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.

摘要

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 micrograms/mL of BFA, which is comparable to the 1 to 10 micrograms/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 [3H]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 [3H]fucose into N-linked glycoproteins, which occurs in trans-Golgi cisternae, appears to be affected to a greater extent than the incorporation of [3H]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 [3H]xylose and [3H]fucose into cell wall hemicelluloses. Taken together, these findings suggest that at concentrations of 2.5 to 7.5 mu g/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 apparatus of plant and animal cels share many functional and structural characteristics, the plant Golgi apparatus possesses properties that make its response to BFA unique.