Handford M G, Sicilia F, Brandizzi F, Chung J H, Dupree P
Department of Biochemistry, University of Cambridge, Building O, Downing Site, Cambridge, CB2 1QW, UK.
Mol Genet Genomics. 2004 Nov;272(4):397-410. doi: 10.1007/s00438-004-1071-z. Epub 2004 Oct 8.
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.
核苷酸糖跨高尔基体膜的转运是多种重要细胞表面成分腔内合成所必需的，并且由核苷酸糖转运蛋白（NSTs）介导，这些转运蛋白是药物/代谢物转运蛋白大家族的成员。尽管这些蛋白在植物中很重要，但到目前为止仅描述了两种，即来自拟南芥的GONST1和AtUTr1。在这项工作中，我们的目标是从拟南芥中鉴定出更多的高尔基体核苷酸糖转运蛋白。基于它们与GONST1的序列相似性，我们发现了另外四种蛋白，我们将其命名为GONST2、3、4和5。这些推定的NSTs被分为三个进化枝：GONST2与GONST1；GONST3与GONST4；GONST5与另外六种未鉴定的蛋白。每个进化枝的一个成员与绿色荧光蛋白（GFP）融合后在烟草细胞中的瞬时表达表明，所有这些推定的NSTs都定位于高尔基体。为了获得核苷酸糖转运活性的证据，我们在一个GDP-甘露糖转运缺陷型酵母突变体（vrg4-2）中表达了这些蛋白以及先前已鉴定的GONST1。我们测试了转化体对与该突变相关的两种表型的挽救情况：对潮霉素B的敏感性和细胞外几丁质酶糖基化的降低。GONST1和GONST2挽救了这两种表型，表明GONST2与先前鉴定的GONST1一样，是一种GDP-甘露糖转运蛋白。GONST3、4和5也挽救了抗生素敏感性，但没有挽救几丁质酶糖基化缺陷，这表明它们也可以将GDP-甘露糖转运穿过酵母高尔基体膜，但效率较低。RT-PCR和Affymetrix数据的分析揭示了GONST1-5在多种器官中的部分重叠表达模式。由于挽救vrg4 - 2表型的能力存在差异以及在植物器官中的表达模式不同，我们推测GONST1和GONST2都是GDP-甘露糖转运蛋白，而GONST3、GONST4和GONST5可能在植物中转运其他核苷酸糖。
