FIGURE 1.

atTic40 utilizes a soluble intermediate during targeting to the IEM. A, schematic of the pre-atTic40 protein. B, [³⁵S]pre-atTic40 was imported into isolated chloroplasts for 30 min. The chloroplasts were incubated in the presence (+) or absence (−) of thermolysin (200 μg/μl) on ice for 30 min. Proteolysis was stopped, and the chloroplasts were lysed and separated into soluble (S) and membrane (P) fractions in the presence (+) or absence (−) of 0.2 m Na₂CO₃, pH 11.5. The graphs represent the quantification of lanes 4, 5, 6, and 7 for either mature atTic40 (left) or the int-atTic40 (right). [³⁵S]Pre-atTic40 was imported into chloroplasts for 5 min at 20 °C (C), the reaction was stopped on ice and treated with thermolysin as in B, and import was resumed in the presence of 5 mm ATP (Chase) for the times indicated. Equivalent fractions were collected and separated into membrane and supernatant fractions by osmotic lysis. The graph represents the quantification of the distribution of atTic40 and int-atTic40 during the chase. D, samples from the 60 min time point in C were treated with 0.2 m Na₂CO₃, pH 11.5, and separated into soluble and membrane fractions. Lane 14 (T) contains a sample equivalent to the starting material before alkaline treatment. The graph represents the distribution of atTic40 between the membrane pellet and supernatant fractions.