Figure 8.

Biochemical requirements for BFA-induced Golgi disassembly in microtubule-disrupted semi-intact cells. (A) CHO-GT cells were treated with nocodazole to depolymerize microtubules and then permeabilized by SLO. The drug-treated semi-intact cells were incubated with BFA/ATP (ATP), BFA/ATP/cytosol (cytosol), or BFA/ATP/NEM-treated cytosol (NEM-cyto) at 32°C for 80 min, fixed, and then subjected to morphometric analysis (Golgi disassembly assay). The drug-treated semi-intact cells incubated with BFA/ATP/NEM-treated cytosol at 32°C for 80 min were washed and then further incubated with BFA/ATP/NEM-treated cytosol/His-NSF at 32°C for 60 min. Again, morphometric analysis was performed (NEM-cyto+His-NSF). The protein concentration of the cytosol used was 2.5 mg/ml. At least 300 cells were counted for each sample. Cells were counted in three randomly selected fields. Three independent experiments were performed, and means and SDs were calculated. (B) Golgi tubules in the presence or absence of nocodazole treatment. Nocodazole-treated (+Noc) or -untreated (−Noc) semi-intact cells were incubated with BFA/ATP/NEM-treated cytosol at 32°C for 80 min. Then, microtubules (MT) were visualized by the immunofluorescence method with the use of anti-α-tubulin antibody. Bar, 10 μm.