Mannosidase complexes involved in glycoprotein degradation

In all eukaryotic cells, the endoplasmic reticulum (ER) has a central role in protein biosynthesis and maturation. Folding of newly synthesized secretory and membrane proteins takes place in the lumen of the ER. Properly folded and assembled proteins exit the ER and continue their journey in the secretory pathway. However, protein folding is error-prone and the accumulation of misfolded or surplus proteins endangers the cellular homeostasis and subsequently the survival of organisms especially under adverse environmental conditions. Consequently, cells have established sophisticated quality control processes that ensure the export of biologically active proteins and the elimination of non- native ones. These quality control processes involve proteins that sense folding-defective proteins and target them for destruction by a precisely controlled protein breakdown process known as ER- associated degradation (ERAD). The degradation of folding-defective glycoproteins is initiated in the lumen of the ER and the specific proteins that recognize the misfolded glycoproteins and determine their fate are unknown. We hypothesize that protein complexes consisting of -mannosidases and thioredoxin-fold containing proteins are key factors in this process. The role of these c omplexes w ill be investigated using genetic, biochemical and cell biology approaches. The project will help to better understand protein quality control mechanisms in plants, which, in the long run, will lead to new strategies to improve plant fitness under constantly changing environmental conditions.

As sessile organisms, plants are constantly exposed to extreme environmental conditions that impact cellular processes and endanger survival. Salinity, heat and other environmental stresses can cause protein misfolding in the endoplasmic reticulum (ER), the main organelle responsible for protein folding and maturation in eukaryotes. Misfolded proteins must be rapidly cleared from the ER to prevent their aggregation or secretion as harmful aberrant proteins. To this end, plant cells have evolved sophisticated quality control mechanisms to ensure the export of biologically active proteins and the elimination of non-native ones. These processes involve proteins that recognise folding-defective proteins and target them for destruction via a precisely controlled process of protein breakdown known as ER-associated degradation (ERAD). The degradation of folding-defective glycoproteins is initiated in the lumen of the ER; however, the specific proteins that initiate this process and recognise the misfolded glycoproteins, determining their fate, remain unknown in plants. Two Arabidopsis thaliana -mannosidases play a key role in these initial recognition steps, generating a glycan degradation signal on misfolded glycoproteins that triggers ERAD. In this project, we investigated whether these -mannosidases form distinct protein complexes with thioredoxin-like domain-containing proteins, and how these mannosidase-interacting proteins contribute to ERAD. We identified distinct thioredoxin-mannosidase complexes and determined the protein domains and motifs important for interaction. We also analysed their contribution to the ERAD of selected misfolded glycoproteins. Our data show that a thioredoxin-like protein of unknown function and a protein disulfide isomerase contribute to ERAD in a specific manner. We found that the mannosidase-interacting protein disulfide isomerase interacts with ERAD substrates and affects their clearance via an unknown mechanism. The other newly characterised protein has a less direct impact on the degradation of ERAD substrates but shows genetic interaction with the protein disulfide isomerase. In summary, our data confirm the existence of distinct protein complexes in the endoplasmic reticulum of plants that contribute to the clearance of misfolded glycoproteins. Our findings will help improve our understanding of protein quality control mechanisms in plants and, in the long term, lead to new strategies to enhance plant resilience in response to changing environmental conditions.