ARTICLES https://doi.org/10.1038/s41477-018-0235-5 1 School of Biosciences, University of Melbourne, Melbourne, Victoria, Australia. 2 Metabolomics Australia, School of Biosciences, University of Melbourne, Melbourne, Victoria, Australia. 3 ARC Centre of Excellence in Plant Cell Walls, School of Biosciences, University of Melbourne, Melbourne, Victoria, Australia. 4 Joint BioEnergy Institute and Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. 5 Department of Plant and Microbial Biology, University of California, Berkeley, CA, USA. 6 These authors contributed equally to this work: Berit Ebert, Carsten Rautengarten. *e-mail: jheazlewood@unimelb.edu.au G lycosylation is a fundamental co-translational and/or post- translational modification process that occurs in all eukary- otes. The attachment of sugars onto either proteins or lipids can alter their biological function, which can affect the development and physiology of an organism 1,2 . The monosaccharide N-acetyl- d-glucosamine (GlcNAc) is one of the most abundant sugars found in biology and is a major constituent of polysaccharides present in organisms ranging from prokaryotes to Eukarya. However, in plants, GlcNAc is a relatively minor residue that is mainly found in evolutionary conserved structures, such as N-linked and O-linked glycoproteins, glycophosphatidylinositol anchors and the glyco- syl inositol phosphorylceramide (GIPC) class of sphingolipids (Supplementary Fig. 1). The addition of GlcNAc to maturing N-glycans and GIPCs occurs within the Golgi lumen and requires the translocation of the activated nucleotide sugar UDP-GlcNAc from the cytosol into the Golgi lumen. GIPCs are major class of lipids found in plasma membranes of plants 3,4 and seem to play important roles in cell wall anchoring 5 . They may also work as precursors of signalling molecules involved in programmed cell death as part of the plant defence machinery 6 . The structure of plant GIPCs consists of a hydroxylated long-chain base moiety amidated by a hydroxylated very-long-chain fatty acid, which is linked to an inositol and a glucuronic acid (GlcA). The core α-1,4-linked GlcA can be modified by the addition of GlcNAc, man- nose (Man), galactose (Gal), arabinose (Ara) and fucose (Fuc) resi- dues using their respective activated nucleotide sugars as the donor molecule 7–11 . These glycan modifications to GIPCs are presumed to occur within the Golgi apparatus 4 . In recent years, core processes involved in the glycosylation of GIPCs in Arabidopsis have been elucidated, including the identification of enzymes involved in the attachment of GlcA 4 , Man 12 and GlcNAc 13 . Like GIPCs, N-linked glycoproteins are associated with various biological processes, including the biosynthesis of the extracellular matrix 14,15 and contributing to plant stress tolerance 16 . N-glycans are initially assembled as a dolichol-linked precursor oligosaccha- ride at the cytoplasmic face of the endoplasmic reticulum (ER), which is then transferred to the lumen to complete the biosynthe- sis of the N-glycan structure to form Glc 3 Man 9 GlcNAc 2 -PP-Dol 17 . Following its transfer to the nascent polypeptide, the N-linked gly- can is processed by α-glucosidases and then by α-mannosidases in the ER and cis-Golgi to remove Glc α-1–2/3 and Man α-1–2 linkages, respectively 18 . The resultant N-linked oligosaccharide Man 5 GlcNAc 2 represents the acceptor substrate for the β-1,2-N- acetylglucosaminyltransferase I (GnTI) within the cis-Golgi. GnTI initiates the rebuilding process through the addition of a single GlcNAc residue to α-1–3 Man residues using UDP-GlcNAc as the donor to generate the intermediate GlcNAcMan 5 GlcNAc 2 N-glycan structure 19 . This structure is further processed within the Golgi lumen by α-mannosidase II before GnTII adds a second GlcNAc residue using UDP-GlcNAc 20,21 . Further decorations of the N-glycan occur with the addition of xylose (Xyl) and Fuc to generate the arche- typal complex N-glycan structure found in plants, with the glycan composition of GlcNAc 2 XylFucMan 3 GlcNac 2 (ref. 20 ). Modifications to this complex N-glycan structure are also known to occur. The addition of Gal and Fuc results in the occurrence of the less common and organ-specific Lewis, an epitope in Arabidopsis 22 , whereas trim- ming of terminal GlcNAc residues by β-N-acetyl-hexosaminidases (HEXO) to produce paucimannose structures can occur in the A Golgi UDP-GlcNAc transporter delivers substrates for N-linked glycans and sphingolipids Berit Ebert 1,6 , Carsten Rautengarten 1,6 , Heather E. McFarlane 1 , Thusitha Rupasinghe 2 , Wei Zeng 1,3 , Kristina Ford 1,3 , Henrik V. Scheller 4,5 , Antony Bacic 1,3 , Ute Roessner 2 , Staffan Persson 1 and Joshua L. Heazlewood  1 * Glycosylation requires activated glycosyl donors in the form of nucleotide sugars to drive processes such as post-translational protein modifications and glycolipid and polysaccharide biosynthesis. Most of these reactions occur in the Golgi, requiring cyto- solic-derived nucleotide sugars, which need to be actively transferred into the Golgi lumen by nucleotide sugar transporters. We identified a Golgi-localized nucleotide sugar transporter from Arabidopsis thaliana with affinity for UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) and assigned it UDP-GlcNAc transporter 1 (UGNT1). Profiles of N-glycopeptides revealed that plants carrying the ugnt1 loss-of-function allele are virtually devoid of complex and hybrid N-glycans. Instead, the N-glycopeptide population from these alleles exhibited high-mannose structures, representing structures prior to the addition of the first GlcNAc in the Golgi. Concomitantly, sphingolipid profiling revealed that the biosynthesis of GlcNAc-containing glycosyl inositol phosphoryl- ceramides (GIPCs) is also reliant on this transporter. By contrast, plants carrying the loss-of-function alleles affecting ROCK1, which has been reported to transport UDP-GlcNAc and UDP-N-acetylgalactosamine, exhibit no changes in N-glycan or GIPC profiles. Our findings reveal that plants contain a single UDP-GlcNAc transporter that delivers an essential substrate for the maturation of N-glycans and the GIPC class of sphingolipids. NATURE PLANTS | www.nature.com/natureplants