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Plant J. 2018 Apr;94(1):131-145. doi: 10.1111/tpj.13847. Epub 2018 Mar 12.

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

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Department of Horticultural Sciences, Texas A&M University, College Station, TX, 77843, USA.
Department of Biomedical Engineering College of Creative Convergence Engineering, Catholic Kwandong University, Gangneung, Gangwon-do, 25601, South Korea.
Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon, Gangwon-do, 24341, South Korea.
Molekulare Physiologie der Pflanzen, Institut für Biologie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität Münster, Schlossplatz 7, D-48149, Münster, Germany.
Department of Chemistry, Texas A&M University, College Station, TX, 77843, USA.
Division of Applied Life Science and Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, 52828, South Korea.
Faculty of Biology, Medicine and Health, University of Manchester, Oxford Rd, Manchester, M13 9PT, UK.
Department of Biochemistry and Molecular Biology, University of Texas-Medical Branch, Oxford Rd, Galveston, TX, 77555, USA.


The oligosaccharyltransferase (OT) complex catalyzes N-glycosylation of nascent secretory polypeptides in the lumen of the endoplasmic reticulum. Despite their importance, little is known about the structure and function of plant OT complexes, mainly due to lack of efficient recombinant protein production systems suitable for studies on large plant protein complexes. Here, we purified Arabidopsis OT complexes using the tandem affinity-tagged OT subunit STAUROSPORINE AND TEMPERATURE SENSITIVE3a (STT3a) expressed by an Arabidopsis protein super-expression platform. Mass-spectrometry analysis of the purified complexes identified three essential OT subunits, OLIGOSACCHARYLTRANSFERASE1 (OST1), HAPLESS6 (HAP6), DEFECTIVE GLYCOSYLATION1 (DGL1), and a number of ribosomal subunits. Transmission-electron microscopy showed that STT3a becomes incorporated into OT-ribosome super-complexes formed in vivo, demonstrating that this expression/purification platform is suitable for analysis of large protein complexes. Pairwise in planta interaction analyses of individual OT subunits demonstrated that all subunits identified in animal OT complexes are conserved in Arabidopsis and physically interact with STT3a. Genetic analysis of newly established OT subunit mutants for OST1 and DEFENDER AGAINST APOTOTIC DEATH (DAD) family genes revealed that OST1 and DAD1/2 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 in DGL1, OST3/6 and STT3a.


Arabidopsis thaliana ; oligosaccharyltransferase complex; protein N-glycosylation; ribosome; tandem affinity purification; transmission electron microscopy

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