Subcelluar localization of two plant-derived glycosyltransferases and identification of their Golgi retention signals

Research project P 13828 Subcellular localisation of plant glycosyltransgerases Herta STEINKELLNER 28.06.1999 Glycosidases and glycosyltransferases that respectively catalyse the stepwise trimming and addition of sugar residues are generally considered as working in a co-ordinated and highly ordered fashion during protein N- glycosylation in vivo. In animals, glycosylation enzymes are located within the Golgi cisternae in the same sequence in which they act to modify oligosaccharide substrates. Although the Golgi retention mechanism(s) is (are) not known it appears to be conserved to some extend between species as a mammalian glycosyltransferase is retained in the Golgi of transgenic plants. The lack of sequence similarities between well characterised animal glycosyltransferases suggest that multiple signals may be involved in their specific localisation. Very little information is currently available on the targeting of proteins to the plant Golgi. The plant Golgi apparatus consists of many stacks of membrane cisternae distributed throughout the cell. The distribution of the stacks is distinct from that of the mammalian Golgi. Although there are probably three distinct compartments in the mammalian and yeast Golgi complex and it is possible to define morphologically three types of cisternae (cis-, medial, and trans-cisternae), the functional equivalents of these compartments in plants are unknown. The overall aim of this project is to characterise the subcellular localisation of two recently cloned plant glycosyltransferases betha1 -2N-acetylglucosaminyl-transferase I (GlcNAc-TI ) and core alpha1-3 fucosyltransferase (FucT), and to identify the sequences of these enzymes involved in targeting/retention to the plant Golgi apparatus. To reach this goal, the two enzymes will be overexpressed in the model plant Nicotiana benthamiana. Immuno electronmicroscopy studies will be used to elucidate subcellular localisation. Subcellular co- localisation experiments of GIcNAc-TI and FucT will allow to adress questions concerning (1) compartmentation of the Golgi, (2) the topological distribution of plant glycosyltransferases and (3) will be of general interest since they provide the first endogenous markers for the plant Golgi apparatus. Furthermore, we want to determine sequences involved in Golgi targeting/retention. The cytoplasmic- transmembrane-stem-(CTS-) region of the enzymes and truncations thereof will be fused to a reporter gene (gfp), and subcellular localisaton of the recombinant expressed proteins will be determined by confocal laserscan microscopy. The use of gfp constructs with different wavelengths will allow the colocalisation of different GIcNAc-TI-gfp and FucT-gfp constructs. The restults will provide information about signals involved in plant Golgi targeting/retention.