Purification and characterization of Arabidopsis thaliana oligosaccharyltransferase complexes from the native host

a protein super-expression system for structural studies

In Sil Jeong, Sangmin Lee, Florian Bonkhofer, Jordan Tolley, Akihito Fukudome, Yukihiro Nagashima, Kimberly May, Stephan Rips, Sang Y. Lee, Patrick Gallois, William Russell, Hyun Suk Jung, Antje von Schaewen, Hisashi Koiwa

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)131-145
Number of pages15
JournalPlant Journal
Volume94
Issue number1
DOIs
StatePublished - Apr 1 2018

Fingerprint

Arabidopsis
Arabidopsis thaliana
mutation
Proteins
proteins
glycosylation
plant proteins
Plantae
recombinant proteins
endoplasmic reticulum
transmission electron microscopy
genetic techniques and protocols
life cycle (organisms)
polypeptides
production technology
mass spectrometry
death
phenotype
mutants
Arabidopsis Proteins

Keywords

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

ASJC Scopus subject areas

  • Genetics
  • Plant Science
  • Cell Biology

Cite this

Purification and characterization of Arabidopsis thaliana oligosaccharyltransferase complexes from the native host : a protein super-expression system for structural studies. / Jeong, In Sil; Lee, Sangmin; Bonkhofer, Florian; Tolley, Jordan; Fukudome, Akihito; Nagashima, Yukihiro; May, Kimberly; Rips, Stephan; Lee, Sang Y.; Gallois, Patrick; Russell, William; Jung, Hyun Suk; von Schaewen, Antje; Koiwa, Hisashi.

In: Plant Journal, Vol. 94, No. 1, 01.04.2018, p. 131-145.

Research output: Contribution to journalArticle

Jeong, IS, Lee, S, Bonkhofer, F, Tolley, J, Fukudome, A, Nagashima, Y, May, K, Rips, S, Lee, SY, Gallois, P, Russell, W, Jung, HS, von Schaewen, A & Koiwa, H 2018, 'Purification and characterization of Arabidopsis thaliana oligosaccharyltransferase complexes from the native host: a protein super-expression system for structural studies', Plant Journal, vol. 94, no. 1, pp. 131-145. https://doi.org/10.1111/tpj.13847
Jeong, In Sil ; Lee, Sangmin ; Bonkhofer, Florian ; Tolley, Jordan ; Fukudome, Akihito ; Nagashima, Yukihiro ; May, Kimberly ; Rips, Stephan ; Lee, Sang Y. ; Gallois, Patrick ; Russell, William ; Jung, Hyun Suk ; von Schaewen, Antje ; Koiwa, Hisashi. / Purification and characterization of Arabidopsis thaliana oligosaccharyltransferase complexes from the native host : a protein super-expression system for structural studies. In: Plant Journal. 2018 ; Vol. 94, No. 1. pp. 131-145.
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abstract = "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.",
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