Distribution of xylosylation and fucosylation in the plant Golgi apparatus

Abstract

Antibodies have been immunopurified which are specific for carbohydrate epitopes containing the β1→2 xylose or α1→3 fucose residues found on complex N‐linked glycans in plants. The antibody specificity was determined by taking advantage of an Arabidopsis thaliana N‐glycosylation mutant which lacks N‐acetyl‐glucosaminyltransferase 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 freeze‐substituted 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 β→2 xylose and α1→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 anti‐xylose or anti‐fucose antibodies. Labeling for xylose‐containing 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 α1→3 fucose is mainly incorporated in the trans cisternae. Finally, fucose‐ and xylose‐containing 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.