Reig Núria, del Rio César, Casagrande Fabio, Ratera Mercè, Gelpí Josep Lluís, Torrents David, Henderson Peter J F, Xie Hao, Baldwin Stephen A, Zorzano Antonio, Fotiadis Dimitrios, Palacín Manuel
Institute for Research in Biomedicine, Barcelona Science Park and Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, E-08028 Barcelona, Spain.
J Biol Chem. 2007 May 4;282(18):13270-81. doi: 10.1074/jbc.M610695200. Epub 2007 Mar 6.
We have identified YkbA from Bacillus subtilis as a novel member of the L-amino acid transporter (LAT) family of amino acid transporters. The protein is approximately 30% identical in amino acid sequence to the light subunits of human heteromeric amino acid transporters. Purified His-tagged YkbA from Escherichia coli membranes reconstituted in proteoliposomes exhibited sodium-independent, obligatory exchange activity for L-serine and L-threonine and also for aromatic amino acids, albeit with less activity. Thus, we propose that YkbA be renamed SteT (Ser/Thr exchanger transporter). Kinetic analysis supports a sequential mechanism of exchange for SteT. Freeze-fracture analysis of purified, functionally active SteT in proteoliposomes, together with blue native polyacrylamide gel electrophoresis and transmission electron microscopy of detergent-solubilized purified SteT, suggest that the transporter exists in a monomeric form. Freeze-fracture analysis showed spherical particles with a diameter of 7.4 nm. Transmission electron microscopy revealed elliptical particles (diameters 6 x 7 nm) with a distinct central depression. To our knowledge, this is the first functional characterization of a prokaryotic member of the LAT family and the first structural data on an APC (amino acids, polyamines, and choline for organocations) transporter. SteT represents an excellent model to study the molecular architecture of the light subunits of heteromeric amino acid transporters and other APC transporters.
我们已鉴定出枯草芽孢杆菌中的YkbA是氨基酸转运蛋白L-氨基酸转运体（LAT）家族的一个新成员。该蛋白的氨基酸序列与人类异源氨基酸转运蛋白的轻亚基约有30%的同一性。从大肠杆菌膜中纯化的带His标签的YkbA重组到蛋白脂质体中后，对L-丝氨酸和L-苏氨酸以及芳香族氨基酸表现出不依赖钠的强制性交换活性，尽管活性较低。因此，我们建议将YkbA重新命名为SteT（丝氨酸/苏氨酸交换转运体）。动力学分析支持SteT的顺序交换机制。对蛋白脂质体中纯化的、功能活跃的SteT进行冷冻断裂分析，以及对去污剂增溶的纯化SteT进行蓝色原聚丙烯酰胺凝胶电泳和透射电子显微镜分析，表明该转运体以单体形式存在。冷冻断裂分析显示直径为7.4 nm的球形颗粒。透射电子显微镜揭示了椭圆形颗粒（直径6×7 nm），有明显的中央凹陷。据我们所知，这是LAT家族原核成员的首次功能表征，也是关于APC（氨基酸、多胺和有机阳离子的胆碱）转运体的首个结构数据。SteT是研究异源氨基酸转运蛋白轻亚基和其他APC转运体分子结构的一个优秀模型。
