Journal: Nature Communications
Article Title: A modular platform for one-step assembly of multi-component membrane systems by fusion of charged proteoliposomes
doi: 10.1038/ncomms13025
Figure Lengend Snippet: ( a ) Schematic representation of the experiment. (top) Binary system (two components): cationic (PL + ) and anionic proteoliposomes (PL − ) fused together. (bottom) Ternary system (three components): anionic LUV or GUV fused with two types of PL + containing either ATP-synthase or bo 3 -oxidase. The vesicles were fused in 20 mM KCl, 5 mM MOPS pH 7.4, 1 mM MgCl 2 for 5–7 min followed by addition of KCl and MOPS to final concentrations of 100 and 50 mM, respectively, the luciferin-luciferase-ADP cocktail prepared as described in Methods, and oxidized Coenzyme Q 1 . In the assembled electron transport chain energization of the membrane is triggered by addition of dithiothreitol (DTT red ), which reduces Q 1 (Q 1 H) to make it available to bo 3 -oxidase. Oxidation of reduced Q 1 by bo 3 -oxidase pumps protons into each post-fusion vesicle. This builds up a PMF which drives ATP synthesis by ATP-synthase in the same vesicle. ATP synthesis is initiated by adding potassium phosphate (KP i ) to energized vesicles 1 min after addition of DTT. The ATP synthesized is detected by the luciferin-luciferase system, where conversion of synthesized ATP into pyrophosphate and AMP by luciferase is followed by light emission registered in a luminometer. ( b ) ATP synthesis by binary system. Blue trace: ATP synthesis by F 1 F o PL − fused with bo 3 PL + ; grey: same as blue but with nigericin; pink: same as blue but with DCCD-treated F 1 F o PL − ; green: same as blue but in 200 mM KCl; black: same as blue, but with bo 3 PL 0 ;. Red trace: ATP synthesis by F 1 F o PL + fused with bo 3 PL − . ( c , d ) ATP synthesis by ternary system. Red trace: ATP synthesis by F 1 F o PL + and bo 3 PL + fused with LUV − or GUV; green: same as red but in 200 mM KCl. Black: PL − were used instead of PL + in the fusion reaction.
Article Snippet: The ATP synthesized in the course of the reaction is detected by the luciferin-luciferase system, where conversion of synthesized ATP into pyrophosphate and AMP by luciferase is followed by light emission registered in a luminometer (Sirius-L single tube luminometer, Titertek).
Techniques: Luciferase, Membrane, Synthesized
