A SINK-SPECIFIC H+/MONOSACCHARIDE COTRANSPORTER FROM NICOTIANA-TABACUM - CLONING AND HETEROLOGOUS EXPRESSION IN BAKERS-YEAST
A SINK-SPECIFIC H+/MONOSACCHARIDE COTRANSPORTER FROM NICOTIANA-TABACUM - CLONING AND HETEROLOGOUS EXPRESSION IN BAKERS-YEAST
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DOI：
10.1046/j.1365-313x.1993.04040601.x
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发表时间：
1993-10-01
期刊：
影响因子：
7.2
通讯作者：
STADLER, R
中科院分区：
文献类型：
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作者：
SAUER, N;STADLER, R
从烟草中分离到一个单糖转运蛋白(MST1)的cDNA克隆，该基因在成熟烟草的根、花和幼叶等库区组织中表达最强。其开放阅读框为1569个碱基，编码523个氨基酸，计算的相对分子质量为57 717 Da。该蛋白与拟南芥和小球藻的其他一组植物单糖转运蛋白、人类葡萄糖转运蛋白、酿酒酵母的单糖转运蛋白以及几种细菌糖转运蛋白同源。与其他转运蛋白一样，MST1蛋白具有极强的亲脂性，并具有12个假定的跨膜结构域。MST1基因克隆在酿酒酵母中的异源表达使其成为一种H+/单糖共转运蛋白，催化己糖(如D-葡萄糖和D-半乳糖)或戊糖(如D-木糖)的摄取和非偶联剂敏感的非代谢底物(如D-木糖或3-O-甲基葡萄糖)的积累。针对β-半乳糖苷酶和MST1蛋白C末端最后27个氨基酸的融合蛋白，制备了多克隆抗体。在转化的酵母细胞的十二烷基硫酸钠提取物中，这些抗体识别一种表观分子量为42 kDa的多肽，而未转化的对照细胞的提取物中没有这种多肽。
A cDNA clone for a monosaccharide transporter (MST1) was isolated from tobacco, which is most strongly expressed in the various sink tissues of mature tobacco plants: roots, flowers, and young leaves. An open reading frame of 1569 bp codes for a protein with 523 amino acids and a calculated molecular weight of 57 717 Da. The protein is homologous to a group of other plant monosaccharide transport proteins from Arabidopsis thaliana and Chlorella kessleri, to human glucose transporters and to Saccharomyces cerevisiae and several bacterial sugar transport proteins. As with these other transporters, the MST1 protein is extremely lipophilic and has 12-putative membrane-spanning domains. Heterologous expression of the MST1 cDNA clone in Saccharomyces cerevisiae allowed its characterization as a putative H+/monosaccharide co-transporter, catalyzing the uptake of hexoses (e.g. D-glucose and D-galactose) or pentoses (e.g. D-xylose) and the energy dependent and uncoupler sensitive accumulation of non-metabolizable substrates (e.g. D-xylose or 3-O-methyl-glucose). Polyclonal antibodies were raised against a fusion protein of beta-galactosidase and the last 27 amino acids of the C-terminus of the MST1 protein. In SDS extracts of transformed yeast cells these antibodies recognize a polypeptide with an apparent molecular weight of 42 kDa, which is absent in extracts from untransformed control cells.