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. 2015 Jan;66(1):85-97.
doi: 10.1093/jxb/eru401. Epub 2014 Oct 18.

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

Christian H Lund  1 , Jennifer R Bromley  2 , Anne Stenbæk  1 , Randi E Rasmussen  1 , Henrik V Scheller  3 , Yumiko Sakuragi  4
Affiliations

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

Christian H Lund et al. J Exp Bot. 2015 Jan.

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.

Keywords: Arabidopsis thaliana; Golgi apparatus; Nicotiana benthamiana; Renilla luciferase; glycosyltransferase; plant cell wall; polysaccharides; protein–protein interaction; type II membrane protein; xyloglucan..

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Figures

Fig. 1.
Fig. 1.
Schematic representation of the reversible Renilla luciferase protein complementation assay (Rluc-PCA) to study Golgi lumenal protein interactions. Membrane proteins with a type II membrane topology, spanning the membrane once with the N-terminus (N) in the cytosol and a lumenal C-terminus, are shown fused to the N-terminal domain (F1) and C-terminal domain (F2) of human-codon optimized Renilla luciferase (hRluc). Arrows denote the dynamics of the protein interaction, the coupling and decoupling of the two domains of hRluc. (This figure is available in colour at JXB online.)
Fig. 2.
Fig. 2.
Localization and activity of Golgi lumenal localized human-codon optimized Renilla luciferase (hRluc). Rat sialyltransferase transmembrane domain (ST) fused to hRluc was used to target hRluc to the Golgi apparatus. (A) ST–hRluc-YFP co-localized with the cis-Golgi marker α-mannosidase-CFP. Scale bar=20 µm. (B) ST–hRluc and hRluc activities in transiently expressing N. benthamiana crude leaf protein extracts. The silencing suppressor p19 was co-expressed with ST–hRluc and hRluc constructs and alone is used as a negative control for background luminescence. Error bars represent 95% confidence interval, n=3. Log10(RLU); relative luminescence units transformed to Log10. (This figure is available in colour at JXB online.)
Fig. 3.
Fig. 3.
Schematic protocol for Rluc-PCA. Genes of interest (GOIs) are PCR amplified and recombined into pDONRTM/Zeo vector by BP cloning (BP). The GOI-containing entry clone can be recombined by LR cloning (LR) to insert GOI into desired destination vectors, which here is the Gateway-compatible phRluc[F1] and phRluc[F2] belonging to Rluc-PCA. Alternatively, GOI can be recombined into other destination vectors, e.g. Gateway-compatible DUALmembrane vectors containing the amino terminal ubiquitin fragment (NubG) and the carboxy terminal ubiquitin fragment fused to an artificial transcription factor (TF–Cub). In Rluc-PCA, phRluc[F1]- and phRluc[F2]-fused GOIs are individually introduced into different Agrobacterium and then co-infiltrated into the leaves of N. benthamiana in desired combinations to test PPIs. The assay is performed 3–4 d post infiltration by harvesting three leaf discs and transferring to tubes containing 200 µl assay buffer and a chrome ball. Leaf discs are macerated and 100 µl is transferred into a black 96-well plate. Bioluminescence upon addition of coelenterazine-h is monitored in a plate luminometer. (This figure is available in colour at JXB online.)
Fig. 4.
Fig. 4.
Rluc-PCA identifies the GAUT1–GAUT7 core-complex and ARAD1–ARAD1 homodimer. (A) Heat map of Log10 values of RLU where dark grey denotes statistically significant higher Log10 values of RLU above the background level (p19). Statistical analysis was performed on the averages derived from three independent experiments, each consisting of three biological replicates (pools) (see materials and methods). A vector containing the silencing suppressor p19 was co-transfected along with GOI–hRluc[F1] and GOI–hRluc[F2]. Error represents 95% confidence interval, n=3. Asterisk represents extracts where GAUT1 was not detected by immunoblot owing to proteolytic processing and possible degradation (Atmodjo et al., 2011). (B) Immunoblot of expressed proteins probed with anti-HA and anti-FLAG primary antibodies.
Fig. 5.
Fig. 5.
Application of the Rluc-PCA to test PPIs among XyG biosynthetic enzymes. Three independent experiments, each consisting of three biological replicates (pools) were made (see materials and methods). Generated results are shown as heat map of Log10 values of RLU where dark grey denotes samples with all experiments being significantly higher than the background level, whereas light grey denotes samples with two out of three experiments being significantly higher than the background level and white denotes Log10 values of RLU of the background p19 infiltrated control in a complementation assay. Statistical analysis was performed on the averages derived from three independent experiments. Plants were co-transfected with Agrobacteria carrying vectors containing silencing suppressor p19, GOI–hRluc[F1], and GOI–hRluc[F2]. Error represents 95% confidence interval, n=3.
Fig. 6.
Fig. 6.
Split-ubiquitin assay used to detect PPIs among XyG biosynthetic proteins. Transformed yeast containing the indicated combinations of TF–Cub and NubG fused proteins were spotted in an OD546 of 1.5 and up to 1000× dilution on SD-His-Leu and SD-His-Leu-Trp-Ade plates. Growth on SD-His-Leu-Trp-Ade plates indicates a positive interaction. X-Gal assay performed on growing yeast on SD-His-Leu is a test for β-galactosidase activity, a reporter for interaction upon blue colour formation, Ost1p–NubI (NubI) and pPR3-N test for the functionality and random interaction of the Cub-fused proteins, respectively. The type II membrane protein TF–Cub–Anp1p tests for random interaction among NubG-fused proteins. Consensus of three biological replicates is shown. (This figure is available in colour at JXB online.)
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