Abstract
Accumulating evidence suggests that the H+-ATPase of the plant plasma membrane is activated by a direct, reversible interaction with 14-3-3 proteins involving the displacement of the C-terminal autoinhibitory domain of the enzyme. The fungal phytotoxin fusicoccin (FC) appears to stabilize this H+-ATPase.14-3-3 complex, thus leading to a persistent activation of the H+-ATPase in vivo. In this study we show that functional replacement of the Saccharomyces cerevisiae H+-ATPase genes by a Nicotiana plumbaginifolia H+-ATPase (pma2) results in the generation of a high affinity fusicoccin binding site that is exceptionally abundant. Acquisition of FC binding capacity is accompanied by a significant increase in the amount of plasma membrane-associated yeast 14-3-3 homologs. The existence of a (plant) PMA2.(yeast)14-3-3 complex was demonstrated using two-dimensional gel systems (native/denaturing). After expression of PMA2 lacking most of its C-terminal region, neither H+-ATPase.14-3-3 complex formation nor FC binding activity could be observed. Furthermore, we obtained direct biochemical evidence for a minimal FC binding complex consisting of the C-terminal PMA2 domain and yeast 14-3-3 homologs. Thus we demonstrated unambiguously the relevance of this regulatory ATPase domain for 14-3-3 interaction as well as its requirement for FC binding.
Highlights
At the C terminus of plant and yeast Hϩ-ATPases, an autoinhibitory domain is present that is thought to play a role in regulation of enzyme activity
PMA2 Generates a Highly Abundant FC Binding Site—Based on sequence information obtained from peptides, one component of the FC-binding protein (FCBP) purified from plant plasma membranes has been identified as a 14-3-3 protein
Interaction of the C-terminal Domain of the Plant Hϩ-ATPase with a 14-3-3 Dimer Creates a FC-binding Protein— the fusicoccin-binding protein initially has been identified as a 14-3-3 protein [5,6,7], several observations indicate that plant 14-3-3 homologs themselves are not able to bind fusicoccin
Summary
Nomenclature—pma and PMA2 designate the N. plumbaginifolia Hϩ-ATPase gene and gene product, respectively. Solubilization of plasma membrane proteins using dodecyl--D-maltoside as detergent was performed in 10 mM MOPS-Bis/Tris, 5 mM EDTA, 0.1 mM dithiothreitol, 0.5 mM ATP, 20% (w/v) glycerol, pH 7.0 (solubilization buffer). Plasma membranes (2.5 mg mlϪ1 in 25 mM MOPS-KOH, 250 mM sucrose, 4 mM ATP, 5 mM EDTA, 2 mM dithiothreitol, pH 7.5) were mixed with an equal volume of trypsin (100 g mlϪ1) in 25 mM MOPS-KOH, 250 mM sucrose, 5 mM EDTA, pH 7.5 (protein to trypsin ratio 25:1, w/w). Immunodetection of the plant HϩATPase (N terminus), yeast Hϩ-ATPase [30], and yeast 14-3-3 homologs [17, 18] followed standard procedures [31] with goat anti-rabbit/mouse immunglobulin-conjugated alkaline phosphatase and the enzyme substrates 4-nitrotetrazolium blue and 5-bromo-4-chloro-3-indolyl phosphate.
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