Plant Physiol. (1998) 118: 551-555

A Phosphothreonine Residue at the C-Terminal End of the Plasma Membrane H⁺-ATPase Is Protected by Fusicoccin-Induced 14-3-3 Binding¹

Anne Olsson^*, Fredrik Svennelid, Bo Ek, Marianne Sommarin, and Christer Larsson

Department of Plant Biochemistry, Lund University, P.O. Box 117, SE-221 00 Lund, Sweden (A.O., F.S., M.S., C.L.); and Uppsala Genetic Center, Department of Plant Biology, Swedish University of Agricultural Sciences, P.O. Box 7080, SE-750 07 Uppsala, Sweden (B.E.)

	ABSTRACT

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We have isolated the plasma membrane H⁺ATPase in a phosphorylated form from spinach (Spinacia oleracea L.) leaf tissue incubated with fusicoccin, a fungal toxin that induces irreversible binding of 14-3-3 protein to the C terminus of the H⁺-ATPase, thus activating H⁺ pumping. We have identified threonine-948, the second residue from the C-terminal end of the H⁺-ATPase, as the phosphorylated amino acid. Turnover of the phosphate group of phosphothreonine-948 was inhibited by 14-3-3 binding, suggesting that this residue may form part of a binding motif for 14-3-3. This is the first identification to our knowledge of an in vivo phosphorylation site in the plant plasma membrane H⁺-ATPase.

	INTRODUCTION

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The plant plasma membrane H⁺-ATPase provides the driving force for secondary active transport, and controls intra- and extracellular pH, as well as cell turgor. Considering that the H⁺-ATPase plays a major role in the control of many cell processes, it is not surprising that it is regulated by a number of factors, including hormones, light, and fungal toxins (for review, see Michelet and Boutry, 1995).

The only mechanism for regulation so far demonstrated is that ATP hydrolytic activity and H⁺ pumping may be controlled via an autoinhibitory domain located in the C-terminal region of the H⁺-ATPase (Palmgren et al., 1990, 1991). Removal of this domain by proteolysis or deletion at the gene level results in an activated form of the enzyme with a higher V_max, a lower K_m for ATP, a more alkaline pH optimum, and a more efficient coupling between ATP hydrolysis and H⁺ pumping (Palmgren et al., 1990, 1991; Regenberg et al., 1995). Very similar results were obtained after incubation of intact tissue with the fungal toxin fusicoccin (Johansson et al., 1993; Rasi-Caldogno et al., 1993; Lanfermeijer and Prins, 1994), a treatment suggested to cause a displacement of the C-terminal autoinhibitory domain.

Fusicoccin was initially suggested to activate the plasma membrane H⁺-ATPase by direct interaction with the enzyme (Marrè, 1979). Later, fusicoccin was demonstrated to bind to a "receptor" belonging to a family of proteins designated 14-3-3 proteins (Korthout and de Boer, 1994; Marra et al., 1994; Oecking et al., 1994). 14-3-3 proteins constitute a highly conserved family of eukaryotic proteins with multiple regulatory functions (for review, see Aitken, 1996). Recently, it was shown that 14-3-3 proteins bind directly to the C-terminal region of the H⁺-ATPase and that fusicoccin stabilizes the H⁺-ATPase/14-3-3 complex formed, rendering the association irreversible (Jahn et al., 1997; Oecking et al., 1997; Baunsgaard et al., 1998). This explains earlier observations that plasma membranes isolated from fusicoccin-treated material contained several times more 14-3-3 than plasma membranes isolated from the corresponding controls (Korthout and de Boer, 1994; Oecking et al., 1994). The strong interaction induced by fusicoccin also allowed the H⁺-ATPase/14-3-3 complex to be solubilized and purified from isolated plasma membranes (Jahn et al., 1997; Oecking et al., 1997). Binding of 14-3-3 proteins to the C-terminal region of the H⁺-ATPase was also shown to occur in the absence of fusicoccin, and it was suggested that 14-3-3 proteins are natural ligands of the H⁺-ATPase, regulating H⁺ pumping by displacing the autoinhibitory domain of the enzyme (Jahn et al., 1997; Oecking et al., 1997; Baunsgaard et al., 1998).

