Figure 7.

The TOC/TIC Pore Size Is Greater Than 25.6 Å.

Biotinylated RSSUHC (RSSUHC-Bt, 8.3 μg/mL in import reaction) was incubated with 17.5- to 70-fold molar excess mSA in import buffer prior to import reactions. Thermolysin treatments were conducted as in Figure 6. Stromal localization was determined as in Figure 6, except that the membrane/stroma fractionation was done on thermolysin-treated chloroplasts. For trypsin (Tryp) treatments, postimport chloroplasts were resuspended in import buffer containing 240 μg/mL trypsin, incubated 30 min at 25°C, resuspended again in 1 mg/mL soybean trypsin inhibitor, reisolated on 40% Percoll, and washed with import buffer. Additionally, protease treatments were conducted in the presence of 1% Triton X-100 (TX). RSSUHC-Bt and mSA were detected on α-biotin and α-FLAG blots, respectively, along with standards that represent a molar percentage of RSSUHC-Bt input. Error bars indicate se (n = 3). mSA-labeled mature RSSUHC or mSA from the Therm treatment were normalized to 1. pr, RSSUHC-Bt precursor; m, mature RSSUHC-Bt. Asterisk indicates nonspecific chloroplast protein.