Department of Biology (Area 10), University of York, York YO10 5YW, United Kingdom.
J Biol Chem. 2012 Jan 27;287(5):3598-608. doi: 10.1074/jbc.M111.281030. Epub 2011 Dec 13.
Tripartite ATP-independent periplasmic (TRAP) transporters are widespread in bacteria but poorly characterized. They contain three subunits, a small membrane protein, a large membrane protein, and a substrate-binding protein (SBP). Although the function of the SBP is well established, the membrane components have only been studied in detail for the sialic acid TRAP transporter SiaPQM from Haemophilus influenzae, where the membrane proteins are genetically fused. Herein, we report the first in vitro characterization of a truly tripartite TRAP transporter, the SiaPQM system (VC1777-1779) from the human pathogen Vibrio cholerae. The active reconstituted transporter catalyzes unidirectional Na(+)-dependent sialic acid uptake having similar biochemical features to the orthologous system in H. influenzae. However, using this tripartite transporter, we demonstrate the tight association of the small, SiaQ, and large, SiaM, membrane proteins that form a 1:1 complex. Using reconstituted proteoliposomes containing particular combinations of the three subunits, we demonstrate biochemically that all three subunits are likely to be essential to form a functional TRAP transporter.
三磷酸非依赖周质（TRAP）转运蛋白在细菌中广泛存在，但研究甚少。它们包含三个亚基，一个小的膜蛋白，一个大的膜蛋白和一个底物结合蛋白（SBP）。尽管 SBP 的功能已经得到很好的证实，但只有在流感嗜血杆菌的唾液酸 TRAP 转运蛋白 SiaPQM 中对膜成分进行了详细研究，其中膜蛋白在遗传上融合在一起。在这里，我们报告了第一个真正的三部分 TRAP 转运蛋白的体外特性，即人类病原体霍乱弧菌的 SiaPQM 系统（VC1777-1779）。活性重建的转运蛋白催化单向依赖 Na+的唾液酸摄取，其生化特征与流感嗜血杆菌的同源系统相似。然而，使用这种三联体转运蛋白，我们证明了形成 1:1 复合物的小的 SiaQ 和大的 SiaM 膜蛋白的紧密关联。使用包含三个亚基的特定组合的重建的脂囊泡，我们从生物化学上证明所有三个亚基都可能是形成功能性 TRAP 转运蛋白所必需的。