Binding of 14-3-3 proteins to their target proteins has been shown to involve a phosphorylated motif in the target (Muslin et al., 1996). Two optimal motifs have been identified, RSXpSXP, based on the binding motif in the animal Ser/Thr protein kinase Raf-1 (Muslin et al., 1996), and RXY/FXpSXP (Yaffe et al., 1997), identified by using phosphoserine-oriented peptide libraries to probe mammalian and yeast 14-3-3 proteins. There is, however, no amino acid sequence in the C-terminal region of the H⁺-ATPase with obvious similarity to any of the two optimal binding motifs, suggesting that 14-3-3 binding to this region involves a somewhat different motif. In the present study we identify a Thr at the C-terminal end of the H⁺-ATPase, the phosphorylation of which is affected by fusicoccin-dependent binding of 14-3-3 to this region.

	MATERIALS AND METHODS

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Plant Material

In Vivo Phosphorylation

Trypsin Treatment

SDS-PAGE and Western Blotting

Protein Digestion and Amino Acid Sequencing

Protein Determination

	RESULTS AND DISCUSSION

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Fusicoccin-Dependent ³²P-Labeling of the H⁺-ATPase

View larger version (84K): [in this window] [in a new window]   Figure 1. In vivo phosphorylation of spinach leaf plasma membrane polypeptides. Polypeptides were stained with Coomassie brilliant blue R (A), and a phosphor imager was used to visualize 32P-polypeptides in the dried gel (B). Immunostaining of the H+-ATPase (C) shows that the 100-kD H+-ATPase band coincides with the 32P-polypeptide, as visualized with a phosphor imager (D). E, Immunostaining of 14-3-3 protein. Pieces of spinach leaves were infiltrated with medium containing 32P-orthophosphate in the absence (lanes 1) or presence (lanes 2 and 3) of fusicoccin and incubated for 90 min. In lanes 2, leaves were incubated for 1 h with 32P-orthophosphate before addition of fusicoccin, whereas in lanes 3, leaves were incubated with fusicoccin for 15 min before addition of 32P-orthophosphate. Plasma membranes were isolated and polypeptides were separated by SDS-PAGE (20 µg of protein per lane). Polypeptides were either stained with Coomassie brilliant blue R, or detected by immunoblotting using antibodies raised against a maize H+-ATPase or a barley 14-3-3 protein. Arrowheads mark the position of the 100-kD H+-ATPase. Numbers at left refer to molecular mass standards.

In vivo activation of spinach leaf H⁺-ATPase by fusicoccin is a rapid process and reaches a plateau within minutes (Johansson et al., 1993). This means that formation of stable H⁺-ATPase/14-3-3 complexes (Jahn et al., 1997; Oecking et al., 1997) is completed within minutes, locking the enzyme in an activated and phosphorylated (Fig. 1, lanes 2) form. In the absence of fusicoccin, no phosphorylation of the H⁺-ATPase was observed (Fig. 1, B and D, lanes 1). This may indicate that the level of phosphorylation, as well as the level of H⁺-ATPase/14-3-3 complex formation, is normally very low, and that phosphorylation may be observed only when the equilibrium is shifted toward complex formation by the action of fusicoccin. Alternatively, the in vivo level of H⁺-ATPase/14-3-3 complex formation is high, but the binding is readily reversible and the complexes are disrupted and the phosphate lost due to endogenous protein phosphatase activity during homogenization of the tissue.

That 14-3-3 binding to the H⁺-ATPase protects the phosphorylated amino acid residue from dephosphorylation was demonstrated by incubating the leaf tissue with fusicoccin before addition of ³²P-orthophosphate (i.e. H⁺-ATPase/14-3-3 complex formation preceded ³²P-labeling of the ATP pool). In that experiment, no ³²P-labeling of the H⁺-ATPase was observed, suggesting that the fusicoccin-induced binding of 14-3-3 somehow shielded the phosphorylated amino acid residue from protein phosphatase/kinase activity (Fig. 1, B and D, lanes 3). Thus, taken together, our results suggest that binding of 14-3-3 to the H⁺-ATPase protects a phosphorylated amino acid residue in the enzyme from being turned over.

