FIGURE 6.

The atTic40 Ser/Pro-rich region and TMD function as a stop-transfer signal when they replace the APG1 TMD. A, schematic of the pre-APG1-atTic40SP-TM protein. B, [³⁵S]pre-APG1-atTic40SP-TM 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 0.1 m Na₂CO₃, pH 11.5. The graph represents the quantification of the distribution of mature APG1-atTic40SP-TM in the soluble supernatant and membrane pellet fractions before (control) or after (pH 11.5) alkaline treatments (lanes 4–7). C, [³⁵S]pre-APG1-atTic40SP-TM was imported into chloroplasts for 2, 5, and 30 min at 20 °C. The reactions were stopped on ice and treated with thermolysin as in B, and equivalent fractions were collected and separated into membrane and supernatant fractions by osmotic lysis. The graph represents the quantification of APG1-atTic40SP-TM distribution in the soluble supernatant and membrane pellet fractions. D, [³⁵S]pre-APG1-atTic40SP-TM was imported into chloroplasts for 5 min at 20 °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 APG1-atTic40SP-TM during the chase. E, samples from the 60 min time point in D were treated with 0.1 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 APG1-atTic40SP-TM between the membrane pellet and supernatant fractions.