Marianne Sommarin

The 14-3-3 protein interacts directly with the C-terminal region of the plant plasma membrane H(+)-ATPase.

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.

Phosphorylation of Thr-948 at the C Terminus of the Plasma Membrane H+-ATPase Creates a Binding Site for the Regulatory 14-3-3 Protein

The plant plasma membrane H+-ATPase is activated by the binding of 14-3-3 protein to the C-terminal region of the enzyme, thus forming an H+-ATPase–14-3-3 complex that can be stabilized by the fungal toxin fusicoccin. A novel 14-3-3 binding motif, QQXYpT948V, at the C terminus of the H+-ATPase is identified and characterized, and the protein kinase activity in the plasma membrane fraction that phosphorylates this threonine residue in the H+-ATPase is identified. A synthetic peptide that corresponds to the C-terminal 16 amino acids of the H+-ATPase and that is phosphorylated on Thr-948 prevents the in vitro activation of the H+-ATPase that is obtained in the presence of recombinant 14-3-3 and fusicoccin. Furthermore, binding of 14-3-3 to the H+-ATPase in the absence of fusicoccin is absolutely dependent on the phosphorylation of Thr-948, whereas binding of 14-3-3 in the presence of fusicoccin occurs independently of phosphorylation but still involves the C-terminal motif YTV. Finally, by complementing yeast that lacks its endogenous H+-ATPase with wild-type and mutant forms of the Nicotiana plumbaginifolia H+-ATPase isoform PMA2, we provide physiological evidence for the importance of the phosphothreonine motif in 14-3-3 binding and, hence, in the activation of the H+-ATPase in vivo. Indeed, replacing Thr-948 in the plant H+-ATPase with alanine is lethal because this mutant fails to functionally replace the yeast H+-ATPase. Considering the importance of the motif QQXYpTV for 14-3-3 binding and yeast growth, this motif should be of vital importance for regulating H+-ATPase activity in the plant and thus for plant growth.

Evolution of the 14-3-3 protein family: does the large number of isoforms in multicellular organisms reflect functional specificity?

Abstract. 14-3-3 proteins constitute a family of eukaryotic proteins that are key regulators of a large number of processes ranging from mitosis to apoptosis. 14-3-3s function as dimers and bind to particular motifs in their target proteins. To date, 14-3-3s have been implicated in regulation or stabilization of more than 35 different proteins. This number is probably only a fraction of the number of proteins that 14-3-3s bind to, as reports of new target proteins have become more frequent. An examination of 14-3-3 entries in the public databases reveals 153 isoforms, including alleloforms, reported in 48 different species. The number of isoforms range from 2, in the unicellular organism Saccharomyces cerevisiae, to 12 in the multicellular organism Arabidopsis thaliana. A phylogenetic analysis reveals that there are four major evolutionary lineages: Viridiplantae (plants), Fungi, Alveolata, and Metazoa (animals). A close examination of the aligned amino acid sequences identifies conserved amino acid residues and regions of importance for monomer stabilization, dimer formation, target protein binding, and the nuclear export function. Given the fact that 53% of the protein is conserved, including all amino acid residues in the target binding groove of the 14-3-3 monomer, one might expect little to no isoform specificity for target protein binding. However, using surface plasmon resonance we show that there are large differences in affinity between nine 14-3-3 isoforms of A. thaliana and a target peptide representing a novel binding motif present in the C terminus of the plant plasma membrane H+ATPase. Thus, our data suggest that one reason for the large number of isoforms found in multicellular organisms is isoform-specific functions.

Fusicoccin Activates the Plasma Membrane H+-ATPase by a Mechanism Involving the C-Terminal Inhibitory Domain.

