COMPLEMENTATION OF THE SACCHAROMYCES-CEREVISIAE PLASMA-MEMBRANE H-ATPASE BY A PLANT H+-ATPASE GENERATES A HIGHLY ABUNDANT FUSICOCCIN BINDING-SITE()

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 d omain 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 functi onal replacement of the Saccharomyces cerevisiae H+-ATPase genes by a Nicotiana plumbaginifolia H+-ATPase (pma2) results in the generation o f a high affinity fusicoccin binding site that is exceptionally abunda nt. Acquisition of FC binding capacity is accompanied by a significant increase in the amount of plasma membrane-associated yeast 14-3-3 hom ologs, The existence of a (plant) PMA2. (yeast)14-3-3 complex was demo nstrated using two-dimensional gel systems (native/denaturing). After expression of PMA2 lacking most of its C-terminal region, neither H+-A TPase 14-3-3 complex formation nor FC binding activity could be observ ed. 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 re quirement for FC binding.