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Purification and characterization of Arabidopsis thaliana oligosaccharyltransferase complexes from the native host: a protein super‐expression system for structural studies
Author(s) -
Jeong In Sil,
Lee Sangmin,
Bonkhofer Florian,
Tolley Jordan,
Fukudome Akihito,
Nagashima Yukihiro,
May Kimberly,
Rips Stephan,
Lee Sang Y.,
Gallois Patrick,
Russell William K.,
Jung Hyun Suk,
Schaewen Antje,
Koiwa Hisashi
Publication year - 2018
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/tpj.13847
Subject(s) - arabidopsis , protein subunit , biology , arabidopsis thaliana , ribosome , biochemistry , eukaryotic large ribosomal subunit , mutant , microbiology and biotechnology , endoplasmic reticulum , gene , rna
Summary 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 SENSITIVE 3a ( STT 3a) expressed by an Arabidopsis protein super‐expression platform. Mass‐spectrometry analysis of the purified complexes identified three essential OT subunits, OLIGOSACCHARYLTRANSFERASE 1 ( OST 1), HAPLESS 6 ( HAP 6), DEFECTIVE GLYCOSYLATION 1 ( DGL 1), and a number of ribosomal subunits. Transmission‐electron microscopy showed that STT 3a 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 STT 3a. Genetic analysis of newly established OT subunit mutants for OST 1 and DEFENDER AGAINST APOTOTIC DEATH ( DAD ) family genes revealed that OST 1 and DAD 1/2 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 DGL 1, OST 3/6 and STT 3a .