led to increased binding of yeast 14-3-3 protein to the plasma membrane of transformed cells. Taken together, our data suggest that modification of residues in two regions of the C-terminal regulatory domain exposes a latent binding site for activatory 14-3-3 proteins.
authors.affiliation: Department of Plant Biology, The Royal Veterinary and Agricultural University, Copenhagen, Denmark.
authors.forename: K B
authors.initials: KB
authors.lastname: Axelsen
authors.forename: K
authors.initials: K
authors.lastname: Venema
authors.forename: T
authors.initials: T
authors.lastname: Jahn
authors.forename: L
authors.initials: L
authors.lastname: Baunsgaard
authors.forename: M G
authors.initials: MG
authors.lastname: Palmgren
mesh_terms: D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D002462:Cell Membrane / Q000201:enzymology; D004789:Enzyme Activation / Q000235:genetics; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D010449:Peptide Mapping; D011506:Proteins / Q000378:metabolism / Q000502:physiology; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D014446:Tyrosine 3-Monooxygenase
chemical_list: D048948:14-3-3 Proteins; D004791:Enzyme Inhibitors; D007527:Isoenzymes; C434364:PMA2 protein, S cerevisiae; D010446:Peptide Fragments; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D014446:Tyrosine 3-Monooxygenase; C092477:PMA1 protein, S cerevisiae; D006180:Proton-Translocating ATPases