EFFECT OF BREFELDIN-A ON THE STRUCTURE OF THE GOLGI-APPARATUS AND ON THE SYNTHESIS AND SECRETION OF PROTEINS AND POLYSACCHARIDES IN SYCAMORE MAPLE (ACER-PSEUDOPLATANUS) SUSPENSION-CULTURED CELLS
EFFECT OF BREFELDIN-A ON THE STRUCTURE OF THE GOLGI-APPARATUS AND ON THE SYNTHESIS AND SECRETION OF PROTEINS AND POLYSACCHARIDES IN SYCAMORE MAPLE (ACER-PSEUDOPLATANUS) SUSPENSION-CULTURED CELLS
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DOI：
10.1104/pp.101.4.1363
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发表时间：
1993-04-01
期刊：
影响因子：
7.4
通讯作者：
STAEHELIN, LA
中科院分区：
文献类型：
--
作者：
DRIOUICH, A;ZHANG, GF;STAEHELIN, LA
Brefeldin A （BFA）是动物细胞中高尔基蛋白介导分泌的特异性抑制剂，已被用于研究植物细胞中高尔基体分泌途径的组织和功能。为此，我们采用电子显微镜、免疫细胞化学和生化技术相结合的方法，研究了该药物对悬浮液培养的梧桐槭细胞中高尔基体结构以及蛋白质和复杂细胞壁多糖分泌的影响。我们使用了2.5和7.5马克杯/毫升的BFA，这与动物细胞实验中使用的1到10马克杯/毫升相当。高压冷冻和冷冻替代细胞的电子显微照片显示，尽管BFA引起内质网池肿胀，但与动物细胞不同，它不会引起枫树高尔基堆积的分解。相反，BFA诱导了大量高尔基堆积的形成，反式高尔基池数量的增加，以及细胞质中非常密集的囊泡的积累，这些囊泡似乎来自反式高尔基池。这些囊泡含有大量的木葡聚糖（XG），主要的半纤维素细胞壁多糖，免疫细胞化学标记抗XG抗体显示。所有这些结构变化在药物去除后120分钟内消失。使用[H-3]亮氨酸进行的体内标记实验表明，在BFA的存在下，蛋白质分泌到培养基中，而不是蛋白质合成，被抑制了大约80%。相反，反式高尔基池中[H-3]焦点与n -连接糖蛋白的结合，似乎比[H-3]木糖的结合受到更大程度的影响，[H-3]木糖的结合已定位于内侧高尔基池。BFA还影响复合多糖的分泌，[H-3]木糖和[H-3]焦块掺入细胞壁半纤维素的比例下降了约50%。综上所述，这些发现表明，浓度为2.5至7.5马克杯/毫升的BFA对梧桐枫细胞的分泌途径产生了以下主要变化：(a)抑制分泌蛋白向细胞表面的运输约80%，抑制半纤维素向细胞表面的运输约50%；(b)它改变了n -连接糖蛋白和半纤维素的糖基化模式；(c)减少反式高尔基池和分泌囊之间的交通；(d)它对含有xg的致密分泌囊泡向细胞表面的运输产生主要阻碍；(e)它诱导在核附近形成大量高尔基堆，可能是由高尔基基质区的融合介导的。因此，尽管植物和动物细胞的高尔基体具有许多共同的功能和结构特征，但植物高尔基体具有使其对BFA反应独特的特性。
Brefeldin A (BFA), a specific inhibitor of Golgi-mediated secretion in animal cells, has been used to study the organization of the secretory pathway and the function of the Golgi apparatus in plant cells. To this end, we have employed a combination of electron microscopical, immunocytochemical, and biochemical techniques to investigate the effects of this drug on the architecture of the Golgi apparatus as well as on the secretion of proteins and complex cell wall polysaccharides in sycamore maple (Acer pseudoplatanus) suspension-cultured cells. We have used 2.5 and 7.5 mug/mL of BFA, which is comparable to the 1 to 10 mug/mL used in experiments with animal cells. Electron micrographs of high-pressure frozen and freeze-substituted cells show that although BFA causes swelling of the endoplasmic reticulum cisternae, unlike in animal cells, it does not induce the disassembly of sycamore maple Golgi stacks. Instead, BFA induces the formation of large clusters of Golgi stacks, an increase in the number of trans-like Golgi cisternae, and the accumulation in the cytoplasm of very dense vesicles that appear to be derived from trans Golgi cisternae. These vesicles contain large amounts of xyloglucan (XG), the major hemicellulosic cell wall polysaccharide, as shown by immunocytochemical labeling with anti-XG antibodies. All of these structural changes disappear within 120 min after removal of the drug. In vivo labeling experiments using [H-3]leucine demonstrate that protein secretion into the culture medium, but not protein synthesis, is inhibited by approximately 80% in the presence of BFA. In contrast, the incorporation of [H-3]fucose into N-linked glycoproteins, which occurs in trans-Golgi cisternae, appears to be affected to a greater extent than the incorporation of [H-3]Xylose, which has been localized to medial Golgi cisternae. BFA also affects secretion of complex polysaccharides as evidenced by the approximate 50% drop in incorporation of [H-3]xylose and [H-3]fucose into cell wall hemicelluloses. Taken together, these findings suggest that at concentrations of 2.5 to 7.5 mug/mL BFA causes the following major changes in the secretory pathway of sycamore maple cells: (a) it inhibits the transport of secretory proteins to the cell surface by about 80% and of hemicelluloses by about 50%; (b) it changes the patterns of glycosylation of N-linked glycoproteins and hemicelluloses; (c) it reduces traffic between trans Golgi cisternae and secretory vesicles; (d) it produces a major block in the transport of XG-containing, dense secretory vesicles to the cell surface; and (e) it induces the formation of large aggregates of Golgi stacks in the vicinity of the nucleus, possibly mediated by the fusion of Golgi matrix zones. Thus, although the Golgi apparatus of plant and animal cells share many functional and structural characteristics, the plant Golgi apparatus possesses properties that make its response to BFA unique.