title: Complementation of the Saccharomyces cerevisiae plasma membrane H+-ATPase by a plant H+-ATPase generates a highly abundant fusicoccin binding site.
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.
authors.affiliation: Lehrstuhl für Pflanzenphysiologie, Ruhr-Universität, D-44780 Bochum, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Piotrowski
authors.forename: P
authors.initials: P
authors.lastname: Morsomme
authors.forename: M
authors.initials: M
authors.lastname: Boutry
authors.forename: C
authors.initials: C
authors.lastname: Oecking
mesh_terms: D048948:14-3-3 Proteins; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D019281:Dimerization; D005816:Genetic Complementation Test; D006027:Glycosides / Q000378:metabolism*; D010947:Plants, Toxic; D011506:Proteins / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014026:Nicotiana / Q000201:enzymology*; D014446:Tyrosine 3-Monooxygenase
chemical_list: D048948:14-3-3 Proteins; D006027:Glycosides; D011506:Proteins; C007808:fusicoccin; D014446:Tyrosine 3-Monooxygenase; D006180:Proton-Translocating ATPases