Bayer Pharmaceuticals PhD Prize:
Structural basis for coupling protein transport and N-glycosylation at the mammalian endoplasmic reticulum
Asparagine-linked protein glycosylation is the most abundant post-translational modification of eukaryotic secretory proteins with crucial implications for protein folding, trafficking and functionality. Glycan transfer is catalyzed by the oligosaccharyltransferase (OST) - a complex of at least eight protein subunits which exists in two different isoforms (STT3A- and STT3B-OST) in higher eukaryotes. The process is tightly coupled to co-translational protein transport across or insertion into the endoplasmic reticulum membrane by the Sec61 protein-conducting channel. Formation of ribosome-translocon complexes (RTCs), including the OST has been described but the mode of OST-integration as well as the arrangement of OST subunits have remained elusive.
We used cryo-electron tomography to obtain structural evidence for exclusive incorporation of STT3A-OST in mammalian translocon-associated OST complexes. Additionally, we successfully determined the first high-resolution structure of an OST-containing RTC using single-particle cryo-EM. The structure has elucidated the subunit arrangement of mammalian STT3A-OST and includes molecular models for two core complex components. Furthermore, dissecting the interfaces between STT3A-OST and Sec61 or the ribosome, respectively, has enabled us to propose a model for isoform specificity of mammalian OST.
Our work substantially improves the understanding of mammalian N-glycosylation and the complex interplay of OST with the translation and translocation machineries.