Abstract
The plasma membrane H+-ATPase is a proton pump essential for several physiological important processes in plants. Through the extrusion of protons from the cell, the PM H+-ATPase establishes and maintains a proton gradient used by proton coupled transporters and secondary active transport of nutrients and metabolites across the plasma membrane. Additional processes involving the PM H+-ATPase includes plant growth, development, and response to biotic and abiotic stresses. Extensive efforts have been made in attempts to elucidate the detailed physiological role and biochemical characteristics of plasma membrane H+-ATPases. Studies on the plasma membrane H+-ATPases have involved both in vivo and in vitro approaches, with the latter employing either solubilisation by detergent micelles, or reconstitution into lipid vesicles. Despite resulting in a large body of information on structure, function, and regulation of H+-ATPases, key questions, in particular concerning the detailed interaction of regulator proteins with the H+-ATPases, remains answering that may require the use of new approaches.
In this work the proton pump Arabidopsis thaliana plasma membrane H+-ATPase isoform 2 has been reconstituted into soluble nanoscale lipid bilayers, also termed nanodiscs. Extensive analysis confirms the correct assembly and reconstitution of active proton pump into nanodiscs. The pump inserts as a monomer, which through activity analysis confirms this as the minimal functional unit of the plasma membrane H+-ATPase. Reconstitution of the H+-ATPase into nanodiscs has the potential to enable structural and functional characterization using various techniques, exemplified by the specific immobilization of reconstituted proton pump using surface plasma resonance. The ability to efficiently separate empty from membrane protein-containing nanodiscs and obtain monodispersity is important when considering down-stream analysis and still a major challenge during sample preparation. In this work a novel approach to this challenge is presented, based on the application of Free Flow Electrophoresis (FFE) in an interval zone electrophoresis mode. The use of FFE for nanodisc purification is exemplified from the separation of two fundamentally different membrane proteins reconstituted in nanodiscs; the Arabidopsis thaliana plasma membrane H+-ATPase isoform 2 and the NADPH-dependent cytochrome P450 oxidoreductase from Sorghum bicolor. The reconstitution procedures used for both membrane proteins results in a mixture of empty and protein-containing nanodiscs, which together with their individual characteristics makes them ideal for displaying the flexibility of the Free Flow Electrophoresis based separation. Several opportunities to further unravel the secrets of the P-type ATPases originate from the reconstitution of the proton pump into nanodiscs.
In this work the proton pump Arabidopsis thaliana plasma membrane H+-ATPase isoform 2 has been reconstituted into soluble nanoscale lipid bilayers, also termed nanodiscs. Extensive analysis confirms the correct assembly and reconstitution of active proton pump into nanodiscs. The pump inserts as a monomer, which through activity analysis confirms this as the minimal functional unit of the plasma membrane H+-ATPase. Reconstitution of the H+-ATPase into nanodiscs has the potential to enable structural and functional characterization using various techniques, exemplified by the specific immobilization of reconstituted proton pump using surface plasma resonance. The ability to efficiently separate empty from membrane protein-containing nanodiscs and obtain monodispersity is important when considering down-stream analysis and still a major challenge during sample preparation. In this work a novel approach to this challenge is presented, based on the application of Free Flow Electrophoresis (FFE) in an interval zone electrophoresis mode. The use of FFE for nanodisc purification is exemplified from the separation of two fundamentally different membrane proteins reconstituted in nanodiscs; the Arabidopsis thaliana plasma membrane H+-ATPase isoform 2 and the NADPH-dependent cytochrome P450 oxidoreductase from Sorghum bicolor. The reconstitution procedures used for both membrane proteins results in a mixture of empty and protein-containing nanodiscs, which together with their individual characteristics makes them ideal for displaying the flexibility of the Free Flow Electrophoresis based separation. Several opportunities to further unravel the secrets of the P-type ATPases originate from the reconstitution of the proton pump into nanodiscs.
| Original language | English |
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| Publisher | Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen |
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| Number of pages | 99 |
| Publication status | Published - 2012 |