Oral Presentation 23rd Annual Lorne Proteomics Symposium 2018

Identification of the plant Golgi localized UDP-GlcNAc transporter and its role in endomembrane lipid and protein glycosylation (#52)

Joshua Heazlewood 1 , Carsten Rautengarten 1 , Wei Zeng 1 , Tony Bacic 1 , Ute Roessner 1 , Staffan Persson 1 , Berit Ebert 1
  1. The University of Melbourne, Melbourne, VIC, Australia

Glycosylation reactions require activated glycosyl donors in the form of nucleotide sugars to drive processes such as post-translational modifications and polysaccharide biosynthesis. Many of these reactions often occur in the endomembrane using cytosolic-derived nucleotide sugars, which are actively transported into the lumen by nucleotide sugar transporters (NSTs). We recently identified a plant UDP-GlcNAc transporter responsible for the delivery of substrate for the maturation of N-glycans and sphingolipids within the endomembrane. To determine the biochemical phenotype of the UDP-GlcNAc transporter loss-of-function mutants, we have applied both proteomic and metabolomic approaches. Initially we developed a reliable N-glycopeptide enrichment and mass spectrometry-based analytical workflow to detect, identify and quantify N-glycopeptides. Next, we applied lipidomic approaches to profile sphingolipids from loss-of-function mutants. Analysis of omic data indicated that that N-glycopeptides containing complex N-glycans (e.g. GlcNAc) only comprise about 5% of the N-glycopeptide population in mutant lines. In contrast, N-glycans from wild-type plants are comprised of around 35% complex-type N-glycans i.e. those containing GlcNAc. While sphingolipid analysis indicated that GlcNAc containing lipids comprised less than 10% of that observed in wild type plants. Our findings indicate that the reference plant Arabidopsis contains a single UDP-GlcNAc transporter responsible for the maturation of complex N-glycans and sphingolipids in the Golgi lumen. The work also highlights the detailed insight and resolution that can be achieved using modern omic approaches.