Nup153 in Nuclear Transport: Plasticity of Nucleoporin/Transport-Receptor Complexes

Abstract

The MDa sized nuclear pore complexes (NPCs) are among the largest molecular machines in eukaryotic cells and constitute a vital transport conduit between nucleoplasm and cytoplasm. Intrinsically disordered and phenylalanine glycine rich nucleoporins (FG-Nups) form a selective permeability barrier in the center of the NPC, through which large molecules can only pass when piggybacked by nuclear transport receptors (NTRs) that specifically interact with FG-motifs. FG-Nups constitute a complex and distinct non-random amino acid composition of these FG-motifs and inter-FG linkers, but how such heterogeneous sequence composition relates to function and how homotypic interactions between disordered stretches, and transient heterotypic interactions with folded transport receptors could give rise to a transport mechanism is still unclear. This holds true in particular since NTRs seem to bind tightly to FG-Nups (Kd in the nanomolar range), but at the same time move through a pore that is densely filled with FG-motifs (> 50 mM FG) within only a few milliseconds. We have now developed an integrated chemical biology-fluorescence approach that allows us to study the molecular plasticity of FG-Nups on the single-molecule level using multi-parameter fluorescence spectroscopy. Despite its inhomogeneous primary sequence, the FG-domain of Nup153 displays a collapsed coil behavior across its entire amino acid sequence. Surprisingly, it retains this collapsed conformation even when bound to NTRs as shown by single-molecule Forster resonance energy transfer and fluorescence anisotropy. However, ultrasensitive photo-induced electron transfer experiments combined with picosecond resolved fluorescence fluctuation analysis revealed the formation of very flexible and dynamic complexes. Screening different labeling sites allowed us to detect differential binding modes of the Nup153·NTR interaction. These results have wide implications on how nuclear transport can pursue specifically and fast inside the nuclear pore complex.