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

Abstract

Brefeldin A (BFA), a specific inhibitor of Colgi-mediated secretion in animal cells, has been used to study the organization of the secretory pathway and the function of the Colgi 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 Colgi apparatus as well as on the secretion of proteins and complex cell wall polysaccharides in sycamore maple (Acerpseudoplatanus) suspension-cultured cells. We have used 2.5 and 7.5 pg/mL of BFA, which is comparable to the 1 to 10 pg/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 Colgi stacks. Instead, BFA induces the formation of large clusters of Colgi stacks, an increase in the number of trans-like Colgi cisternae, and the accumulation in the cytoplasm of very dense vesicles that appear to be derived from trans Colgi cisternae. These vesicles contain large amounts of xyloglucan (XC), the major hemicellulosic cell wall polysaccharide, as shown by immunocytochemical labeling with anti-XC 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 [3Hlfucose into N-linked glycoproteins, which occurs in transColgi cisternae, appears to be affected to a greater extent than the incorporation of [3H]xylose, which has been localized to media1 Colgi 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 pg/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

Short-Lived and Phosphorylated Proteins Contribute to Carrier-Mediated Efflux, but Not to Influx, of Auxin in Suspension-Cultured Tobacco Cells1

“…Because this drug reversibly blocks the secretory pathway from the Golgi to the plant cell surface (Driouich et al, 1993;Satiat-Jeunemaitre and Hawes, 1993;Schindler et al, 1994;Satiat-Jeunemaitre et al, 1996), our data were consistent with one or more proteins of the efflux system being rapidly turned over at the PM, as proposed by Wilkinson and Morris (1994), on the basis of monensin action. By contrast, carrier-mediated auxin influx did not involve rapid protein recycling because BFA had no effect on [ 14 C]2,4-D accumulation.…”

The N‐terminal 77 amino acids from tobacco N‐acetylglucosaminyltransferase I are sufficient to retain a reporter protein in the Golgi apparatus of Nicotiana benthamiana cells

“…Although BFA causes swelling of the endoplasmatic reticulum cisternae, unlike in mammalian cells, it does not induce the disassembly of Golgi stacks. Instead, BFA induces the formation of large clusters of Golgi stacks and the accumulation in the cytoplasm of very dense vesicles which contain large amounts of xyloglucan, the major hemicellulosic cell wall polysaccharide [15]. When epidermal tissue expressing CTS-gfp was incubated with brefeldin A the gfp labelling pattern appeared signi¢cantly di¡erent (Fig.…”

Dynamic Response of Prevacuolar Compartments to Brefeldin A in Plant Cells

“…For example, BFA at 2.8 mg mL 21 was sufficient and specific to induce the formation of BFA compartments and block protein traffic from ER to Golgi in animal cells (Klausner et al, 1992), but a higher concentration of BFA (i.e. at 10 mg mL 21 ) was required to induce Golgi changes (Satiat-Jeunemaitre and Hawes, 1992;Driouich et al, 1993) and block ER-to-Golgi protein traffic (Jiang and Rogers, 1998) in plant cells. However, BFA at 10 mg mL 21 did not cause detectable morphological changes for PVCs in BY-2 cells (Tse et al, 2004).…”