A reversible <i>Renilla </i>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

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

Boosting of enzymatic softwood saccharification by fungal GH5 and GH26 endomannanases

Background: Softwood is a promising feedstock for lignocellulosic biorefineries, but as it contains galactoglucomannan efficient mannan-degrading enzymes are required to unlock its full potential. Results: Boosting of the saccharification of pretreated softwood (Canadian lodgepole pine) was investigated for 10 fungal endo-β(1→4)-mannanases (endomannanases) from GH5 and GH26, including 6 novel GH26 enzymes. The endomannanases from Trichoderma reesei (TresMan5A) and Podospora anserina (PansMan26) were investigated with and without their carbohydrate-binding module (CBM). The pH optimum and initial rates of enzyme catalysed hydrolysis were determined on pure β-mannans, including acetylated and deacetylated spruce galactoglucomannan. Melting temperature (Tm) and stability of the endomannanases during prolonged incubations were also assessed. The highest initial rates on the pure mannans were attained by GH26 endomannanases. Acetylation tended to decrease the enzymatic rates to different extents depending on the enzyme. Despite exhibiting low rates on the pure mannan substrates, TresMan5A with CBM1 catalysed highest release among the endomannanases of both mannose and glucose during softwood saccharification. The presence of the CBM1 as well as the catalytic capability of the TresMan5A core module itself seemed to allow fast and more profound degradation of portions of the mannan that led to better cellulose degradation. In contrast, the presence of the CBM35 did not change the performance of PansMan26 in softwood saccharification. Conclusions: This study identified TresMan5A as the best endomannanase for increasing cellulase catalysed glucose release from softwood. Except for the superior performance of TresMan5A, the fungal GH5 and GH26 endomannanases generally performed on par on the lignocellulosic matrix. The work also illustrated the importance of using genuine lignocellulosic substrates rather than simple model substrates when selecting enzymes for industrial biomass applications

GO-PROMTO Illuminates Protein Membrane Topologies of Glycan Biosynthetic Enzymes in the Golgi Apparatus of Living Tissues

The Golgi apparatus is the main site of glycan biosynthesis in eukaryotes. Better understanding of the membrane topology of the proteins and enzymes involved can impart new mechanistic insights into these processes. Publically available bioinformatic tools provide highly variable predictions of membrane topologies for given proteins. Therefore we devised a non-invasive experimental method by which the membrane topologies of Golgi-resident proteins can be determined in the Golgi apparatus in living tissues. A Golgi marker was used to construct a series of reporters based on the principle of bimolecular fluorescence complementation. The reporters and proteins of interest were recombinantly fused to split halves of yellow fluorescent protein (YFP) and transiently co-expressed with the reporters in the Nicotiana benthamiana leaf tissue. Output signals were binary, showing either the presence or absence of fluorescence with signal morphologies characteristic of the Golgi apparatus and endoplasmic reticulum (ER). The method allows prompt and robust determinations of membrane topologies of Golgi-resident proteins and is termed GO-PROMTO (for GOlgi PROtein Membrane TOpology). We applied GO-PROMTO to examine the topologies of proteins involved in the biosynthesis of plant cell wall polysaccharides including xyloglucan and arabinan. The results suggest the existence of novel biosynthetic mechanisms involving transports of intermediates across Golgi membranes