Quaternary structure and regulation of the nicotiana plumbaginifolia plasma membrane proton pum ATPase

The plasma membrane H+-ATPase uses the energy released by ATP hydrolysis to transport protons across the plasma membrane. The resulting electro-chemical gradient is used to energize ion or metabolite transporters. The H+-ATPases are therefore expected to be tightly regulated. At the post-translational level, the plant H+-ATPase is activated by phosphorylation of the ultimate Thr955 and binding of 14-3-3 proteins. Preliminar experiments have shown that PMA2, a Nicotiana plumbaginifolia H+-ATPase, exists as a dimer and is transformed into an oligomeric complex upon activation. Using a PMA2-GFP fusion protein expressed in the yeast Saccharomyces cerevisiae, we have analyzed the effect of independent deletion of the cytosolic regions. The large loop, unlike the other regions, appeared required for dimer formation. Dimerization of the PMA2 large loop expressed in Escherichia coli was demonstrated by size exclusion chromatography. The dimeric and the activated oligomeric form of the His-tagged PMA2 expressed in yeast were separated by affinity and size exclusion chromatography. The latter was examined by single particle cryo-electron microscopy. 3D reconstitution indicated a hexameric organization built up of six H+-ATPase and six 14-3-3 proteins. To study PMA2 regulation in the plant, transgenic plants were obtained, expressing either the wild-type PMA2, a low activity Thr mutant (PMA2-T955A) or a constitutively activated isoform deleted of its auto-inhibitory C-terminal domain. Expression of the truncated isoform was less than 100%, suggesting counter-selection of plants highly expressing this activated isoform. These plants were characterized by abnormal bending of young leaves, suggesting disturbed regulation of cell expansion. Some transgenic lines were characterized by co-suppression of both the ectopic and the resident PMA2 gene, resulting in delayed flowering and male sterility, which suggests a role for PMA2 during developmental stages. Further characterization of these transgenic lines should help in deciphering the regulation and physiological roles of the PMA2 H+-ATPase