Lippincott-Schwartz J, Glickman J, Donaldson J G, Robbins J, Kreis T E, Seamon K B, Sheetz M P, Klausner R D
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
J Cell Biol. 1991 Feb;112(4):567-77. doi: 10.1083/jcb.112.4.567.
Brefeldin A (BFA) causes rapid redistribution of Golgi proteins into the ER, leaving no definable Golgi apparatus, and blocks transport of proteins into post-Golgi compartments in the cell. In this study we follow the disassembly of the Golgi apparatus in BFA-treated, living cells labeled with NBD-ceramide and demonstrate that forskolin can both inhibit and reverse this process. Long, tubular processes labeled with NBD-ceramide were observed emerging from Golgi elements and extending out to the cell periphery in cells treated with BFA for 5 min. With longer incubations in BFA, the NBD label was dispersed in a fine reticular pattern characteristic of the ER. Treatment with forskolin inhibited these effects of BFA as well as BFA's earliest morphologic effect on the Golgi apparatus: the redistribution to the cytosol of a 110-kD Golgi peripheral membrane protein. In addition, forskolin could reverse BFA's block in protein secretion. Forskolin inhibition of BFA's effects was dose dependent and reversible. High concentrations of BFA could overcome forskolin's inhibitory effect, suggesting forskolin and BFA interact in a competitive fashion. Remarkably, in cells already exposed to BFA, forskolin could reverse BFA's effects causing the 110-kD Golgi peripheral membrane protein to reassociate with Golgi membrane and juxtanuclear Golgi complexes to reassemble. Neither membrane permeant cAMP analogues nor cAMP phosphodiesterase inhibitors could replicate or enhance forskolin's inhibition of BFA. 1,9-Dideoxyforskolin, which does not activate adenylyl cyclase, was equally as effective as forskolin in antagonizing BFA. A derivative of forskolin, 7-HPP-forskolin, that is less potent than forskolin at binding to adenylyl cyclase, was also equally effective as forskolin in antagonizing BFA. In contrast a similar derivative, 6-HPP-forskolin, that is equipotent with forskolin at binding to adenylyl cyclase, did not inhibit BFA's effects. These results suggest that forskolin acts as a competitive antagonist to BFA, using a cAMP-independent mechanism to prevent and reverse the morphologic effects induced by BFA.
布雷菲德菌素A（BFA）可使高尔基体蛋白迅速重新分布到内质网中，导致无法明确界定高尔基体，并且阻断蛋白质向细胞内高尔基体后区室的转运。在本研究中，我们追踪了用NBD-神经酰胺标记的经BFA处理的活细胞中高尔基体的解体过程，并证明福斯高林既能抑制又能逆转这一过程。在用BFA处理5分钟的细胞中，观察到从高尔基体元件伸出并用NBD-神经酰胺标记的长管状结构，并延伸至细胞周边。在BFA中孵育时间更长时，NBD标记物以内质网特有的精细网状模式分散。用福斯高林处理可抑制BFA的这些作用以及BFA对高尔基体最早的形态学作用：一种110-kD高尔基体周边膜蛋白重新分布到细胞质中。此外，福斯高林可逆转BFA对蛋白质分泌的阻断作用。福斯高林对BFA作用的抑制呈剂量依赖性且可逆。高浓度的BFA可克服福斯高林的抑制作用，提示福斯高林和BFA以竞争性方式相互作用。值得注意的是，在已经暴露于BFA的细胞中，福斯高林可逆转BFA的作用，使110-kD高尔基体周边膜蛋白重新与高尔基体膜结合，并且使核旁高尔基体复合体重新组装。膜通透性cAMP类似物和cAMP磷酸二酯酶抑制剂均不能复制或增强福斯高林对BFA的抑制作用。1,9-二脱氧福斯高林不激活腺苷酸环化酶，在拮抗BFA方面与福斯高林同样有效。福斯高林的一种衍生物7-HPP-福斯高林与腺苷酸环化酶结合的能力比福斯高林弱，在拮抗BFA方面也与福斯高林同样有效。相比之下，一种类似的衍生物6-HPP-福斯高林与腺苷酸环化酶结合的能力与福斯高林相当，却不能抑制BFA的作用。这些结果提示，福斯高林作为BFA的竞争性拮抗剂，通过一种不依赖cAMP的机制来预防和逆转BFA诱导的形态学作用。
