Large scale expression, purification and 2D crystallization of recombinantplant plasma membrane H+-ATPase

P-type ATPases convert chemical energy into electrochemical gradients that are used to energize secondary active transport. Analysis of the structure and function of P-type ATPases has been limited by the lack of active recom binant ATPases in quantities suitable for crystallographic studies aiming a t solving their three-dimensional structure. We have expressed Arabidopsis thaliana plasma membrane H+-ATPase isoform AHA2 equipped with a His(6)-tag, in the yeast Saccharomyces cerevisiae. The H+-ATPase could be purified bot h in the presence and in the absence of regulatory 14-3-3 protein depending on the presence of the diterpene fusicoccin which specifically induces for mation of the H+-ATPase/14-3-3 protein complex. Amino acid analysis of the purified complex suggested a stoichiometry of two 14-3-3 proteins per H+-AT Pase polypeptide. The purified H+-ATPase readily formed two-dimensional cry stals following reconstitution into lipid vesicles. Electron cryo-microscop y of the crystals yielded a projection map at similar to8 Angstrom resoluti on, the p22(1)2(1) symmetry of which suggests a dimeric protein complex. Th ree distinct regions of density of approximately equal size are apparent an d may reflect different domains in individual molecules of AHA2. (C) 2001 A cademic Press.