A plant plasma membrane H+-ATPase expressed in yeast is activated by phosphorylation at its penultimate residue and binding of 14-3-3 regulatory proteins in the absence of fusicoccin.
The Nicotiana plumbaginifolia plasma membrane H(+)-ATPase isoform PMA2, equipped with a His(6) tag, was expressed in Saccharomyces cerevisiae and purified. Unexpectedly, a fraction of the purified tagged PMA2 associated with the two yeast 14-3-3 regulatory proteins, BMH1 and BMH2. This complex was formed in vivo without treatment with fusicoccin, a fungal toxin known to stabilize the equivalent complex in plants. When gel filtration chromatography was used to separate the free ATPase from the 14-3-3.H(+)-ATPase complex, the complexed ATPase was twice as active as the free form. Trypsin treatment of the complex released a smaller complex, composed of a 14-3-3 dimer and a fragment from the PMA2 C-terminal region. The latter was identified by Edman degradation and mass spectrometry as the PMA2 C-terminal 57 residues, whose penultimate residue (Thr-955) was phosphorylated. In vitro dephosphorylation of this C-terminal fragment prevented binding of 14-3-3 proteins, even in the presence of fusicoccin. Mutation of Thr-955 to alanine, aspartate, or a stop codon prevented PMA2 from complementing the yeast H(+)-ATPase. These mutations were also introduced in an activated PMA2 mutant (Gln-14 --> Asp) characterized by a higher H(+) pumping activity. Each mutation directly modifying Thr-955 prevented 14-3-3 binding, decreased ATPase specific activity, and reduced yeast growth. We conclude that the phosphorylation of Thr-955 is required for 14-3-3 binding and that formation of the complex activates the enzyme.
Maudoux
O
O
Unité de Biochimie Physiologique, Université Catholique de Louvain, Croix du Sud 2-20, B-1348 Louvain-la-Neuve, Belgium.
Batoko
H
H
Oecking
C
C
Gevaert
K
K
Vandekerckhove
J
J
Boutry
M
M
Morsomme
P
P
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism*; D006027:Glycosides / Q000494:pharmacology*; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D010947:Plants, Toxic; D011506:Proteins / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013912:Threonine; D014026:Nicotiana / Q000201:enzymology*; D014446:Tyrosine 3-Monooxygenase
D048948:14-3-3 Proteins; C075852:BMH1 protein, S cerevisiae; C097164:BMH2 protein, S cerevisiae; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D006027:Glycosides; D007527:Isoenzymes; C434364:PMA2 protein, S cerevisiae; D010446:Peptide Fragments; D011506:Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C007808:fusicoccin; D013912:Threonine; D014446:Tyrosine 3-Monooxygenase; C092477:PMA1 protein, S cerevisiae; D006180:Proton-Translocating ATPases