Fig. 4. Insertion of Toc34 into protein-free liposomes under different conditions. (A) [³H]-Leucine-labelled Toc34 was incubated with protein-free liposomes (1 mM final lipid concentration, lanes 2-11) of different lipid composition (for nomenclature, see Table 1) or of outer envelope lipids (OEL, lanes 12-14), followed by thermolysin treatment (Thr, lanes 3, 5, 7, 9, 11, 13 and 14). In lane 14 the membrane was solubilised by TX-100 before thermolysin treatment. Binding (before thermolysin treatment, black bar) and insertion (8 kDa fragment after thermolysin treatment corrected for the number of leucine residues, grey bar) was quantified as described in Materials and Methods and is shown as a histogram. The binding of Toc34 to liposomes of composition C3 was set to 100%. The results represent an average of at least three independent experiments. (B) [³H]-Leucine-labelled Toc34 was incubated with protein-free liposomes (1 mM final lipid concentration, composition C3, lanes 1 and 2) in the presence of 1 mM MgCl₂ (lanes 1-10) and 1 mM GTP (lane 3 and 4), 1 mM GMP-PNP (lanes 5 and 6), 1 mM GDP (lanes 7 and 8) or 1 mM ATP (lanes 9 and 10) followed by quantification of binding and insertion as for Fig. 3. Binding of Toc34 to liposomes in the absence of nucleotides was set to 100%. The results represent an average of at least three independent experiments.