title: Oligosaccharyltransferase subunits bind polypeptide substrate to locally enhance N-glycosylation.
abstract: Oligosaccharyltransferase is a multiprotein complex that catalyzes asparagine-linked glycosylation of diverse proteins. Using yeast genetics and glycoproteomics, we found that transient interactions between nascent polypeptide and Ost3p/Ost6p, homologous subunits of oligosaccharyltransferase, were able to modulate glycosylation efficiency in a site-specific manner in vivo. These interactions were 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 used 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.
authors.affiliation: From the ‡School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: M Fairuz B
authors.initials: MF
authors.lastname: Jamaluddin
authors.affiliation: From the ‡School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Ulla-Maja
authors.initials: UM
authors.lastname: Bailey
authors.affiliation: From the ‡School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland 4072, Australia b.schulz@uq.edu.au.
authors.forename: Benjamin L
authors.initials: BL
authors.lastname: Schulz
mesh_terms: D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D015870:Gene Expression; D006031:Glycosylation; D006602:Hexosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D011485:Protein Binding; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D055672:Static Electricity; D013379:Substrate Specificity
chemical_list: D008565:Membrane Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D001216:Asparagine; D006602:Hexosyltransferases; C507060:OST6 protein, S cerevisiae; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase