Figure 1: Validation of the split-luciferase based extraction assay.
A) Diagram for split-luciferase extraction assay. Note that for properly reconstituted liposomes, both the HiBiT and thrombin sites are in the lumen of the proteoliposome.
B) Workflow for split-luciferase assay.
C) Protease protection assay shows that substrates are properly oriented. Anti-HiBiT western blot of pre-cleared liposomes shows minimal loss of signal upon addition of thrombin protease, but complete loss of signal with both thrombin and detergent.
D) Addition of an inert His6-tagged protein to ultracentrifugation improves liposome pelleting efficiency and reduces background activity.
E) Standard curves used to calculate percent substrate extracted. Four separate reconstitutions of the standard Sumo-Sec22 model substrate in standard liposomes show a linear and reproducible signal. Different concentrations of pre-cleared, but unreacted proteoliposomes were mixed with LgBiT and furimazine reagent in detergent, which permeablizes the proteoliposomes. The X-axis refers to the percent of substrate in the extraction assay.
F) The split-luciferase extraction assay shows ATP-dependent and physiological substrate extraction activity. The known substrate Sec22 is extracted whereas the native mitochondrial protein Fis1 is not extracted unless a hydrophobic patch is added, which serves as an Msp1 recognition sequence. Error bars show standard error of the mean.