Interactions between a fluctuating polymer barrier and transport factors together with enzyme action are sufficient for selective and rapid transport through the nuclear pore complex

Abstract

The nuclear pore complex, the only pathway for transport between the nucleus and cytoplasm, functions as a highly selective gate that blocks nonspecific macromolecules while allowing the rapid transport of tagged [transport factor (TF) bound] cargo up to an order of magnitude larger. The mechanism of this gate's operation is not yet fully understood and progress has been primarily hindered by the inherent complexity and multiscale nature of the problem. One needs to consider the hundreds of disordered proteins (phenylalanine glycine nucleoporins or FG nups) lining the pore, as well as their overall architecture and dynamics at the microsecond scale, while also accounting for transport at the millisecond scale across the entire pore. Here we formulate an approach that addresses transport properties over a large range of length and time scales. We do this by incorporating microscopic biophysical details, such as charge and specific TF-FG nup interactions, to compute the free energy landscape encountered by the cargo. We connect this to macroscopic transport by treating cargo translocation as a stochastic barrier crossing process and computing the current and the translocation time. We then identify distinct transport regimes (fast permeable, slow permeable, and impermeable) determined by the cargo size, TF affinity for FG nups, and the activity of the enzymes that cleave TFs from cargo. Our results, therefore provide an integrated picture of transport through the NPC, while highlighting how FG nup interactions with TFs and enzyme activity cooperate to produce selectivity and efficiency.

Intrinsically disordered nuclear pore proteins show ideal-polymer morphologies and dynamics

“…As demonstrated here, this phenomenon is particularly articulated when the majority of the polymer become confined in the nanopore confinement. Hence in the physiologically relevant parameter range as determined above, we observe that FG nup assemblies in a nanopore can undergo major conformational changes for only minor changes in intermolecular interactions [15,45]. The polymer systems investigated thus far in this work have contained only one type of FG nup.…”

Bound-state diffusion due to binding to flexible polymers in a selective biofilter

“…Using the upper bound of N t = 0.67 mM for both FSFG 6 and FSFG 12 gels, we calculated a dissociation constant of K D = 300 ± 80 µM and an off-rate of k off = 3.0 × 10 5 ± 0.8 × 10 5 s −1 for FSFG 6 . The corresponding values for FSFG 12 are K D = 180 ± 20 µM and k off = 1.8 × 10 5 ± 0.2 × 10 5 s −1 .…”

“…We aimed to to use proteins with rapid dynamics, including diffusion-limited on-rates, and well-established binding affinities. For this reason, we chose yeast nuclear transport factor 2 (NTF2) and synthetic fragments derived from the yeast FG Nup Nsp1 ( FSFG 6 and FSFG 12 (23,24)). NTF2 is of similar size to the red fluorescent protein mCherry, which is inert to the FG-Nup-based protein fragments.…”

“…Using this model, FSFG 6 is about 10 nm across and FSFG 12 14 nm. We confirmed significant incorporation of the protein into the gel by 1) the binding of NTF2 to the gel, which predicts that all of the protein is incorporated and 2) by digesting the protein out of the gel using trypsin and measuring the resulting concentration in equilibrated solution.…”

“…In contrast, non-binding proteins larger than about 30 kDa have much lower flux through the pore than similarly-sized transport factor-cargo complexes (4)(5)(6). While models of diffusion within the NPC have been developed to explain selectivity, many reduce the problem to that of a particle diffusing within an effective energy landscape (6,(9)(10)(11)(12), an approach which does not address the molecular mechanisms of diffusion that could lead to selectivity. Therefore, we sought to investigate mechanisms of binding and mobility that can lead to selective transport.…”