Biochemical requirements for BFA-induced Golgi
disassembly (A) and Golgi tubule fusion (B) in semi-intact cells. (A)
Semi-intact CHO-GT cells were incubated with either BFA/ATP/cytosol
(cyto), BFA/ATP/NEM-treated cytosol (NEM-cyto), or BFA/ATP/NEM-treated
cytosol/DTT (NEM-cyto+DTT) at 32°C for 80 min, fixed, and then
subjected to morphometric analysis (Golgi disassembly assay). The
protein concentration of the cytosol used in this experiment was 1.7
mg/ml. (B) At first, semi-intact cells were incubated with BFA/ATP at
32°C for 80 min to generate the Golgi tubules. About 65% of cells
had Golgi tubules. Then the cells were further incubated with
BFA/ATP/NEM-treated cytosol (NEM-cyto), BFA/ATP/NEM-treated
cytosol/myc-NSF (myc-NSF), BFA/ATP/NEM-treated cytosol/His-NSF
(His-NSF), or BFA/ATP/NEM–treated cytosol/K266Q-NSF (inactive NSF) at
32°C for 80 min, and morphometric analysis was performed (Golgi
tubule fusion assay). The protein concentration of the cytosol used was
2.3 mg/ml. Three hundred 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.