Localization of the Phosphorylation Site

Plasma membranes labeled with ³²P as in Figure 1 (lanes 2) were treated with trypsin in the presence of Brij 58, a detergent producing 100% sealed, cytoplasmic-side-out vesicles (Johansson et al., 1995), thus making the C-terminal region of all H⁺-ATPase molecules accessible for trypsin cleavage. Mild trypsin treatment resulted in a partial removal of the native 100-kD H⁺-ATPase band and the appearance of a 90-kD band (Fig. 2A). This 90-kD band, representing the H⁺-ATPase lacking the C-terminal region, was not phosphorylated (Fig. 2B), indicating that the phosphorylated amino acid residue was located in the C-terminal region; the remaining 100-kD H⁺-ATPase species was not labeled. The latter result is in agreement with the recent finding (Jahn et al., 1997; Oecking et al., 1997) that trypsin treatment causing removal of the C-terminal region from about 50% of the H⁺-ATPase molecules was sufficient for the removal of all bound 14-3-3, indicating that the C termini of H⁺-ATPase molecules binding 14-3-3 are more susceptible to proteolysis.

View larger version (32K): [in this window] [in a new window]   Figure 2. Trypsin treatment of isolated plasma membrane vesicles removes all 32P label from the H+-ATPase. A, Immunostaining of the H+-ATPase; B, 32P-polypeptides in A, as visualized with a phosphor imager. Plasma membranes containing [32P]H+-ATPase were treated with trypsin in the presence of Brij 58, thus making the C-terminal region of all H+-ATPase molecules accessible for proteolysis. Polypeptides were separated by SDS-PAGE and transferred to a membrane for immunostaining. The positions of the native 100-kD H+-ATPase and the 90-kD species resulting from proteolytic removal of the C-terminal region of the H+-ATPase are indicated.

To determine the position of the phosphorylation site in the C terminus, the labeled H⁺-ATPase was excised from the gel and digested with the proteinase LysC, cleaving polypeptides C-terminal to Lys residues. The resulting peptides were separated by reversed-phase HPLC and the radioactivity in each fraction determined. Only two radiolabeled peaks, with the sequences GLDIDTIQQNYT and GLDIDTXQQAYX, were found (data not shown). These amino acid sequences are homologous to the relatively conserved C-terminal end of the plant plasma membrane H⁺-ATPase (Fig. 3), except that the C-terminal Val residue was missing in both sequences (this residue probably fell under the detection limit). The sequences obtained differ in at least one position and probably represent two different H⁺-ATPase isoforms in spinach leaves. To identify the phosphorylated amino acid residue, the radioactivity released during each sequencing cycle was measured. Most of the radioactivity applied was released with the 12th amino acid (Fig. 4), i.e. the second Thr in the sequence corresponding to Thr-948 in the Arabidopsis isoform AHA 1 (compare with Fig. 3). Thr-948 is a highly conserved residue, present in all H⁺-ATPases found in the database (Fig. 3).

View larger version (55K): [in this window] [in a new window]   Figure 3. Alignment of the amino acid sequences obtained for the two 32P]H+-ATPase peptides with C-terminal H+-ATPase sequences. The amino acid sequences obtained for the two 32P-peptides (32PC-terminal1 and 32PC-terminal2) are compared with C-terminal sequences (the last 15-21 amino acids, depending on isoform) of plant plasma membrane H+-ATPases found in the database. GeneWorks (IntelliGenetics, Oxford Molecular Group, Oxford, UK) was used for the multiple alignment. Sequence identity is indicated with boxes and dots. Dashes indicate gaps introduced to maximize sequence identity. Numbering in the consensus sequence is according to the Arabidopsis isoform AHA1.

