DISTRIBUTION OF XYLOSYLATION AND FUCOSYLATION IN THE PLANT GOLGI-APPARATUS

Antibodies have been immunopurified which are specific for carbohydrat e epitopes containing the beta 1-->2 xylose or alpha 1-->3 fucose resi dues found on complex N-linked glycans in plants. The antibody specifi city was determined by taking advantage of an Arabidopsis thaliana N-g lycosylation mutant which tacks N-acetyl-glucosaminyltransferase I and is unable to synthesize complex glycans. These antibodies were used t o immunolocalize xylose- and fucose-containing glycoproteins in suspen sion-cultured sycamore cells (Acer pseudoplatanus). By mapping the enz ymatic reaction products within the Golgi apparatus, the fucosyl- and xylosyltransferase subcellular localization was made possible using im munocytochemistry on thin sections of high-pressure frozen and freeze- substituted sycamore cells. This procedure allows a much better preser vation of organelles, and particularly of the Golgi stack morphology, than that obtained with conventionally fixed samples. Glycoproteins co ntaining beta 1-->2 xylose and alpha 1-->3 fucose residues were immuno detected in the cell wall, the vacuole, and the Golgi cisternae. The e xtent of immunolabeling over the different cisternae of 50 Golgi stack s was quantified after treatment with anti-xylose or anti-fucose antib odies. Labeling for xylose-containing glycoproteins was predominent in the medial cisternae, while fucose-containing glycoproteins were main ly detected in the trans compartment. Therefore, in plants, complex N- linked glycan xylosylation probably occurs mostly at the medial Golgi level and alpha 1-->3 fucose is mainly incorporated in the trans ciste rnae. Finally, fucose- and xylose-containing glycoproteins were also i mmunolocalized, albeit to a lesser extent, in earlier Golgi compartmen ts. This indicates that the glycosylation events are a continuous proc ess with some maxima in given compartments, rather than a succession o f discrete and compartment-dependent steps.