Login / Signup

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

Inda SetyawatiWeronika K StanekMaria MajsnerowskaLotteke J Y M SwierEls PardonJan SteyaertAlbert GuskovDirk Jan Slotboom
Published in: eLife (2020)
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.
Keyphrases
  • molecular dynamics
  • high density
  • single cell
  • room temperature
  • mass spectrometry
  • transcription factor
  • clinical evaluation