Specific protein interactions between rice members of the GT43 and GT47 families form various central cores of putative xylan synthase complexes

Abstract

SUMMARYMembers of the glycosyltransferase (GT)43 and GT47 families have been associated with heteroxylan synthesis in both dicots and monocots and are thought to assemble into central cores of putative xylan synthase complexes (XSCs). Currently, it is unknown whether protein–protein interactions within these central cores are specific, how many such complexes exist, and whether these complexes are functionally redundant. Here, we used gene association network and co‐expression approaches in rice to identify four OsGT43s and four OsGT47s that assemble into different GT43/GT47 complexes. Using two independent methods, we showed that (i) these GTs assemble into at least six unique complexes through specific protein–protein interactions and (ii) the proteins interact directly in vitro. Confocal microscopy showed that, when alone, all OsGT43s were retained in the endoplasmic reticulum (ER), while all OsGT47s were localized in the Golgi. co‐expression of OsGT43s and OsGT47s displayed complexes that form in the ER but accumulate in Golgi. ER‐to‐Golgi trafficking appears to require interactions between OsGT43s and OsGT47s. Comparison of the central cores of the three putative rice OsXSCs to wheat, asparagus, and Arabidopsis XSCs, showed great variation in GT43/GT47 combinations, which makes the identification of orthologous central cores between grasses and dicots challenging. However, the emerging picture is that all central cores from these species seem to have at least one member of the IRX10/IRX10‐L clade in the GT47 family in common, suggesting greater functional importance for this family in xylan synthesis. Our findings provide a new framework for future investigation of heteroxylan biosynthesis and function in monocots.

Streamlining assays of glycosyltransferases activity using in vitro GT-array (i-GT-ray) platform: Application to family GT37 fucosyltransferases

“…In this work, we developed a platform in vitro GT-array ( i -GT-ray) that simplifies the screening of transferase activity in vitro . The i -GT-ray platform is based on a recently developed method called nucleic acid programmable protein array (NAPPA), which has been widely used in protein-protein interactions (PPIs) studies in animals ( 28 , 29 , 30 , 31 ), Arabidopsis ( 32 ), and more recently, was applied to demonstrate PPIs between GTs on microplates ( 33 ). In the original NAPPA method, glass slides are coated with N-terminal Halo- or C-terminal GST-tagged GT plasmid DNA and an anti-tag capture antibody (CAb), which allows protein production using cell-free in vitro protein synthesis and protein capture on glass slide.…”