FUNCTIONAL RECONSTITUTION OF THE SOLUBILIZED ARABIDOPSIS-THALIANA STP1 MONOSACCHARIDE-H+ SYMPORTER IN LIPID VESICLES AND PURIFICATION OF THE HISTIDINE TAGGED PROTEIN FROM TRANSGENIC SACCHAROMYCES-CEREVISIAE
FUNCTIONAL RECONSTITUTION OF THE SOLUBILIZED ARABIDOPSIS-THALIANA STP1 MONOSACCHARIDE-H+ SYMPORTER IN LIPID VESICLES AND PURIFICATION OF THE HISTIDINE TAGGED PROTEIN FROM TRANSGENIC SACCHAROMYCES-CEREVISIAE
复制标题
DOI：
10.1046/j.1365-313x.1994.6020225.x
复制
发表时间：
1994-08-01
期刊：
影响因子：
7.2
通讯作者：
SAUER, N
中科院分区：
文献类型：
--
作者：
STOLZ, J;STADLER, R;SAUER, N
编码拟南芥 STP1 单糖/H+ 同向转运蛋白或组氨酸标记的 STP1-His6 蛋白的完整 DNA 序列在面包酵母酿酒酵母中表达。野生型 STP1 和重组组氨酸标签蛋白均位于转化酵母细胞的质膜中。与野生型蛋白相比，C 端修饰没有导致转运活性损失。使用抗 STP1 抗体来确认酵母中蛋白质的身份，并比较酵母或拟南芥膜提取物中 STP1 蛋白质的表观分子量。纯化的酵母质膜与由大肠杆菌脂质和牛心细胞色素 e 氧化酶组成的脂蛋白体融合。向这些融合的囊泡中添加抗坏血酸/TMPD/细胞色素-c，导致膜电位立即形成（内部为负值；用[H-3]四苯基鏻阳离子监测），同时解偶联剂敏感的 D-葡萄糖流入赋能的囊泡中。 STP1-His6 蛋白用辛基-PD-葡萄糖苷溶解后具有功能活性，通过洗涤剂稀释将该蛋白插入脂蛋白体并测定所得转运能力即可证明这一点。使用来自转基因酵母细胞的总膜或质膜的洗涤剂提取物在 Ni2+-NTA 柱上一步纯化 STP1-His6 蛋白。通过免疫印迹和 N 末端测序检查纯化蛋白的身份。
Complete DNA sequences encoding the Arabidopsis thaliana STP1 monosaccharide/H+ symporter or a histidine-tagged STP1-His6 protein were expressed in baker's yeast Saccharomyces cerevisiae. Both wild-type STP1 and the recombinant his-tagged protein were located in the plasma membranes of transformed yeast cells. The C-terminal modification caused no loss of transport activity compared with the wild-type protein. Anti-STP1-antibodies were used to confirm the identity of the protein in yeast and to compare the apparent molecular weights of STP1 proteins in membrane extracts from yeast or Arabidopsis thaliana. Purified yeast plasma membranes were fused with proteoliposomes consisting of Escherichia coli lipids and beef heart cytochrome-e oxidase. Addition of ascorbate/TMPD/cytochrome-c to these fused vesicles caused an immediate formation of membrane potential (inside negative; monitored with [H-3]tetraphenylphosphonium cations) and a simultaneous, uncoupler-sensitive influx of D-glucose into the energized vesicles. STP1-His6 protein is functionally active after solubilization with octyl-PD-glucoside, which was shown by insertion of the protein into proteoliposomes by detergent dilution and determination of the resulting transport capacity. Detergent extracts from either total membranes or plasma membranes of transgenic yeast cells were used for one-step purification of the STP1-His6 protein on Ni2+-NTA columns. The identity of the purified protein was checked by immunoblotting and N-terminal sequencing.