The 14-3-3 Protein Interacts Directly with the C-Terminal Region of the Plant Plasma Membrane H + -ATPase

Abstract

Accumulating evidence suggests that 14-3-3 proteins are involved in the regulation of plant plasma membrane H(+)-ATPase activity. However, it is not known whether the 14-3-3 protein interacts directly or indirectly with the H(+)-ATPase. In this study, detergent-solubilized plasma membrane H(+)-ATPase isolated from fusicoccin-treated maize shoots was copurified with the 14-3-3 protein (as determined by protein gel blotting), and the H(+)-ATPase was recovered in an activated state. In the absence of fusicoccin treatment, H(+)-ATPase and the 14-3-3 protein were well separated, and the H(+)-ATPase was recovered in a nonactivated form. Trypsin treatment removed the 10-kD C-terminal region from the H(+)-ATPase as well as the 14-3-3 protein. Using the yeast two-hybrid system, we could show a direct interaction between Arabidopsis 14-3-3 GF14-phi and the last 98 C-terminal amino acids of the Arabidopsis AHA2 plasma membrane H(+)-ATPase. We propose that the 14-3-3 protein is a natural ligand of the plasma membrane H(+)-ATPase, regulating proton pumping by displacing the C-terminal autoinhibitory domain of the H(+)-ATPase.

Fusicoccin Effect on the in Vitro Interaction between Plant 14-3-3 Proteins and Plasma Membrane H+-ATPase

“…In fact, whereas contrasting models have been put forward (8,13), our data appear to be fully compatible with that proposed by Oecking et al (13), according to which FC strengthens the interaction between 14-3-3 and H ϩ -ATPase, thus shifting cytosolic isoforms to the plasma membrane and locking H ϩ -ATPase in its activated state. The identification of the C terminus as the 14-3-3-interacting region appears to be in accordance with the well documented regulatory function of this domain and with recent evidence indicating its direct interaction with 14-3-3 proteins (13,29).…”

The Binding Site for Regulatory 14-3-3 Protein in Plant Plasma Membrane H+-ATPase

“…The segment encoding residues Ala-890 to Val-948 of AHA2 was amplified using the forward primer 5Ј-CCG GAA TTC GAA GCT GTT AAC ATC TTC-3Ј. The forward primers 5Ј-CCG GAA TTC AGT TAC AGA GAA TTG TCT-3Ј, 5Ј-CCG GAA TTC CAA GCT AAA AGA AGA GCT GAG-3Ј, 5Ј-CCG GAA TTC GCT GCT GAG ATG GCT-3Ј, 5Ј-CCG GAA TTC CTT AGG GAG CTG CAC AC-3Ј were used to amplify AHA2 cDNA fragments encoding Tyr-900 to Val-948, Ala-910 to Val-948, Ala-914 to Val-948, and Leu-919 to Val-948, respectively. SDS-PAGE and 14-3-3 Protein Overlay Assay-SDS-PAGE and Western blotting was performed as described (12). Equal amounts of plasma membrane protein were loaded in each well.…”

“…14-3-3 proteins regulate the activity of several important plant enzymes. Among them are nitrate reductase (9, 10), F 0 F 1 -ATP synthase (11), and the plasma membrane H ϩ -ATPase (12,13). Most secondary transporters in plant cells depend upon the activity of the plasma membrane H ϩ -ATPase, which is essential for generating the electrochemical gradient across the plasma membrane (for a review, see Ref.…”

Binding of 14-3-3 Protein to the Plasma Membrane H+-ATPase AHA2 Involves the Three C-terminal Residues Tyr946-Thr-Val and Requires Phosphorylation of Thr947

“…Double transformants were grown on solid medium lacking histidine but containing 25 mM 3-aminotriazole to determine whether the encoded fusion proteins interacted. ␤-Galactosidase activity was measured in liquid cultures as described (18).…”

“…However, other mechanisms are known to regulate the 14-3-3 protein/target protein association. Fusicoccin (FC), 1 a wilt-inducing toxin produced by the fungus Fusicoccum amygdali, induces a tight association between 14-3-3 proteins and the plant plasma membrane H ϩ -ATPase (18,19). This proton pump generates H ϩ -gradients across the plant plasma membrane, which are used for driving solute uptake through H ϩ -coupled symporters (20).…”