In vitro reconstitution of dynamically interacting integral membrane subunits of energy-coupling factor transporters

Inda Setyawati, Weronika Karoli Stanek, Maria Dosz-Majsnerowska, Lotteke Ziel-Swier, Els Pardon, Jan Steyaert, Albert Guskov*, Dirk Slotboom*

*Corresponding author for this work

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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.
Original languageEnglish
Article numbere64389
Pages (from-to)1-21
Number of pages21
Publication statusPublished - 22-Dec-2020

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