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.
Highlights
In eukaryotes, biosynthetic machineries of extracellular matrix polysaccharides and N- and O-protein glycosylation are located in the endomembrane system, in the Golgi apparatus
Transfection of ST–human-codon optimized Renilla luciferase (hRluc) with a C-terminal YFP fusion alongside the Golgi marker α-mannosidase (Nelson et al, 2007), with C-terminal CFP fusion, into N. benthamiana confirmed the targeting of hRluc to the Golgi apparatus (Fig. 2A)
ST–hRluc and hRluc were transiently expressed in N. benthamiana and activity assayed by the conversion of coelenterazine-h to coelenteramide, which
Summary
Biosynthetic machineries of extracellular matrix polysaccharides (e.g. cell wall polysaccharides, proteoglycans) and N- and O-protein glycosylation are located in the endomembrane system, in the Golgi apparatus It is known in plants, yeast, and mammals that the Golgi-localization and function of some of glycan biosynthetic enzymes, including glycosyltransferases (GTs), are regulated through protein–protein interactions (PPIs). In situ binary PPI assays, such as bimolecular fluorescence complementation (BiFC), and Förster resonance energy transfer (FRET) are non-invasive and can facilitate identification of PPIs in living tissues, and in addition can provide information about the subcellular localization of PPIs in cells. Using Rluc-PCA enabled successful identification of novel candidates for PPIs amongst XyG biosynthetic enzymes
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