Reconstitution of brefeldin A-induced Golgi tubulation and fusion with theendoplasmic reticulum in semi-intact Chinese hamster ovary cells

The fungal metabolite brefeldin A (BFA) induces the disassembly of the Golg i complex in mammalian cells. The drug seems to accentuate tubule formation and causes the subsequent fusion with the endoplasmic reticulum (ER). To i nvestigate the biochemical requirements and kinetics of BFA-induced Golgi d isassembly, we have reconstituted the process of green fluorescent protein- tagged Golgi complex disassembly in streptolysin O-permeabilized semi-intac t Chinese hamster ovary cells. For quantitative analysis of the morphologic al changes to the Golgi complex in semi-intact cells, we developed a novel morphometric analysis. Based on this analysis, we have dissected the BFA-in duced Golgi disassembly process biochemically into two processes, Golgi tub ule formation and fusion with the ER, and found that the formation is induc ed by only ATP and the residual factors in the cells and that the subsequen t fusion is mediated in an N-ethylmaleimide-sensitive factor-dependent mann er via Golgi tubules. Tubulation occurs by two pathways that depend on eith er microtubule integrity or exogenously added cytosol. In the presence of G TP gamma S, coat protein I inhibited the Golgi tubule fusion with the ER bu t showed no apparent effect on tubulation. Additionally, we analyzed the ki netics of tubulation and fusion independently in nocodazole-treated and -un treated semi-intact cells and found that tubulation is a rate-limiting step of the Golgi disassembly.