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Arabidopsis thaliana expresses multiple Golgi-localised nucleotide-sugar transporters related to GONST1

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Abstract

Transport of nucleotide-sugars across the Golgi membrane is required for the lumenal synthesis of a variety of essential cell surface components, and is mediated by nucleotide sugar transporters (NSTs) which are members of the large drug/metabolite superfamily of transporters. Despite the importance of these proteins in plants, so far only two have been described, GONST1 and AtUTr1 from Arabidopsis thaliana. In this work, our aim was to identify further Golgi nucleotide-sugar transporters from Arabidopsis. On the basis of their sequence similarity to GONST1, we found four additional proteins, which we named GONST2, 3, 4 and 5. These putative NSTs were grouped into three clades: GONST2 with GONST1; GONST3 with GONST4; and GONST5 with six further uncharacterized proteins. Transient expression in tobacco cells of a member of each clade, fused to the Green Fluorescent Protein (GFP), suggested that all these putative NSTs are localised in the Golgi. To obtain evidence for nucleotide sugar transport activity, we expressed these proteins, together with the previously characterised GONST1, in a GDP-mannose transport-defective yeast mutant ( vrg4-2). We tested the transformants for rescue of two phenotypes associated with this mutation: sensitivity to hygromycin B and reduced glycosylation of extracellular chitinase. GONST1 and GONST2 complemented both phenotypes, indicating that GONST2, like the previously characterized GONST1, is a GDP-mannose transporter. GONST3, 4 and 5 also rescued the antibiotic sensitivity, but not the chitinase glycosylation defect, suggesting that they can also transport GDP-mannose across the yeast Golgi membrane but with a lower efficiency. RT-PCR and analysis of Affymetrix data revealed partially overlapping patterns of expression of GONST1–5 in a variety of organs. Because of the differences in ability to rescue the vrg4 -2 phenotype, and the different expression patterns in plant organs, we speculate that GONST1 and GONST2 are both GDP-mannose transporters, whereas GONST3, GONST4 and GONST5 may transport other nucleotide-sugars in planta.

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Acknowledgments

We thank Ariel Orellana for advice on the chitinase studies and for helpful discussions, and Chris Barton for contributing to the Affymetrix analysis. The Biotechnology and Biological Sciences Research Council supported this research. M.G.H. was the recipient of a Broodbank Trust Fellowship; F.S. was supported by the Institute Pasteur-Fondazione Cenci Bolognetti and F.B. was the recipient of a Canadian Research Chair Grant

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  1. M. G. Handford

    Present address: Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425, Ñuñoa, Santiago, Chile

  2. J. H. Chung

    Present address: Paterson Institute for Cancer Research, Wilmslow Road, Manchester, M20 4BX, UK

Authors and Affiliations

  1. Department of Biochemistry, University of Cambridge, Building O, Downing Site, Cambridge, CB2 1QW, UK

    M. G. Handford, F. Sicilia, J. H. Chung & P. Dupree

  2. Department of Biology, University of Saskatchewan, 112 Science Place, Saskatoon, Saskatchewan, S7N 5E2, Canada

    F. Brandizzi

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  1. M. G. Handford
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Correspondence to P. Dupree.

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Communicated by G. Jürgens

The first two authors contributed equally to this work

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Handford, M.G., Sicilia, F., Brandizzi, F. et al. Arabidopsis thaliana expresses multiple Golgi-localised nucleotide-sugar transporters related to GONST1. Mol Genet Genomics 272, 397–410 (2004). https://doi.org/10.1007/s00438-004-1071-z

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