Single-Molecule Analysis of the Recognition Forces Underlying Nucleo-Cytoplasmic Transport

Abstract

Macromolecular exchange between the nucleus and cytoplasm of cells is gated at nuclear pores by a family of intrinsically-disordered nucleoporins (nups). These feature phenylalanine-glycine repeats in ‘cohesive’ domains (FG domains) that interact to form a physical barrier. Through unknown mechanisms, karyopherins (importins, exportins, transportins, NTRs) penetrate this barrier to facilitate the movement of large proteins and RNPs across without paying an external energetic cost, simply by interacting with FG domains. To address the molecular binding and dissociation mechanisms involved in this coupled gating-translocation process, single molecule force spectroscopy was used here to measure the interaction force between nup FG repeats, and between importin beta and nup FG repeats. As predicted, cohesive FG domains bound each other through multiple FG repeat interactions. In contrast importin bound only relaxed coil multiple FG repeats simultaneously, whereas just one FG binding site was assessed in collapsed coil multiple FG domains. Most importantly, the interaction forces and fast dissociation rate constants measured between two FG repeats, and between importin and one FG repeat, were almost identical. This suggests that the force needed to separate interactions between FG repeats of nups at the NPC (i.e. for kaps to penetrate the gate and translocate across) could be provided in full by the enthalpy gained through the formation of karyopherin-FG repeat interactions.