Journal article
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
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.
Language: | English |
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Year: | 1999 |
Pages: | 7227-7234 |
ISSN: | 15204995 and 00062960 |
Types: | Journal article |
DOI: | 10.1021/bi982482l |
ORCIDs: | 0000-0002-9982-6114 |
14-3-3 Proteins Amino Acid Sequence Amino Acid Substitution Arabidopsis Cell Membrane Enzyme Activation Enzyme Inhibitors Isoenzymes Kinetics Molecular Sequence Data Mutagenesis, Insertional Mutagenesis, Site-Directed PMA1 protein, S cerevisiae PMA2 protein, S cerevisiae Peptide Fragments Peptide Mapping Proteins Proton-Translocating ATPases Saccharomyces cerevisiae Saccharomyces cerevisiae Proteins Tyrosine 3-Monooxygenase