Project Details
Projekt
Structural basis of protein O-mannosylation
Applicant
Professorin Dr. Irmgard Sinning
Subject Area
Structural Biology
Biochemistry
Biochemistry
Term
since 2017
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 289991887
The endoplasmic reticulum (ER) is the first station of membrane proteins and secretory proteins entering the secretory pathway. Protein glycosylation of these proteins is the most abundant and diverse post-translational modification, in which specific amino acids are covalently linked to carbohydrates. It impacts a large number of processes in the cell, and is conserved in evolution. The most prominent types are asparagine-linked glycosylation (N-glycosylation) and protein O-mannosylation, which is essential in fungi and animals. Defects in the mannosylation machinery interfere e.g. ER homeostasis in yeast, and often cause growth defects, embryonic lethality or severe congenital disorders of glycosylation (CDG). O-mannosyltransferases (PMTs, POMTs) are integral membrane proteins of the ER that catalyze mannosyltransfer to Ser or Thr residues. PMTs form homo- or heterodimers and structural information is so far not available. The yeast Pmt1:Pmt2 dimer can associate with the oligosaccharyl-transferase complex (OST), the Sec translocon (Sec61) as well as with the tetrameric Sec63 complex (Sec62,63,71,72). Based on these observations, it is likely that these PMT interactions are dynamic and depend on the translocated protein and/or the lipid environment. Despite the importance of O-mannosylation and increasing efforts to understand its mechanisms, our understanding of the molecular details of the machinery is still surprisingly limited. Therefore, we aim to unravel the PMT structure and interactions by an integrated structural biology approach focused on PMT4. We will analyse the oligomeric state of Pmt4, its interaction with /and regulation by lipids, and its interplay with the translocation machinery. Embedded in this consortium we aim to integrate atomic knowledge into a detailed and global understanding of the function of O-mannosylation, its specific adaptations, and its dynamic interactions. As crystallization and structure determination of PMTs is a high risk project, we want to biochemically characterize the Alg1,2 and 11 proteins with respect to their interactions first via their soluble domains as a backup project (with P9 Thiel). The ALG (asparagine-linked glycosylation) proteins are involved in the synthesis of the final oligosaccharide tree, where five mannose residues are consecutively added by the activity of Alg1, Alg2 and Alg11. Finally, this will show how mutations found in CDG patients affect the enzymatic cascade.
DFG Programme
Research Units
