FIGURE 4.

Effect of 3AB protein on reconstitution of synthetic liposomes by direct mixing and dialysis. A, following dialysis, 3AB-containing proteoliposomes were fractionated by ultracentrifugation through discontinuous sucrose gradients. The arrowhead denotes the position of liposomes in the gradient at the 20–40% sucrose interface. B, fractions obtained from the indicated sucrose gradient position were analyzed by SDS-PAGE and silver staining to detect poliovirus 3AB. M designates molecular weight markers. C, cryo-electron micrograph of liposomes lacking 3AB protein displaying single-membraned liposomes of various sizes. Scale bar = 100 nm for C and D. D, cryo-electron micrograph of reconstituted 3AB-containing proteoliposomes. Selected double-membraned vesicles from this image (arrows) are enlarged 2-fold in the right panel. E, additional images of double-membraned vesicles and more complex structures after reconstitution in the presence of 3AB. White dots mark the presumed directionality of the membrane invagination. Scale bar = 50 nm. F, frequencies of different membrane topologies formed in the absence (white bar) and presence (gray bar) of 3AB protein. The asterisk denotes a statistically significant difference. G, cumulative frequency plot of vesicle diameters for liposomes alone (▿) or proteoliposomes containing purified 3AB protein (●) as visualized by cryo-electron microscopy. Data show significant statistical differences for single-, double-, and multimembraned vesicles (p < 0.1, Kolmogorov-Smirnov test).