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A Golgi UDP-GlcNAc transporter delivers substrates for N-linked glycans and sphingolipids

Abstract

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 cytosolic-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 phosphorylceramides (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.

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Fig. 1: In vitro UGNT1 transport activity.
Fig. 2: Developmental expression and subcellular localization of UGNT1.
Fig. 3: Plants carrying the ugnt1-mutant alleles are devoid of complex N-glycans.
Fig. 4: GIPCs derived from UGNT1-mutant plants display altered glycosylation profiles.
Fig. 5: UDP-GlcNac levels from plants carrying mutant alleles.

Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE65 partner repository with the data set identifier PXD006635. All filtered peptide spectrum matches are available in Supplementary Dataset 1. All filtered and quantified sphingolipid data are available in Supplementary Table 5. A.thaliana WT (Col-0) and mutant seeds were obtained from the Arabidopsis Biological Resource Center (http://abrc.osu.edu/). Protein sequences for UGNT1 (At4G32272.1) and ROCK1 (At5g65000.1) are available from The Arabidopsis Information Resource (http://www.arabidopsis.org/). The following T-DNA insertion lines were used in this study: ugnt1-1 (SAIL_1262_C12), ugnt1-2 (SAIL_134_E12C1), rock1-2 (SALK_001259), rock1-4 (SALK_112086) and cgl1-T/cgl1-3 (SALK_073650). The data that support the findings of this study are available from the corresponding author upon request.

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Acknowledgements

We thank the Biological Optical Microscopy Platform (BOMP) at the University of Melbourne and acknowledge access to the Mass Spectrometry and Proteomics Facility (MSPF) at the Bio21 Institute. Lipid analysis was performed by Metabolomics Australia at the University of Melbourne, a NCRIS initiative under Bioplatforms Australia Pty. The research was supported by an Australian Research Council Discovery Project (DP180102630). B.E. and S.P. are supported by Australian Research Council Future Fellowships (FT160100276 and FT160100218) and H.E.M. is supported by an Australian Research Council Discovery Early Career Researcher Award (DE170100054). This study was also supported by the Mizutani Foundation for Glycoscience (160151) and the DOE Joint BioEnergy Institute (http://www.jbei.org) supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the U.S. Department of Energy. W.Z., K.F. and A.B. were supported by the Australia Research Council Centre of Excellence in Plant Cell Walls (CE110001007). The substrates obtained from Carbosource Services (Athens, GA, USA) were supported in part by the NSF-RCN grant no. 0090281.

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B.E., C.R., H.V.S. and J.L.H. designed the project. B.E., C.R., H.E.M., T.R., W.Z. and K.F. performed the experiments. B.E., C.R., H.E.M., T.R., K.F., H.V.S., A.B., U.R., S.P. and J.L.H. analysed the results. B.E., C.R. and J.L.H. wrote the manuscript.

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Correspondence to Joshua L. Heazlewood.

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Supplementary Figures 1–6 and Supplementary Tables 1–5

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Supplementary Data 1

N-glycopeptides identified by tandem mass spectrometry for plant lines used in this study. Matched glycopeptides from Arabidopsis rossette material from wild-type and mutant lines

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Ebert, B., Rautengarten, C., McFarlane, H.E. et al. A Golgi UDP-GlcNAc transporter delivers substrates for N-linked glycans and sphingolipids. Nature Plants 4, 792–801 (2018). https://doi.org/10.1038/s41477-018-0235-5

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