Figure 5.

Effect of CPN20 mutation at the O-fucosylation sites or SPY mutation on chloroplast localization or stability of CPN20. (A) CPN20 mutation at the O-fucosylation sites affects the localization of CPN20 in the chloroplast. Plasmids of GFP fused downstream of wild-type full-length ORF of CPN20 and mutated CPN20 with T116a and T119A were transformed into mesophyll protoplasts prepared from leaves of 30-day-old Col-0 plants. Auto, GFP, and Merge indicate chlorophyll autofluorescence, the fluorescence of CPN20-GFP, and merged image of Auto and GFP. (B) the stability of CPN20 is unaltered by SPY mutation. Two-week-old seedlings of 35S:CPN20-GFP in Col-0 and spy-3 backgrounds were incubated in liquid MS medium supplemented with 40 μM MG132 for 16 h and washed five times before being transferred to liquid MS supplemented with 100 μM cycloheximide for further incubation. Proteins were extracted at the indicated time points and detected by an anti-GFP antibody. Rubisco levels as detected by ponceau staining were used as loading controls.