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Title: Purification and characterization of Arabidopsis thaliana oligosaccharyltransferase complexes from the native host: a protein super‐expression system for structural studies

The oligosaccharyltransferase (OT) complex catalyzesN‐glycosylation of nascent secretory polypeptides in the lumen of the endoplasmic reticulum. Despite their importance, little is known about the structure and function of plantOTcomplexes, mainly due to lack of efficient recombinant protein production systems suitable for studies on large plant protein complexes. Here, we purified ArabidopsisOTcomplexes using the tandem affinity‐taggedOTsubunitSTAUROSPORINE AND TEMPERATURE SENSITIVE3a (STT3a) expressed by an Arabidopsis protein super‐expression platform. Mass‐spectrometry analysis of the purified complexes identified three essentialOTsubunits,OLIGOSACCHARYLTRANSFERASE1 (OST1),HAPLESS6 (HAP6),DEFECTIVE GLYCOSYLATION1 (DGL1), and a number of ribosomal subunits. Transmission‐electron microscopy showed thatSTT3a becomes incorporated intoOT–ribosome super‐complexes formedin vivo, demonstrating that this expression/purification platform is suitable for analysis of large protein complexes. Pairwisein plantainteraction analyses of individualOTsubunits demonstrated that all subunits identified in animalOTcomplexes are conserved in Arabidopsis and physically interact withSTT3a. Genetic analysis of newly establishedOTsubunit mutants forOST1andDEFENDER AGAINST APOTOTIC DEATH(DAD) family genes revealed thatOST1 andDAD1/2 subunits are essential for the plant life cycle. However, mutations in these individual isoforms produced much milder growth/underglycosylation phenotypes than previously reported for mutations inDGL1,OST3/6andSTT3a.

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Author(s) / Creator(s):
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Publisher / Repository:
Date Published:
Journal Name:
The Plant Journal
Page Range / eLocation ID:
p. 131-145
Medium: X
Sponsoring Org:
National Science Foundation
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