title: A reversible Renilla luciferase protein complementation assay for rapid identification of protein-protein interactions reveals the existence of an interaction network involved in xyloglucan biosynthesis in the plant Golgi apparatus.
abstract: A growing body of evidence suggests that protein-protein interactions (PPIs) occur amongst glycosyltransferases (GTs) required for plant glycan biosynthesis (e.g. cell wall polysaccharides and N-glycans) in the Golgi apparatus, and may control the functions of these enzymes. However, identification of PPIs in the endomembrane system in a relatively fast and simple fashion is technically challenging, hampering the progress in understanding the functional coordination of the enzymes in Golgi glycan biosynthesis. To solve the challenges, we adapted and streamlined a reversible Renilla luciferase protein complementation assay (Rluc-PCA), originally reported for use in human cells, for transient expression in Nicotiana benthamiana. We tested Rluc-PCA and successfully identified luminescence complementation amongst Golgi-localizing GTs known to form a heterodimer (GAUT1 and GAUT7) and those which homooligomerize (ARAD1). In contrast, no interaction was shown between negative controls (e.g. GAUT7, ARAD1, IRX9). Rluc-PCA was used to investigate PPIs amongst Golgi-localizing GTs involved in biosynthesis of hemicelluloses. Although no PPI was identified among six GTs involved in xylan biosynthesis, Rluc-PCA confirmed three previously proposed interactions and identified seven novel PPIs amongst GTs involved in xyloglucan biosynthesis. Notably, three of the novel PPIs were confirmed by a yeast-based split-ubiquitin assay. Finally, Gateway-enabled expression vectors were generated, allowing rapid construction of fusion proteins to the Rluc reporters and epitope tags. Our results show that Rluc-PCA coupled with transient expression in N. benthamiana is a fast and versatile method suitable for analysis of PPIs between Golgi resident proteins in an easy and mid-throughput fashion in planta.
authors.affiliation: University of Copenhagen, Department of Plant Biology and Biotechnology, Frederiksberg, DK-1871, Denmark.
authors.forename: Christian H
authors.initials: CH
authors.lastname: Lund
authors.affiliation: University of Copenhagen, Department of Plant Biology and Biotechnology, Frederiksberg, DK-1871, Denmark Joint BioEnergy Institute, Feedstocks Division, Emeryville, CA 94608, USA Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Bromley
authors.affiliation: University of Copenhagen, Department of Plant Biology and Biotechnology, Frederiksberg, DK-1871, Denmark.
authors.forename: Anne
authors.initials: A
authors.lastname: Stenbæk
authors.affiliation: University of Copenhagen, Department of Plant Biology and Biotechnology, Frederiksberg, DK-1871, Denmark.
authors.forename: Randi E
authors.initials: RE
authors.lastname: Rasmussen
authors.affiliation: Joint BioEnergy Institute, Feedstocks Division, Emeryville, CA 94608, USA Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA.
authors.forename: Henrik V
authors.initials: HV
authors.lastname: Scheller
authors.affiliation: University of Copenhagen, Department of Plant Biology and Biotechnology, Frederiksberg, DK-1871, Denmark ysa@plen.ku.dk.
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Sakuragi
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics; D005818:Genetic Engineering; D005936:Glucans / Q000096:biosynthesis*; D006056:Golgi Apparatus / Q000378:metabolism; D049410:Luciferases, Renilla / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism; D014990:Xylans / Q000096:biosynthesis*
chemical_list: D005936:Glucans; D010940:Plant Proteins; D014990:Xylans; C029353:xyloglucan; D049410:Luciferases, Renilla
keywords: Arabidopsis thaliana; Golgi apparatus; Nicotiana benthamiana; Renilla luciferase; glycosyltransferase; plant cell wall; polysaccharides; protein–protein interaction; type II membrane protein; xyloglucan.