FIGURE 2.

Topology and membrane integration of SYT1 and SYT1-EmGFP proteins. A, protease protection assay. The right-side-out plasma membrane vesicles were treated with various concentrations of a low specificity protease thermolysin. After digestion of the plasma membrane proteins, each sample was analyzed by immunoblotting with anti-SYT1 and anti-GFP antibodies. B, degradation pattern of plasma membrane-type H⁺-ATPase. After receiving the same treatment as the right-side-out plasma membrane vesicles in A, the degradation of the plasma membrane-type H⁺-ATPase was detected with anti-PMA2 antibody. C, membrane integration of SYT1 and SYT1-EmGFP. Two micrograms of crude membrane vesicle proteins isolated from seedlings of wild-type plants and transgenic plants expressing SYT1-EmGFP were treated with Na₂CO₃. After ultracentrifugation, the supernatant (Sup) and precipitate (Ppt) of each membrane sample were analyzed by immunoblotting with anti-SYT1, anti-GFP, and anti-PAQs antibodies. The separated proteins were also silver-stained.