(A) Toc75ΔP1 and Toc75ΔP1-2 accumulate as their mature forms in TOC75ΔP1#2 and TOC75ΔP1-2#2 plants. In vitro translated mature [35S]Toc75ΔP1 and [35S]Toc75ΔP1-2 were mixed with chloroplasts extracts from TOC75ΔP1#2 and TOC75ΔP1-2#2 plants and analyzed by SDS-PAGE. The mobility of [35S]Toc75ΔP1 and [35S]Toc75ΔP1-2, detected by phosphorimaging (Lanes 1 and 2), was compared to the mobility of endogenous Toc75ΔP1 and Toc75ΔP1-2, detected by immunoblotting with anti-atToc75 serum (Lanes 3 and 4). (B) Toc75ΔP1 and Toc75ΔP1-2 are integrated into the chloroplast envelope. Isolated chloroplasts (T) from heterozygous toc75-III-1, TOC75, TOC75ΔP1#2 and TOC75ΔP1-2#2 plants were hypotonically lysed and fractionated by centrifugation at 18,000 × g for 30 min at 4°C into membrane pellet (M) and soluble (S) fractions. Equivalent samples of each fraction from chloroplasts corresponding to 10 μg chlorophyll was resolved by SDS-PAGE and immunoblotted with anti-atToc75 serum. (C) Toc75myc and Toc75ΔP1myc constructs used in this study. The dashed line represents the deleted POTRA domain region in POTRA1-deleted constructs. The site of insertion of myc tag is shown in each panel. (D) Protease sensitivity of Toc75myc, and Toc75ΔP1myc proteins in isolated intact chloroplasts. Intact chloroplasts from TOC75, TOC75myc and TOC75ΔP1myc seedlings were treated with trypsin or thermolysin in the absence (-) or presence (+) of 1% Triton X-100. Reactions were incubated on ice for 30 min, and proteolysis was stopped with 2.5 mM PMSF, 0.05 mg/mL Nα-Tosyl-L-lysine chloromethyl ketone (TLCK), 0.25 mg/mL soybean trypsin inhibitor, and 2 μg/mL aprotinin (for trypsin) or 20 mM EDTA (for thermolysin). Chloroplasts were analyzed by immunoblotting with antibodies against various proteins as indicated. The asterisk indicates the position of a non-specific immunoreactive band. a and b denote bands corresponding to the ~55 kDa and ~46 kDa trypsin fragments of Toc75myc and Toc75ΔP1myc, respectively.