Figure 2. Reconstitution of l-Opa1.

(A) Representative size-exclusion chromatograph and SDS-PAGE gel of human l-Opa1 purified from P. pastoris. (B) SDS-PAGE gel of human s-Opa1 purified from P. pastoris. l-Opa1 activity, with velocity (C) and specific activity (D) of GTP hydrolysis in the presence and absence of cardiolipin, while varying protein concentration of Opa1. Data are shown as mean ± SD, with error bars from three independent experiments. Representative single-liposome photobleaching steps (E and F) and histogram of step sizes (distribution for 110 liposomes shown) (G).

Figure 2—figure supplement 1. GTP hydrolysis activity.

GTP hydrolysis (GTPase) activity of l-Opa1 (A) and s-Opa1 (B) in the presence and absence of cardiolipin. Both G300E l-Opa1 and G300E s-Opa1 do not show any GTPase activity (C and D). Mixing G300E s-Opa1 with WT l-Opa1 at 1:1 molar ratio (E) does not alter the GTPase activity of, detergent solubilized, WT l-Opa1 significantly (E and A, p>0.2, t-test). A similar effect is seen upon addition of G300E l-Opa1 to WT s-Opa1 at 1:1 ratio (F). Under these conditions, s-Opa1 GTPase activity is similar to s-Opa1 alone (F and B, p>0.2, t-test). Data shown as mean ± SD, error bars from three experiments.

Figure 2—figure supplement 2. Liposome co-flotation.

Liposome co-flotation analysis: Reconstituted l-Opa1 co-floats with liposomes both with and without cardiolipin (A and D). Liposomes were labeled with 0.2% (mol) TexasRed-DHPE, and their distribution was confirmed by liposome dye absorbance at 590 nm. Opa1 distribution was analyzed by western blot. Opa1/liposome fractions was mostly found near 15 ~ 30% sucrose. This reconstitution is stable under high salt (B and E) or carbonate conditions (C and F). s-Opa1 interacts with liposomes in a cardiolipin-dependent manner (G-L). This interaction is resistant to high salt (H) but sensitive to carbonate treatment (I), where the protein was found in the bottom fractions lacking liposome (60% sucrose). s-Opa1 does not associate with DOPC liposomes (J-L). These results indicate that l-Opa1 was successfully reconstituted through integral transmembrane region, whereas the s-Opa1 bilayer-association is through a cardiolipin:s-Opa1 peripheral membrane interaction.

Figure 2—figure supplement 3. Bilayer homogeneity and FCS.

Epifluorescence image of polymer-tethered lipid bilayers before (A) and after Opa1 reconstitution (B), showing a homogeneous lipid bilayer. Scale bar: 10 µm. FCS profiles of TexasRed-PE and TexasRed labeled anti-Opa1 antibody show slower diffusion for reconstituted l-Opa1 (C), indicating successful reconstitution, and that the reconstituted l-Opa1 diffuses freely.

Figure 2—figure supplement 4. Blue native gels.

(A) Blue native (BN-PAGE) gels show WT l-Opa1 and s-Opa1 can self-assemble as oligomers in DDM. (B) Mixtures of WT l-Opa1 and WT s-Opa1 show a range of species from ~480 KDa - ~ 1 MDa. G300E l-Opa1, in the presence of WT s-Opa1, does not alter this gel migration pattern. In contrast, complexes comprising WT l-Opa1 and G300E s-Opa1 show a slight shift to a population mainly containing a ~ 480 Kda and 720 KDa species.

Figure 2—figure supplement 5. FCS.

Fluorescence autocorrelation profiles of TexasRed labeled anti-His antibody in the presence of unlabeled liposomes (A), and TexasRed-PE-labeled liposomes (B), showing diffusion coefficients of unbound antibody versus liposomes. FCS profile of reconstituted l-Opa1 (detected with a TexasRed labeled antibody) (C) is similar to that of dye-labeled liposomes (B), indicating successful reconstitution of Opa1.