Large scale expression, purification and 2D crystallization of recombinant plant 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 recombinant ATPases in quantities suitable for crystallographic studies aiming at 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 both in the presence and in the absence of regulatory 14-3-3 protein depending on the presence of the diterpene fusicoccin which specifically induces formation 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+-ATPase polypeptide. The purified H(+)-ATPase readily formed two-dimensional crystals following reconstitution into lipid vesicles. Electron cryo-microscopy of the crystals yielded a projection map at approximately 8 A resolution, the p22(1)2(1) symmetry of which suggests a dimeric protein complex. Three distinct regions of density of approximately equal size are apparent and may reflect different domains in individual molecules of AHA2.
Jahn
T
T
Department of Plant Biology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark.
Dietrich
J
J
Andersen
B
B
Leidvik
B
B
Otter
C
C
Briving
C
C
Kühlbrandt
W
W
Palmgren
M G
MG
D048948:14-3-3 Proteins; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D002462:Cell Membrane / Q000201:enzymology*; D020285:Cryoelectron Microscopy; D003460:Crystallization; D019281:Dimerization; D006027:Glycosides / Q000494:pharmacology; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013329:Structure-Activity Relationship; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D048948:14-3-3 Proteins; D006027:Glycosides; D007527:Isoenzymes; D008081:Liposomes; D011993:Recombinant Fusion Proteins; C007808:fusicoccin; D014446:Tyrosine 3-Monooxygenase; D006180:Proton-Translocating ATPases