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Fig. 6. Protein extracts of Physcomitrella patens protoplasts, transformed with RB60:GFPK, D 28RB60:GFP, L50:GFPK, mGFP, Chit:GFPK constructs or without DNA (control), were incubated in SDS sample buffer and fractionated by SDS/PAGE. Proteins were electroblotted onto nitrocellulose membranes. Membranes were incubated with specific anti-GFP antibodies and visualized by chemiluminescence. The size of the various GFP fusion proteins matched their predicted size, indicating their authentic size and localization. Two forms of the L50:GFPK fusion protein, showing an ≈2.5-kDa size difference, were found, suggesting that the upper form is chloroplastic and that the lower form is the leaderless form of the microsomal protein. Using this assay, we could not detect the L28:GFP fusion protein, suggesting that it is unstable. The estimated molecular mass (in kDa) of protein standards is shown on the right.

Fig. 7. Brefeldin A (BFA) does not inhibit RB60 targeting to the chloroplast. The effect of the secretory system inhibitor BFA on the accumulation of RB60:GFPK was monitored in selected Physcomitrella patens protoplasts. Fluorescence images of RB60:GFPK that were taken from the chloroplast (a and c) and cortical (b and d) planes of the cell before the addition of BFA (BFA-0') and after 120 min (BFA-120') showed that RB60:GFPK accumulation in chloroplasts is not affected by BFA (compare a and b with c and d), suggesting an independent endoplasmic reticulum and chloroplast import mechanisms.