View larger version (21K): [in this window] [in a new window]   Figure 4. Identification of the in vivo-phosphorylated amino acid residue. The graphs show the radioactivity released for each cycle during sequencing of the two 32P-peptides (32PC-terminal1 and 32PC-terminal2 in Fig. 3) and identifies Thr-948 (numbering is according to the Arabidopsis isoform AHA1, Fig. 3) as the phosphorylated amino acid residue. PSL, Photostimulated luminescence.

Role of Phosphothreonine-948

	CONCLUDING REMARKS

Phosphorylation of an amino acid residue in the C-terminal region of the H⁺-ATPase has been suggested as a possible mechanism to regulate H⁺-ATPase activity (Palmgren et al., 1991). In vivo phosphorylation of the oat root plasma membrane H⁺-ATPase, as well as a pH- and Ca²⁺-dependent in vitro phosphorylation of Ser and Thr residues of the H⁺-ATPase in isolated plasma membrane vesicles, was shown by Schaller and Sussman (1988), but no effect on activity was found. More recently, Vera-Estrella et al. (1994) and Xing et al. (1996) have described an elicitor-induced, in vivo dephosphorylation of the plasma membrane H⁺-ATPase of cultured tomato cells, a dephosphorylation that caused activation of the enzyme. Furthermore, Sekler et al. (1994) have demonstrated in vivo phosphorylation of the H⁺-ATPase of the acidophilic alga Dunaliella acidophila. In none of these cases was the phosphorylated amino acid identified. However, for D. acidophila, the phosphorylation site was clearly localized to the C-terminal part of the enzyme.

In this work we present the first identification to our knowledge of an in vivo phosphorylation site in the plant plasma membrane H⁺-ATPase. We show that after incubation of spinach leaf tissue with fusicoccin, the plasma membrane H⁺-ATPase may be isolated in a phosphorylated form (Fig. 1, B and D, lanes 2). Furthermore, we show that the phosphorylated amino acid is located in the C-terminal region of the H⁺-ATPase (Figs. 2 and 3) and is identical to Thr-948 (Fig. 4), the second amino acid from the C-terminal end in most isoforms of the H⁺-ATPase (Fig. 3). Because phosphothreonine-948 is protected from turnover by 14-3-3 binding, it may be part of a binding motif for 14-3-3 protein. Alternatively, phosphothreonine-948 may be shielded from protein phosphatase/kinase activity due to a conformational change induced by 14-3-3 binding. The putative 14-3-3-binding motif in which phosphothreonine-948 resides is far from a perfect match to any of the recently identified optimal binding motifs for 14-3-3 proteins (Muslin et al., 1996; Yaffe et al., 1997). However, as proposed by Yaffe et al. (1997), "The nature of 14-3-3 interaction is determined by the extent to which the binding motif matches the optimal consensus sequence", and "determines if the role of 14-3-3 is to act as a sequestering molecule, a chaperone, or an adaptor." For the H⁺-ATPase/14-3-3 interaction, where the 14-3-3 protein acts as a regulatory protein, a relatively weak and readily reversible binding should be expected. A weak interaction is supported by experimental data showing that the H⁺-ATPase when isolated is devoid of 14-3-3 unless the tissue has been incubated with fusicoccin to induce a stronger interaction (Jahn et al., 1997; Oecking et al., 1997). Thus, the irreversible binding of 14-3-3 induced by fusicoccin is lethal; the hosts of the fungus Fusicoccum amygdali, peach and almond trees, wilt and die because the irreversible activation of the H⁺-ATPase in stomatal cells results in a permanent opening of the stomata (for review, see de Boer, 1997).

	FOOTNOTES

   Received April 24, 1998; accepted July 1, 1998.

	ACKNOWLEDGMENTS

We wish to thank Adine Karlsson for excellent technical assistance and Dr. Louise Race (Department of Plant Cell Biology, Lund University, Sweden) for critically reading the manuscript. We thank Dr. Wolfgang Michalke (Institut für Biologie III, Albert-Ludwigs-Universität, Freiburg, Germany) for kindly providing the monoclonal antibody to the H⁺-ATPase and Dr. David B. Collinge (Department of Plant Biology, Royal Veterinary and Agricultural University, Frederiksberg C, Denmark) for a generous gift of the antiserum to the 14-3-3 protein.

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