(A) Controls for intra-membrane and inter-membrane FRET: When both TexasRed and Cy5 PE are present in the same bilayer, high FRET efficiency is observed. When TexasRed and Cy5 PE are present in two different bilayers, with a ~ 7 nm tethering distance (from bilayer center to bilayer center in the double bilayer stack), FRET efficiency was low (data analyzed from 10 random spots in two bilayers (p<0.0001, t test). Analysis of ~20 particles shows ~40% FRET efficiency for both homotypic and heterotypic tethering. This indicates that l-Opa1 is able to bring the two membranes within close proximity (<7 nm) without mixing the two membranes. (B) Quantification of DOPC liposomes tethered to a DOPC bilayer containing reconstituted l-Opa1. Liposomes do not tether to the supported bilayer, indicating that in the absence of cardiolipin, l-Opa1 does not tether liposomes alone. The lack of liposome docking to exposed regions also argues that few defects were introduced into the bilayer following reconstitution. Data from three different bilayers.
Figure 4—figure supplement 1—source data 1. Docking.