Binding to FG-Nups Is Insufficient to Promote Nuclear Transport

Abstract

Nuclear pore complexes (NPCs) mediate all cargo traffic between the nucleus and the cytoplasm of eukaryotic cells. The central channel is occupied by a network of intrinsically disordered polypeptides that contain thousands of phenylalanine-glycine (FG) repeats. Transport factors bind to the FG-network and carry cargos through the NPC. The precise mechanism by which transport factors allow cargos to overcome and migrate through the permeability barrier is unknown. Single particle tracking experiments were used to determine the transport properties of a large cargo, the tetrameric protein beta-galactosidase (∼500 kDa), with four M9 signal sequences (M9-βGal). With 1 μM transportin and 100 pM M9-βGal, the cargo transport efficiency was 24±4% and the NPC interaction time was 8.7±0.8 ms. When the transportin concentration was reduced to 25 pM, these parameters were 3±2% and 4.8±0.4 ms, respectively. These data indicate that multiple transportin molecules are required for efficient transport and a single transport factor allows binding but not transport. Particle tracking data indicate a central barrier to translocation, and therefore support the hypothesis that transport factors promote translocation by increasing cargo solubility within the central FG-network. The weak dependence of interaction time on the number of bound transport cofactors indicates that avidity effects are low, implying both a low effective concentration of free FG repeats and a millimolar FG-transportin affinity. A weak FG-transportin affinity implies that any FG-FG interactions must also be weak, otherwise transport receptor interactions would be insufficient to overcome the permeability barrier. These results are consistent with a model in which the permeability barrier is comprised of a denser central FG-network and cargo recognition occurs within a sparser peripheral FG-network.