Mapping the Interaction of the STT3 Subunit of the Oligosaccharyl Transferase Complex with Nascent Polypeptide Chains

Andrey L Karamyshev,Daniel J Kelleher,Reid Gilmore,Arthur E Johnson,Gunnar Von Heijne,Ingmarie Nilsson

doi:10.1074/jbc.m509168200

Abstract

Many secretory and membrane proteins are N-glycosylated by the oligosaccharyl transferase complex during their translocation across the endoplasmic reticulum membrane. Several experimental observations suggest that the highly conserved STT3 subunit contains the active site of the oligosaccharyl transferase. Here, we report a detailed study of the interaction between the active site of the STT3 protein and nascent polypeptide chains using an in vitro photocrosslinking technique. Our results show that the addition of a glycan moiety in a stretch of approximately 15 residues surrounding a QK(*)T cross-linking site impairs the interaction between the nascent chain and STT3.

Highlights

N-Linked glycosylation is one of the most common types of protein modification in eukaryotic cells
To further characterize the co-translational interaction between STT3 and nascent polypeptide chains, we have studied the effect on nascent chain cross-linking of introducing bona fide Asn-Xaa-Thr glycosylation sites close to the Gln-Lys*-Thr cross-linking site
Using photocrosslinking from an engineered Gln-Lys*-Thr (QK*T) site in a nascent polypeptide chain, we have shown previously [11] that nascent secretory proteins are adjacent to the oligosaccharyl transferase (OST) component STT3 during their translocation across the endoplasmic reticulum membrane

Summary

Introduction

N-Linked glycosylation is one of the most common types of protein modification in eukaryotic cells. We report a detailed study of the interaction between the active site of the STT3 protein and nascent polypeptide chains using an in vitro photocrosslinking technique.

Results

Conclusion