Composition, Assembly, and Trafficking of a Wheat Xylan Synthase Complex  

Abstract

Xylans play an important role in plant cell wall integrity and have many industrial applications. Characterization of xylan synthase (XS) complexes responsible for the synthesis of these polymers is currently lacking. We recently purified XS activity from etiolated wheat (Triticum aestivum) seedlings. To further characterize this purified activity, we analyzed its protein composition and assembly. Proteomic analysis identified six main proteins: two glycosyltransferases (GTs) TaGT43-4 and TaGT47-13; two putative mutases (TaGT75-3 and TaGT75-4) and two non-GTs; a germin-like protein (TaGLP); and a vernalization related protein (TaVER2). Coexpression of TaGT43-4, TaGT47-13, TaGT75-3, and TaGT75-4 in Pichia pastoris confirmed that these proteins form a complex. Confocal microscopy showed that all these proteins interact in the endoplasmic reticulum (ER) but the complexes accumulate in Golgi, and TaGT43-4 acts as a scaffold protein that holds the other proteins. Furthermore, ER export of the complexes is dependent of the interaction between TaGT43-4 and TaGT47-13. Immunogold electron microscopy data support the conclusion that complex assembly occurs at specific areas of the ER before export to the Golgi. A di-Arg motif and a long sequence motif within the transmembrane domains were found conserved at the NH 2 -terminal ends of TaGT43-4 and homologous proteins from diverse taxa. These conserved motifs may control the forward trafficking of the complexes and their accumulation in the Golgi. Our findings indicate that xylan synthesis in grasses may involve a new regulatory mechanism linking complex assembly with forward trafficking and provide new insights that advance our understanding of xylan biosynthesis and regulation in plants.

Asparagus IRX9, IRX10, and IRX14A Are Components of an Active Xylan Backbone Synthase Complex that Forms in the Golgi Apparatus

“…2). Our observations also are consistent with recent findings by Jiang et al (2016), who showed that wheat TaGT43 plays a critical role in initiating XSC assembly in the ER prior to the transport of the XSC to the GA. Heterologous expression studies have shown that IRX10 proteins from multiple species have distributive xylan XylT activity (Jensen et al, 2014;Urbanowicz et al, 2014), while our observation is that AoIRX10 heterologous expression alone in N. benthamiana leaves was only capable of adding a single Xyl residue to the Xyl 5 -AA acceptor (Fig. 2).…”

“…Many plant cell wall polysaccharides are synthesized by an enzyme complex located at either the GA or the plasma membrane . Previous studies have indicated that this is indeed likely to be the case for xylan biosynthesis, with key proteins predicted to be involved in a core XSC (IRX9, IRX10, and/or IRX14; Zeng et al, 2010;Ren et al, 2014;Rennie and Scheller 2014;Jiang et al, 2016). Here, we show that asparagus AoIRX9, AoIRX10, and AoIRX14A are all required to form an active XSC, as only coexpression of all three proteins led to xylan XylT activity at levels similar to that observed using native endogenous asparagus MMs (Fig.…”

“…Different laboratory-based experimental techniques have been utilized to study protein-protein interactions, including yeast two-hybrid approaches (Jones et al, 2014), the reversible Renilla luciferase protein complementation assay (Stefan et al, 2007), BiFC (Bracha-Drori et al, 2004;Wilson et al, 2015;Jiang et al, 2016), and affinity purification followed by mass spectrometrybased proteomics (Van Leene et al, 2008). A comparison of the pros and cons of each strategy can be found in a review by Zhang et al (2010).…”

Integrating cell biology and proteomic approaches in plants

“…CLSM observations of transiently transformed tobacco leaves revealed that this complex is localized in ER. Co-infiltration of tobacco leaves with components of this complex and BiFC analysis revealed that co-expression and interaction of TaGT43-4 and TaGT47-13 targeted the complex for export from ER to Golgi [26]. This approach substantially widened the knowledge about xylan synthesis regulation.…”

“…They are synthetized by xylan synthase complex, which is composed of 2 glycosyltrasferases, two mutases, a germin-like protein and vernalization related protein as found by proteomic analysis of wheat etiolated seedlings [26]. CLSM observations of transiently transformed tobacco leaves revealed that this complex is localized in ER.…”