In vitro reconstitution of dynamically interacting integral membrane subunits of energy-coupling factor transporters
Inda Setyawati,
Weronika K Stanek,
Maria Majsnerowska,
Lotteke J Y M Swier,
Els Pardon,
Jan Steyaert,
Albert Guskov,
Dirk J Slotboom
Affiliations
Inda Setyawati
Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, Netherlands; Biochemistry Department, Bogor Agricultural University, Bogor, Indonesia
Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, Netherlands; Moscow Institute of Physics and Technology, Dolgoprudny, Russian Federation
Energy-coupling factor (ECF) transporters mediate import of micronutrients in prokaryotes. They consist of an integral membrane S-component (that binds substrate) and ECF module (that powers transport by ATP hydrolysis). It has been proposed that different S-components compete for docking onto the same ECF module, but a minimal liposome-reconstituted system, required to substantiate this idea, is lacking. Here, we co-reconstituted ECF transporters for folate (ECF-FolT2) and pantothenate (ECF-PanT) into proteoliposomes, and assayed for crosstalk during active transport. The kinetics of transport showed that exchange of S-components is part of the transport mechanism. Competition experiments suggest much slower substrate association with FolT2 than with PanT. Comparison of a crystal structure of ECF-PanT with previously determined structures of ECF-FolT2 revealed larger conformational changes upon binding of folate than pantothenate, which could explain the kinetic differences. Our work shows that a minimal in vitro system with two reconstituted transporters recapitulates intricate kinetics behaviour observed in vivo.
