Strous G J, van Kerkhof P, van Meer G, Rijnboutt S, Stoorvogel W
Laboratory of Cell Biology, AZU-H02.314, University of Utrecht, The Netherlands.
J Biol Chem. 1993 Feb 5;268(4):2341-7.
Brefeldin A (BFA) rapidly blocks anterograde exocytotic transport through the Golgi complex. Sustained retrograde traffic induced by brefeldin A causes redistribution of constituents of the Golgi, but not the trans-Golgi network (TGN), to the endoplasmic reticulum (ER). In the present study on HepG2 cells, we have observed a differential effect of BFA on transport from the TGN of two soluble proteins: alpha 1-antitrypsin as a representative of secretory proteins and cathepsin D as a prototype of lysosomal enzymes. The Golgi complex of HepG2 cells is sensitive to BFA, as within minutes after its addition nearly all activity of three resident Golgi enzymes was recovered in the ER as monitored by cell fractionation on sucrose density gradients. In accordance with this, "high mannose"-glycosylated alpha 1-antitrypsin was retained in or transported back to the ER. "Complex"-glycosylated alpha 1-antitrypsin was neither secreted into the medium nor transported back to the ER. Most of it was retained in vesicles with the buoyant density of Golgi. These vesicles contained the fluid phase endocytotic marker horseradish peroxidase when this was added to the culture medium prior to the BFA, suggesting that the vesicles derived from the TGN. After BFA addition, the compartment became inaccessible to endocytosed horseradish peroxidase. In contrast to blocking transport of complex alpha 1-antitrypsin, BFA did not affect processing of newly synthesized complex-glycosylated procathepsin D (53 kDa) to the mature 31-kDa form. Neither did it interfere with processing of endocytosed procathepsin D. That the mature cathepsin D had indeed reached the lysosomes was verified by Percoll density gradient fractionation. In conclusion, in HepG2 cells, BFA induces two blocks in the secretory pathway: one at the level of the ER-Golgi juncture and the other in the TGN. In contrast, transport from the Golgi complex to the lysosomes and from the plasma membrane to the lysosomes continued.
布雷菲德菌素A（BFA）可迅速阻断通过高尔基体复合体的顺行胞吐转运。布雷菲德菌素A诱导的持续逆行运输会导致高尔基体成分（但不包括反式高尔基体网络，即TGN）重新分布到内质网（ER）。在本项针对HepG2细胞的研究中，我们观察到BFA对两种可溶性蛋白从TGN转运的不同影响：作为分泌蛋白代表的α1-抗胰蛋白酶和作为溶酶体酶原型的组织蛋白酶D。HepG2细胞的高尔基体复合体对BFA敏感，因为在添加BFA后的几分钟内，通过蔗糖密度梯度细胞分级分离监测发现，三种驻留高尔基体酶的几乎所有活性都在内质网中恢复。与此一致的是，“高甘露糖型”糖基化的α1-抗胰蛋白酶被保留在内质网中或转运回内质网。“复合型”糖基化的α1-抗胰蛋白酶既没有分泌到培养基中，也没有转运回内质网。其中大部分保留在具有高尔基体浮力密度的囊泡中。当在添加BFA之前将液相内吞标记物辣根过氧化物酶添加到培养基中时，这些囊泡含有辣根过氧化物酶，这表明这些囊泡源自TGN。添加BFA后，内吞的辣根过氧化物酶无法进入该区室。与阻断复合型α1-抗胰蛋白酶的转运相反，BFA不影响新合成的复合型糖基化组织蛋白酶原D（53 kDa）加工成成熟的31 kDa形式。它也不干扰内吞的组织蛋白酶原D的加工。通过Percoll密度梯度分级分离证实成熟的组织蛋白酶D确实已到达溶酶体。总之，在HepG细胞中，BFA在分泌途径中诱导两个阻断：一个在ER-高尔基体连接处水平，另一个在TGN中。相比之下，从高尔基体复合体到溶酶体以及从质膜到溶酶体的转运仍在继续。
