Purification and characterization of Arabidopsis thaliana oligosaccharyltransferase complexes from the native host: a protein super-expression system for structural studies. [Reinigung und Charakterisierung des Arabidopsis thaliana Oligosaccharyltransferase-Komplexes aus dem nativen Wirt: Ein Protein Super-Expressionssystem für strukturelle Studien.]

Jeong, IS; Lee, S; Bonkhofer, F; Tolley, J; Fukudome, A; Nagashima, Y; May, K; Rips, S; Lee, SY; Gallois, P; Russell, W; Hyun Suk, J; von Schaewen, A; Koiwa, H

Zusammenfassung

The oligosaccharyltransferase (OT) complex catalyzes N-glycosylation of nascent secretory polypeptides in the lumen of the endoplasmic reticulum (ER). Despite its 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 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 gets 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 reported for mutations in DGL1, OST3/6, and STT3a before.