Fusion (%) = 100 (B-A)/(C-A)

Transcription

1 6 Fusion (%) = 1 (B-A)/(C-A) fluorescence, a.u. x 1 C 1 B A 6 1 A Supplementary Figure 1. Fusion of lipid vesicles studied with cobalt-calcein liquid content transfer assay. An example of fusion % calibration for the red trace shown in Fig. 1b. The difference between the maximal fusion signal B and the background signal A is divided by the equivalent maximal signal C following the release of encapsulated calcein by addition of EDTA/Triton X-1 and is multiplied by 1%. The trace from the shadowed region is shown in Fig. 1b.

2 Fluorescence, (a. u. X 1 ) Supplementary Figure. Effect of external salt addition on vesicles fusion rate after initiation of vesicle fusion reaction studied with cobalt-calcein liquid content transfer assay. Addition of external mm KCl three times (arrows) seconds after complementary vesicles were mixed in 1 mm MOPS, ph 7. does not stop fusion.

3 Liquid content leakage, % SUV + + SUV -, buffer SUV + + SUV -, plus 1 mm KCl SUV + SUV -, buffer Supplementary Figure. Liquid content leakage during fusion of complementary SUV. nm anionic SUV containing 1 mm calcein, 1 mm MOPS, ph 7. were fused with probe-free nm cationic (red trace) or neutral (black) SUV in buffer (1 mm MOPS, ph 7.), or probe-free cationic SUV in buffer supplemented with 1 mm KCl (blue trace)

4 .1 OD 7.1 SUV + SUV -, buffer SUV + SUV -, plus mm KCl SUV + + SUV -, buffer SUV + + SUV -, plus mm KCl. 9 1 Supplementary Figure. Aggregation of complementary SUV. nm anionic SUVs were mixed with nm cationic (red curves) or neutral (black curves) SUV in buffer (1 mm MOPS, ph 7.) (thick curves) or buffer plus mm KCl (thin curves). The signal was followed by light absorption at 7 nm.

5 Inner lipid monolayer mixing, % Intervesicular lipid mixing, % a 6 b 1 SUV + + SUV -, 1 mm KCl SUV + + SUV -, 1 mm KCl SUV + SUV -, 1 mm KCl buffer buffer + 1 mm KCl buffer + mm KCl buffer + mm KCl buffer + mm KCl buffer + 1 M KCl Supplementary Figure. Lipid mixing in complementary SUV. (a) Intervesicular lipid mixing. nm anionic SUV containing % (weight fraction) NBD, % Rho-B, % POPA and 71% PC were mixed with nm probe-free cationic (red trace) or neutral (black) SUV in buffer (1 mm MOPS, ph 7.) containing 1 mm KCl, or with cationic SUV in buffer supplemented with 1 mm KCl (blue trace), at 1: ratio. (b) Inner lipid monolayer mixing. Same anionic SUV treated with sodium dithionite to quench fluorescence of their external lipid monolayer as described in Methods were mixed with probe-free nm cationic SUV in buffer (1 mm MOPS, ph 7.) containing various amounts of KCl, at 1: volume

6 fusion, % glucose, mm glucose, 1 mm glucose 8 mm, KCl mm buffer 1 1 KCl, mm sucrose, mm sucrose, 1 mm KCl, mm 1 6 Supplementary Figure 6. Influence of osmolytes (glucose and sucrose) on vesicle fusion. nm anionic SUV were fused with nm cationic SUV in buffer (1 mm MOPS, ph 7.) in presence of or 1 mm glucose and sucrose as compared to fusion in and mm KCl.

7 Fluorescense (a. u. x 1 ) a b OD.8 DOTAP PL + E-PC PL + PC PL PL + PL +, LDAO PL PL, LDAO 1 6 Supplementary Figure 7. Effect of bilayer charge on hydrolase activity of F 1 F o -synthase in neutral proteoliposomes (PL, PC only) or cationic proteoliposomes (PL +, % PC: % cationic lipids DOTAP or E-PC, by weight). (a) ACMA quenching by DOTAP (orange trace), E-PC (red) and PC (black) PL. The proteoliposomes were formed by using the same amount of the protein and lipids as described in Methods, and the same volumes of proteoliposomes were used in the assay. Proton pumping was initiated by adding. mm to the reaction medium (1 mm KCl, 1 mm MgCl, mm MOPS ph 7.). (b) hydrolysis by PC and E-PC PL + without (black, red) and with (grey, orange).% LDAO. The activity was measured with regenerating system containing 1 mm KCl, mm MOPS, ph 7.,. mm MgCl, 1 mm, µm nigericin, mm phosphoenolpyruvate,. mm NADH, units/ml of pyruvate kinase and lactate dehydrogenase.

8 Fluorescense (a. u. x 1 ) Fluorescense (a. u. x 1 ) a 6 PL, mm KCl PL, mm KCl PL, 1 mm KCl b 6 + mm KCl nigericin 1 nigericin 1 PL +, 1 mm KCl PL +, mm KCl 1 PL +, 1 mm KCl 1 c.8. DTT PL +, 1 mm KCl PL +, 1 mm KCl PL, 1 mm KCl PL, 1 mm KCl Supplementary Figure 8. Dependence of proton pumping by F 1 F o -synthase (a, b) and bo -oxidase (c) in cationic (PL + ) and neutral (PL ) proteoliposomes on KCl concentration. Proteoliposomes were assayed in 1 mm MOPS ph 7., 1 mm MgCl, and indicated concentrations of KCl. Proton gradient was dissipated by µm nigericin. (a) hydrolysis driven ACMA quenching by PL is independent of the salt concentration. (b) hydrolysis driven ACMA quenching by PL + is sensitive to low ionic strength and is minimal in 1 mm KCl (red trace); the pumping can be restored by addition of mm KCl (blue arrow). (c) ACMA quenching driven by coenzyme-q 1 oxidation and triggered by addition of DTT to bo -oxidase proteoliposomes. Proton pumping by PL + is weak (red) and low ionic strength sensitive (pink) in contrast to proton pumping by PL (black, grey), which is not affected by low ionic strength.

9 Fluorescense (a. u. x 1 ) a b c.9 F. 1 F o PL, no DCCD. F 1 F o PL, + DCCD bo PL, no DCCD bo PL, + DCCD DTT OD.7.6 F 1 F o PL, no detergent F 1 F o PL, with detergent Supplementary Figure 9. Control experiments demonstrating unidirectional reconstitution of F 1 F o -synthase into proteoliposomes (a), and DCCD-selective inhibition of proton pumping by F 1 F o (b) but not bo oxidase (c). (a) hydrolase activity of F 1 F o PL with (grey) and without (black) vesicle disruption by the non-denaturing detergent.% sodium cholate measured with -regenerating system as described in Methods. (b) hydrolysis driven ACMA quenching by F 1 F o -synthase PL (black) was completely abolished after 1 hour incubation with µm DCCD (grey). (c) Coenzyme Q 1 driven ACMA quenching by bo PL is not sensitive to µm DCCD.