A. thaliana alpha-mannosidase family

N-glycosylation is one of the major posttranslational modifications of proteins in eukaryotic cells. The biosynthesis of protein N-linked glycans results from a series of highly co-ordinated step-by-step enzymatic conversions occurring in the endoplasmic reticulum (ER) and Golgi apparatus. While the enzymes involved in the formation and processing of complex N-glycans in plants have been cloned and characterised recently, almost nothing is currently known about plant class I alpha-mannosidases, which are involved in the early N-glycan processing steps. In addition, these proteins might also play a role in N-glycan dependent quality control in the ER, where misfolded glycoproteins are destroyed by a process called endoplasmic reticulum-associated degradation (ERAD). The aim of this project is to characterise a family of five Arabidopsis thaliana proteins, which display significant homology to class I alpha-mannosidases present in mammals and yeast. Preliminary results in our laboratory suggest that these putative enzymes are involved in the trimming of Man9GlcNAc2 to Man5GlcNAc2 N-glycans. Man5GlcNAc2 is subsequently the substrate for the formation of hybrid and complex N-glycans in the Golgi. To investigate the actual function of the A. thaliana class I alpha-mannosidase family, the five plant genes will be expressed in insect cells and a comprehensive study of the substrate specificity will be carried out to determine their enzymatic properties in vitro. A reverse genetic approach will be used to investigate the possible role of the five enzymes in the processing of N-glycans in vivo. In this respect the N-glycan profiles will be determined in knockout lines. Since these proteins could also be involved in glycoprotein degradation in the ER, the expression and half-life of a well-characterised glycoprotein will be studied in the knockouts and corresponding overexpression lines. Furthermore subcellular localisation studies of the proteins and phenotypic characterisation of knockout and overexpression lines will provide important information about their putative physiological role. The expected results will help to understand the function of early N-glycan processing steps in plants and elucidate if these class I alpha-mannosidase proteins are involved in N-glycan dependent protein degradation like it has been proposed for mammals and yeast.

In this project we characterized a family of five plant proteins (called MNS1-MNS5), which are involved in fundamental cellular processes in the endoplasmic reticulum. N-glycosylation is a major posttranslational modification of proteins in all eukaryotic cells and plays an essential role in proper folding of secretory glycoproteins and quality control processes that ensure clearance of misfolded or damaged proteins. Three of the characterized MNS proteins remove mannose residues from correctly folded secretory glycoproteins and are essential for further maturation of N-glycans in the Golgi apparatus. A block in these mannose trimming reactions causes a severe root growth defect and the formation of altered cell walls, which highlights the importance of these MNS proteins for plant development. In contrast, the other two members of the MNS protein family were not directly involved in N-glycan processing, but play a critical role in generation of the glycan signal that directs misfolded glycoproteins for degradation by a conserved pathway called endoplasmic reticulum-associated degradation (ERAD). The glycan degradation signal on aberrant proteins is subsequently recognized by a specific carbohydrate binding protein that links the misfolded glycoprotein with the degradation machinery. Our discoveries contribute to a better understanding of N-glycan processing steps and we could decipher the glyco-code involved in degradation of misfolded glycoproteins in plants. These processes are important for the proper development of plants and for their response to different stress situations.