Oligosaccharyltransferase Subunits Bind Polypeptide Substrate to Locally Enhance N-glycosylation*

M Fairuz B Jamaluddin, Ulla-Maja Bailey, Benjamin L Schulz

Oligosaccharyltransferase is a multiprotein complex that catalyses asparagine-linked glycosylation of diverse proteins. Using yeast genetics and glycoproteomics we show that transient interactions between nascent polypeptide and Ost3p/Ost6p, homologous subunits of oligosaccharyltransferase, modulate glycosylation efficiency in a site-specific manner in vivo. These interactions are driven by hydrophobic and electrostatic complementarity between amino acids in the peptide-binding groove of Ost3p/Ost6p and the sequestered stretch of substrate polypeptide. Based on this dependence, we use in vivo scanning mutagenesis and in vitro biochemistry to map the precise interactions that affect site-specific glycosylation efficiency. We conclude that transient binding of substrate polypeptide by Ost3p/Ost6p increases glycosylation efficiency at asparagines proximal and C-terminal to sequestered sequences. We detail a novel mode of interaction between translocating nascent polypeptide and oligosaccharyltransferase in which binding to Ost3p/Ost6p segregates a short flexible loop of glycosylation-competent polypeptide substrate that is delivered to the oligosaccharyltransferase active site for efficient modification.