Plasma membrane vesicles isolated from spinach leaves incubated with the fungal toxin fusicoccin showed a twofold increase in ATP hydrolytic activity and a threefold increase in H+ pumping compared to controls. This increase in H+-ATPase activity was largely completed within 4 min of incubation and was not due to de novo synthesis of H+-ATPase as demonstrated by immunoblotting. Incubation with fusicoccin also resulted in a decrease in the apparent Km for ATP of the H+-ATPase from 0.22 to 0.10 mM. The fusicoccin-mediated activation of H+-ATPase activity and the accompanying decrease in the Km for ATP are changes very similar to those observed upon trypsin activation of the H+-ATPase, where an autoinhibitory domain in the C-terminal region of the H+-ATPase is removed. Thus, trypsin treatment of plasma membrane vesicles from control leaves gave a twofold increase in ATP hydrolytic activity and a threefold increase in H+ pumping, as well as a decrease in the apparent Km for ATP of the H+-ATPase from 0.22 to 0.10 mM. Trypsin treatment of plasma membranes from fusicoccin-incubated leaves did not further enhance the H+-ATPase activity, however, and neither was the Km for ATP further decreased. That trypsin really removed a small segment from the fusicoccin-activated H+-ATPase was confirmed by immunoblotting, which showed the appearance of a 90-kD band in addition to the native 100-kD H+-ATPase band upon trypsin treatment. Taken together, our data suggest that in vivo activation of the H+-ATPase by fusicoccin proceeds by a mechanism involving a displacement of the C-terminal inhibitory domain.

Effect of detergents on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane vesicles

In search for a detergent to be used to assess the sidedness of plant plasma membrane vesicles by enzyme latency we tested the effect of 42 detergents on the ATPase activity of right-side-out and inside-out plasma membrane vesicles from sugar beet leaves. Most of the detergents seemed to inactivate the ATPase in addition to disrupting the permeability barrier to ATP. There were two main exceptions, namely long chain polyoxyethylene acyl ethers, such as detergents of the Brij series and Lubrol WX, and long chain lysophospholipids. These two types of detergents permeabilized the membranes at low concentrations and did not inhibit the ATPase at higher concentrations. Unmasking of latent active sites seemed to explain the activation of the plasma membrane H(+)-ATPase produced by long chain polyoxyethylene acyl ethers. These detergents should therefore be ideal for determination of vesicle orientation based on ATPase latency. By contrast, long chain lysophospholipids were found to be highly specific activators of the enzyme. In addition, long chain fatty acids were found to strongly inhibit ATP-dependent proton accumulation in the vesicles without inhibiting ATP hydrolysis. This uncoupling effect of the fatty acids could be abolished by the addition of fatty acid-free bovine serum albumin (BSA). Similarly, the proton transport capacity of ageing vesicles could be restored by addition of BSA. The latter findings may explain why isolated plasma membranes so often exhibit increased permeability to protons on ageing.

Phosphatidylinositol and phosphatidylinositolphosphate kinases in plant plasma membranes

Both phosphatidylinositol (PI) and phosphatidylinositolphosphate (PIP) kinase activities were present in plasma membrane fractions isolated from shoots and roots of dark-grown wheat (Triticum aestivum L.) by aqueous polymer two-phase partition. The enzymes phosphorylated their respective endogenous substrates as well as exogenously added substrates (PI and phosphatidylinositol 4-monophosphate, PI-4P), to form PIP and phosphatidylinositol diphosphate (PIP2). The reactions were dependent on ATP. Phosphorylation of added PI reached maximum activity around 0.75 mM ATP, while the ATP requirement for maximal activity was higher both for phosphorylation of added PI-4P (1.25 mM ATP) and of endogenous lipids (1.5 mM ATP). Optimal Mg2+ concentration varied between 5 mM (endogenous PI phosphorylation) and 15 mM (phosphorylation of exogenous PI). The Mg2+ requirement could be substituted only partially by Mn2+ and not at all by Ca2+ Phosphorylation of endogenous lipid substrates was inhibited by Triton X-100 concentrations above 0.015%, while phosphorylation of exogenous substrates was stimulated several-fold by up to 0.5% Triton X-100. Triton X-100 also influenced the optimal pH range of the reactions. While phosphorylation of endogenous PI and PIP was optimal at pH 6.5–7 without Triton X-100 in the assay medium, addition of 0.010% Triton X-100 extended the optimal pH range up to pH 8.6. Phosphorylation of exogenous lipids were optimal at pH 7.8–8.2. At optimal conditions and with endogenous substrates, PIP formation was 125–225 and 40–90 pmol/mg protein per min in shoot and root plasma membranes, respectively, and PIP2 formation 10–25 and 4–8 pmol/mg protein per min, respectively. With exogenous substrates, the corresponding rates increased 8–20-times. These results demonstrate the close resemblance between the characteristics of PI and PIP kinase activities in plant membranes with corresponding activities in animal plasma membranes. It is, however, not yet known if polyphosphoinositide metabolism in plant cells resembles the corresponding metabolism in animal cells also in function, that is, in acting as a signal-transducing system for internal Ca2+ mobilization.

