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. 2014 Oct 13;5:535. doi: 10.3389/fpls.2014.00535

Figure 6.

Figure 6

Processing of OEP80 during chloroplast import in vitro. (A) After 10-min incubation with radiolabeled precursors in the import condition in the dark, pea chloroplasts were re-isolated through a Percoll cushion and divided into two samples. The first sample was immediately lysed and separated into soluble (S1), peripheral membrane (S2), and integral membrane (P) fractions as described in the legend to Figure 4A. The second sample was resuspended in import buffer containing 3 mM MgATP for 30 min at room temperature in the light then separated into the three fractions. The OEP80 precursor of 74 kD and imported products ranging from 66–71 kD are labeled as x and y; precursor, intermediate and mature forms of Toc75 are indicated with pr, i, and m, respectively. (B) Intact chloroplasts isolated from pea seedlings and incubated with radiolabeled OEP80 in the import condition for 30 min in light (i) or chloroplasts isolated from A. thaliana seedlings (ii) were analyzed directly (C) or lysed hypotonically and separated into soluble (S) and pellet (P) fractions by centrifugation. Proteins in each fraction were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was incubated with the partially-purified anti-OEP80 antibody and the immunoreactions were visualized by colorimetric assay using alkaline phosphatase (αOEP80), and the radiolabeled proteins in the membrane were visualized by phosphorimager analysis ([35S]). The two images were overlaid using ink spots and radiolabeled marking spots (Overlay). In Overlay, radioactive signals are indicated with magenta and the immunoreactive signals with green. The lane tl was loaded with translation products and pre-stained (blue) molecular weight markers. Endogenous OEP80 of 70 kD and the soluble 63-kD protein of unknown identity in the A. thaliana chloroplasts recognized by the anti-OEP80 antibody are indicated with a number sign (#) and an asterisk (*); the OEP80 precursor of 74 kD and imported products ranging from 66–71 kD are labeled as x and y, respectively.