N-glycosylation pathway in plants

N-glycosylation is one of the most important posttranslational modifications of proteins in eukaryotes. The formation of so called complex N-glycans, the most prevalent structure, is carried out by a series of glycosylation enzymes in a highly coordinated fashion. The processing reactions are fairly well documented in animal cells, and many of the enzymes have been characterised at a molecular level, but they are not nearly as well understood in plant cells. Beside the processing of N-linked oligosaccharides, glycosylation enzymes are valuable general Golgi marker. So far in plants there are no such markers available which strongly affects a variety of fundamental cell biological research, e.g. characterisation of protein transport, Golgi targeting/retention, structure of the plant Golgi The overall aim of the proposal is to reconstruct the processing steps in the formation of complex N-glycans in the plant Golgi. We will investigate the two major forces that are proposed to drive this important biological process: coordination by substrate specificity or by spatial distribution within the Golgi or by a combination of both. However to achieve this we first have to confirm the cloning of Golgi alpha-Mannosidase II (GMII) , the last molecularly uncharacterised of a series of 5 closely coordinated glycosylation enzymes responsible for the formation of complex N-glycans. Substrate specificity studies of GMII will allow to determine at which stage of the pathway GMII can act. Results will be compared with results obtained by determination of the intra-Golgi distribution of three enzymes that work in close proximity. N-glycan profiles of k.o. mutants which specifically lack glycosylation enzymes will further indicate the specific role of each enzyme. By comparison of the results obtained by substrate specificity, intra Golgi localisation and N-glycan profiling of k.o. mutants we expect to elucidate the processing steps that lead to complex N-glycan formation. Furthermore we expect that GMII specific antiserum, which will be produced in this project, will serve as a general Golgi marker in A. thaliana.

N-glycosylation is one of the most important posttranslational modifications of proteins in eukaryotes. The formation of so called complex N-glycans, the most prevalent structure, is carried out by a series of glycosylation enzymes in a highly coordinated fashion. The processing reactions are fairly well documented in animal cells, and many of the enzymes have been characterised at a molecular level, but they are not nearly as well understood in plant cells. Beside the processing of N-linked oligosaccharides, glycosylation enzymes are valuable general Golgi marker. So far in plants there are no such markers available which strongly affects a variety of fundamental cell biological research, e.g. characterisation of protein transport, Golgi targeting/retention, structure of the plant Golgi. The overall aim of the proposal is to reconstruct the processing steps in the formation of complex N-glycans in the plant Golgi. We will investigate the two major forces that are proposed to drive this important biological process: coordination by substrate specificity or by spatial distribution within the Golgi or by a combination of both. However to achieve this we first have to confirm the cloning of Golgi alpha-Mannosidase II (GMII) , the last molecularly uncharacterised of a series of 5 closely coordinated glycosylation enzymes responsible for the formation of complex N-glycans. Substrate specificity studies of GMII will allow to determine at which stage of the pathway GMII can act. Results will be compared with results obtained by determination of the intra-Golgi distribution of three enzymes that work in close proximity. N-glycan profiles of k.o. mutants which specifically lack glycosylation enzymes will further indicate the specific role of each enzyme. By comparison of the results obtained by substrate specificity, intra Golgi localisation and N-glycan profiling of k.o. mutants we expect to elucidate the processing steps that lead to complex N-glycan formation. Furthermore we expect that GMII specific antiserum, which will be produced in this project, will serve as a general Golgi marker in A. thaliana.