Arabidopsis thaliana expresses multiple Golgi-localised nucleotide-sugar transporters related to GONST1.
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.
Handford
M G
MG
Department of Biochemistry, University of Cambridge, Building O, Downing Site, Cambridge, CB2 1QW, UK.
Sicilia
F
F
Brandizzi
F
F
Chung
J H
JH
Dupree
P
P
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D006056:Golgi Apparatus / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D033703:Nucleoside Transport Proteins / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D015003:Yeasts
D029681:Arabidopsis Proteins; D002352:Carrier Proteins; D017931:DNA Primers; C445096:GONST1 protein, Arabidopsis; D026901:Membrane Transport Proteins; D033703:Nucleoside Transport Proteins; D049452:Green Fluorescent Proteins