FIGURE 5.

Reconstitution of TAP into proteoliposomes. A, quantification of lipid and protein after gradient centrifugation. Purified TAP was reconstituted into liposomes prepared from E. coli total lipids. Proteoliposomes were separated by Ficoll density gradient centrifugation (0–10%; left image), and gradient fractions were assayed for lipids (right panels, upper) and TAP (right panels, lower). Empty and filled liposomes are indicated as open and filled circles, respectively. Corresponding fractions are marked by triangles. B, orientation of TAP in proteoliposomes. Proteoliposomes containing TAP (2.5 μg) were incubated with TEV protease in the presence or absence of 1% TX-100. After TEV cleavage, samples were analyzed by SDS-PAGE (10%) and immunoblotting using antibodies specific for TAP1, TAP2, and His₆ tag. P, purified protein; R, reconstituted protein. TX-100, Triton X-100. C, freeze-fracture EM. Particles indicate incorporated TAP complexes.