Reconstitution of brefeldin A-induced golgi tubulation and fusion with the endoplasmic reticulum in semi-intact chinese hamster ovary cells.
The fungal metabolite brefeldin A (BFA) induces the disassembly of the Golgi complex in mammalian cells. The drug seems to accentuate tubule formation and causes the subsequent fusion with the endoplasmic reticulum (ER). To investigate the biochemical requirements and kinetics of BFA-induced Golgi disassembly, we have reconstituted the process of green fluorescent protein-tagged Golgi complex disassembly in streptolysin O-permeabilized semi-intact Chinese hamster ovary cells. For quantitative analysis of the morphological changes to the Golgi complex in semi-intact cells, we developed a novel morphometric analysis. Based on this analysis, we have dissected the BFA-induced Golgi disassembly process biochemically into two processes, Golgi tubule formation and fusion with the ER, and found that the formation is induced by only ATP and the residual factors in the cells and that the subsequent fusion is mediated in an N-ethylmaleimide-sensitive factor-dependent manner via Golgi tubules. Tubulation occurs by two pathways that depend on either microtubule integrity or exogenously added cytosol. In the presence of GTPgammaS, coat protein I inhibited the Golgi tubule fusion with the ER but showed no apparent effect on tubulation. Additionally, we analyzed the kinetics of tubulation and fusion independently in nocodazole-treated and -untreated semi-intact cells and found that tubulation is a rate-limiting step of the Golgi disassembly.
Kano
F
F
Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa, Sakyo-Ku, Kyoto 606-8502, Japan.
Sako
Y
Y
Tagaya
M
M
Yanagida
T
T
Murata
M
M
D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology*; D016466:CHO Cells; D020755:Coat Protein Complex I / Q000378:metabolism; D006224:Cricetinae; D003600:Cytosol / Q000502:physiology; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure*; D017930:Genes, Reporter; D006056:Golgi Apparatus / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure*; D049452:Green Fluorescent Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007700:Kinetics; D008164:Luminescent Proteins / Q000032:analysis; D008561:Membrane Fusion / Q000187:drug effects*; D008870:Microtubules / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure*; D011993:Recombinant Fusion Proteins / Q000032:analysis; D014162:Transfection
D020755:Coat Protein Complex I; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D020126:Brefeldin A; D016244:Guanosine 5'-O-(3-Thiotriphosphate)