Abstract
Plasma membrane H(+)-ATPases form a subfamily of P-type ATPases responsible for pumping protons out of cells and are essential for establishing and maintaining the crucial transmembrane proton gradient in plants and fungi. Here, we report the reconstitution of the Arabidopsis thaliana plasma membrane H(+)-ATPase isoform 2 into soluble nanoscale lipid bilayers, also termed nanodiscs. Based on native gel analysis and cross-linking studies, the pump inserts into nanodiscs as a functional monomer. Insertion of the H(+)-ATPase into nanodiscs has the potential to enable structural and functional characterization using techniques normally applicable only for soluble proteins.
Highlights
The plasma membrane Hϩ-ATPase generates electrochemical gradients in plants and fungi
Plant plasma membrane (PM) Hϩ-ATPase Purifies as a Dimer upon Heterologous Expression in Yeast—The S. cerevisiae strain RS-72 has previously proved to be suitable for the high level expression and purification of A. thaliana PM Hϩ-ATPase Arabidopsis thaliana Hϩ-ATPase isoform 2 (AHA2) [50]
We found that the Vmax values of nanodisc-reconstituted PM Hϩ-ATPase were similar to those measured for asolectin-activated, dodecyl -D-maltoside (DDM)-solubilized PM Hϩ-ATPase
Summary
The plasma membrane Hϩ-ATPase generates electrochemical gradients in plants and fungi. Results: We developed a protocol for reconstitution of active Hϩ-ATPase in nanodiscs. Conclusion: The minimal functional unit of the Hϩ-ATPase is a monomer. Significance: The plasma membrane Hϩ-ATPase functions like well characterized cation pumping P-type ATPases. Plasma membrane H؉-ATPases form a subfamily of P-type ATPases responsible for pumping protons out of cells and are essential for establishing and maintaining the crucial transmembrane proton gradient in plants and fungi. We report the reconstitution of the Arabidopsis thaliana plasma membrane H؉-ATPase isoform 2 into soluble nanoscale lipid bilayers, termed nanodiscs. Based on native gel analysis and cross-linking studies, the pump inserts into nanodiscs as a functional monomer. Insertion of the H؉-ATPase into nanodiscs has the potential to enable structural and functional characterization using techniques normally applicable only for soluble proteins
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