Identification of Ca2+-stimulated polyphosphoinositide phospholipase C in isolated plant plasma membranes

A polyphosphoinositide phospholipase C has been identified in highly purified plasma membranes from shoots and roots of wheat seedlings. The enzyme preferentially hydrolysed phosphatidylinositol 4‐phosphate and phosphatidylinositol 4,5‐bisphosphate and had a different phosphoinositide substrate profile from soluble phospholipase C. The enzyme activity was lower in plasma membranes isolated from light‐grown shoots than from dark‐grown ones, whereas no differences in activity between plasma membranes from light‐ and dark‐grown roots were seen. Maximum activity of the membrane‐bound enzyme was observed around pH 6. It was activated by micromolar concentrations of Ca2+, but not by GTP or GTP analogues. The enzyme may participate in signal transduction over the plant plasma membrane.

Inside-out plant plasma membrane vesicles of high purity obtained by aqueous two-phase partitioning

Highly purified plasma membranes obtained from leaves of sugar beet (Beta vulgaris L.) by aqueous two‐phase partitioning were separated into two fractions by further phase partition steps. The vesicles partitioning to the interface showed an ATP‐dependent H+‐uptake (measured using the pH probe acridine orange) and a negligible K+,Mg2+‐ATPase latency, while the vesicles partitioning in the upper phase showed only slow H+‐uptake and a high ATPase latency on addition of Triton X‐100. Based on these results the material at the interface is estimated to contain ∼90% sealed, inside‐out vesicles, and the material in the upper phase ∼90% sealed, right‐side‐out vesicles

Evolution and isoform specificity of plant 14-3-3 proteins.

The 14-3-3 proteins, once thought of as obscure mammalian brain proteins, are fast becoming recognized as major regulators of plant primary metabolism and of other cellular processes. Their presence as large gene families in plants underscores their essential role in plant physiology. We have examined the Arabidopsis thaliana 14-3-3 gene family, which currently is the largest and most complete 14-3-3 family with at least 12 expressed members and 15 genes from the now completed Arabidopsis thaliana genome project. The phylogenetic branching of this family serves as the prototypical model for comparison with other large plant 14-3-3 families and as such may serve to rationalize clustering in a biological context. Equally important for ascribing common functions for the various 14-3-3 isoforms is determining an isoform-specific correlation with localization and target partnering. A summary of localization information available in the literature is presented. In an effort to identify specific 14-3-3 isoform location and participation in cellular processes, we have produced a panel of isoform-specific antibodies to Arabidopsis thaliana 14-3-3s and present initial immunolocalization studies that suggest biologically relevant, discriminative partnering of 14-3-3 isoforms.

Phosphorylation of phosphatidylinositols in isolated plant membranes

Lipid kinase Phosphatidylinositol phosphorylation Phosphoinositide Polyphosphoinositide Plant membrane Plasma membrane