Figure 4.

Effect of Wortmannin and SYP21 Overexpression on GFP-spo.

(A) A constant amount (10 μg) of GFP-spo was coexpressed with increasing concentrations of SYP21 encoding plasmid as effector. Cells and media were harvested, and equal quantities were analyzed by protein gel blotting and detection with anti-GFP antibodies. The effector plasmid concentration is given above each lane in micrograms. Glycosylated (white arrowheads) and unglycosylated (black arrowheads) precursors are clearly distinguished from the GFP core fragment (indicated by a star). Molecular mass markers are indicated in kilodaltons. Note the accumulation of the glycosylated precursor in the cell with an increasing concentration of SYP21.

(B) Protoplasts were transfected with a constant amount (10 μg) of plasmid encoding for GFP-spo, pooled and distributed in equal portions, followed by incubation in the presence of different concentrations of wortmannin for 24 h. Cells and media were harvested, and equal quantities were analyzed by protein gel blotting and detection with anti-GFP antibodies. The concentration of the drug is given above each lane. Annotations are as in (A). Note that increasing concentrations of wortmannin reduce the formation of the unglycosylated precursor and the GFP core fragment and also decrease cellular levels of the glycosylated precursor.