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

被引:20
作者
Jahn, T
Dietrich, J
Andersen, B
Leidvik, B
Otter, C
Briving, C
Kühlbrandt, W
Palmgren, MG
机构
[1] Royal Vet & Agr Univ, Dept Plant Biol, DK-1870 Frederiksberg C, Copenhagen, Denmark
[2] Max Planck Inst Biophys, D-60528 Frankfurt, Germany
[3] AstraZeneca R&D, S-43183 Molndal, Sweden
关键词
Arabidopsis thaliana; 14-3-3; protein; fusicoccin; proton pump; Saccharomyces cerevisiae;
D O I
10.1006/jmbi.2001.4688
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 similar to8 Angstrom 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. (C) 2001 Academic Press.
引用
收藏
页码:465 / 476
页数:12
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