SummaryAntibodies have been immunopurified which are specific for carbohydrate epitopes containing the pl+2 xylose or a1 4 3 fucose residues found on complex Nlinked glycans in plants. The antibody specificity was determined by taking advantage of an Arabidopsis thaliana N-glycosylation mutant which lacks N-acetylglucosaminyltransferase I and is unable to synthesize complex glycans. These antibodies were used to immunolocalize xylose-and fucose-containing glycoproteins in suspension-cultured sycamore cells (Acer pseudoplatanus). By mapping the enzymatic reaction products within the Golgi apparatus, the fucosyl-and xylosyltransferase subcellular localization was made possible using immunocytochemistry on thin sections of high-pressure frozen and freezesubstituted sycamore cells. This procedure allows a much better preservation of organelles, and particularly of the Golgi stack morphology, than that obtained with conventionally fixed samples. Glycoproteins containing p1+2 xylose and al+3 fucose residues were immunodetected in the cell wall, the vacuole, and the Golgi cisternae. The extent of immunolabeling over the different cisternae of 50 Golgi stacks was quantified after treatment with antixylose or anti-fucose antibodies. Labeling for xylosecontaining glycoproteins was predominent in the medial cisternae, while fucose-containing glycoproteins were mainly detected in the trans compartment. Therefore, in plants, complex N-linked glycan xylosylation probably occurs mostly at the medial Golgi level and a1 4 3 fucose is mainly incorporated in the trans cisternae. Finally, fucose-and xylosecontaining glycoproteins were also immunolocalized, albeit to a lesser extent, in earlier Golgi compartments. This indicates that the glycosylation events are a continuous process with some maxima in given compartments, rather than a succession of discrete and compartment-dependent steps.

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The role of N-glycans in the secretion of glycoproteins by suspension-cultured sycamore cells was studied. The transport of glycoproteins to the extracellular compartment was investigated in the presence of a glycan-processing inhibitor, castanospermine. Castanospermine has been selected because it inhibits homogeneously glycan maturation in sycamore cells and leads to the accumulation of a single immature N-glycan. The structure of this glycan has been identified as Glc3Man7GlcNAc2 by labeling experiments, affinity chromatography on concanavalin A-Sepharose and proton NMR. In contrast with previous results showing that N-glycosylation is a prerequisite for secretion of N-linked glycoproteins, this secretion is not affected by the presence of castanospermine. As a consequence, the presence of this unprocessed glycan is sufficient for an efficient secretion of glycoproteins in the extracellular compartment of suspension-cultured sycamore cells.