Oligosaccharyltransferase subunits bind polypeptide substrate to locally enhance N-glycosylation.

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.
Mesh Terms:
Amino Acid Sequence, Asparagine, Catalytic Domain, Gene Expression, Glycosylation, Hexosyltransferases, Hydrophobic and Hydrophilic Interactions, Membrane Proteins, Molecular Sequence Data, Mutagenesis, Site-Directed, Peptide Mapping, Protein Binding, Protein Structure, Secondary, Protein Subunits, Protein Transport, Recombinant Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Sequence Alignment, Static Electricity, Substrate Specificity
Mol. Cell Proteomics
Date: Dec. 01, 2014
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