Zhang G F, Driouich A, Staehelin L A
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder 80309-0347.
J Cell Sci. 1993 Mar;104 ( Pt 3):819-31. doi: 10.1242/jcs.104.3.819.
We have re-examined the effects of the ionophore monensin on the Golgi apparatus of sycamore maple suspension-cultured cells using a combination of high pressure freezing, immunocytochemical and biochemical techniques. Exposure of the cells to 10 microM monensin, which reduces protein secretion by approximately 90%, resulted first in the swelling of the trans-Golgi network, then of the trans-most trans-cisterna, the remaining trans-cisternae, and finally of the cis and medial cisternae. We postulate that these different rates of swelling reflect an underlying hierarchy of compartmental acidification with the trans-Golgi network being the most acidic compartment. Recovery occurred in the reverse sequence. Previous studies have suggested that the large swollen vesicles that accumulate in the cytoplasm of monensin-treated cells arise from the swelling and detachment of entire trans-cisternae. However, based on the many membrane blebbing configurations seen in association with the trans-Golgi network and the trans-Golgi cisternae of monensin-treated cells, and the fact that the surface area of the trans-Golgi cisternae is about five times greater than the surface area of the swollen vesicles, it appears that the swollen vesicles are produced by a budding mechanism. After 35-40 min of monensin treatment, cells with smaller, non-swollen, compact Golgi stacks began to appear and rapidly increased in number, contributing > 60% of the cell population after 60 min and > 80% after 100 min. In contrast, large numbers of swollen vesicles persisted in the cytoplasm of all cells for over 100 min. Since azide treatment of monensin-treated cells can prematurely induce the unswelling response and cellular ATP levels drop substantially after 45 min of monensin treatment, we propose that un-swelling of the Golgi stacks is due to a monensin-induced decline in ATP levels in the cells. Immunocytochemical labeling of the high pressure frozen cells with anti-xyloglucan antibodies demonstrated that the concentration of xyloglucan, a hemicellulose, in the swollen vesicles increased with time. This increase in vesicle contents may explain why these swollen vesicles do not contract in parallel with the Golgi stacks. In vivo labeling experiments with [3H]fucose, [3H]UDP-glucose and [3H]leucine demonstrated that monensin-treatment not only inhibited protein secretion, but also cellulose synthesis. Protein synthesis, on the other hand, was reduced only slightly during the first 30 min of treatment, but quite strongly between 30 and 60 min, consistent with the observed drop in ATP levels after > 40 min of exposure to monensin.(ABSTRACT TRUNCATED AT 400 WORDS)
我们运用高压冷冻、免疫细胞化学和生化技术相结合的方法，重新研究了离子载体莫能菌素对梧桐悬浮培养细胞高尔基体的影响。将细胞暴露于10微摩尔的莫能菌素中，蛋白质分泌减少了约90%，首先导致反式高尔基体网络肿胀，接着是最靠反式一侧的反式扁平囊肿胀，其余反式扁平囊肿胀，最后顺式和中间扁平囊也肿胀。我们推测这些不同的肿胀速率反映了区室酸化的潜在层级结构，其中反式高尔基体网络是酸性最强的区室。恢复过程则按相反顺序发生。先前的研究表明，在经莫能菌素处理的细胞细胞质中积累的大的肿胀囊泡源自整个反式扁平囊的肿胀和脱离。然而，基于在经莫能菌素处理的细胞的反式高尔基体网络和反式高尔基体扁平囊上观察到的许多膜泡形成形态，以及反式高尔基体扁平囊的表面积约为肿胀囊泡表面积五倍这一事实，似乎肿胀囊泡是通过出芽机制产生的。在莫能菌素处理35 - 40分钟后，具有较小的、未肿胀的、紧密高尔基体堆叠的细胞开始出现且数量迅速增加，60分钟后占细胞群体的比例超过60%，100分钟后超过80%。相比之下，大量肿胀囊泡在所有细胞的细胞质中持续存在超过10分钟。由于用叠氮化物处理经莫能菌素处理的细胞可过早诱导消肿反应，且在莫能菌素处理45分钟后细胞ATP水平大幅下降，我们提出高尔基体堆叠的消肿是由于莫能菌素诱导细胞内ATP水平下降所致。用抗木葡聚糖抗体对高压冷冻细胞进行免疫细胞化学标记表明，木葡聚糖（一种半纤维素）在肿胀囊泡中的浓度随时间增加。囊泡内容物的这种增加可能解释了为什么这些肿胀囊泡不会与高尔基体堆叠同步收缩。用[³H]岩藻糖、[³H]UDP - 葡萄糖和[³H]亮氨酸进行的体内标记实验表明，莫能菌素处理不仅抑制蛋白质分泌，还抑制纤维素合成。另一方面，蛋白质合成在处理的前30分钟仅略有减少，但在30至60分钟之间减少相当明显，这与在暴露于莫能菌素40分钟后观察到的ATP水平下降一致。（摘要截断于400字）
