A physical model describing the interaction of nuclear transport receptors with FG nucleoporin domain assemblies.

Raphael Zahn,Dirk Görlich,Dino Osmanović,Ralf P Richter,Murray Stewart,Carolina Araya Callis,Bart W Hoogenboom,Severin Ehret,Steffen Frey,Changjiang You

doi:10.7554/elife.14119

Abstract

The permeability barrier of nuclear pore complexes (NPCs) controls bulk nucleocytoplasmic exchange. It consists of nucleoporin domains rich in phenylalanine-glycine motifs (FG domains). As a bottom-up nanoscale model for the permeability barrier, we have used planar films produced with three different end-grafted FG domains, and quantitatively analyzed the binding of two different nuclear transport receptors (NTRs), NTF2 and Importin β, together with the concomitant film thickness changes. NTR binding caused only moderate changes in film thickness; the binding isotherms showed negative cooperativity and could all be mapped onto a single master curve. This universal NTR binding behavior - a key element for the transport selectivity of the NPC - was quantitatively reproduced by a physical model that treats FG domains as regular, flexible polymers, and NTRs as spherical colloids with a homogeneous surface, ignoring the detailed arrangement of interaction sites along FG domains and on the NTR surface.

Highlights

In eukaryotic organisms, the nuclear envelope separates the nucleoplasm from the cytoplasm and encloses most of the genetic material in the cell
We monitored the formation of FG domain films and their interaction with nuclear transport factor 2 from Homo sapiens (NTF2) and Importin b from Saccharomyces cerevisiae (Impb) by spectroscopic ellipsometry (SE) and quartz crystal microbalance (QCM-D), simultaneously and on the same sample (Figure 1—figure supplement 3), to quantify areal protein densities, G, and effective film thicknesses, d, respectively
We have used a bottom-up nanoscale system for a quantitative study of how nuclear transport receptors (NTRs) interact with FG domains from the nuclear pore complexes (NPCs)

Summary

Introduction

The nuclear envelope separates the nucleoplasm from the cytoplasm and encloses most of the genetic material in the cell. The ordered course of gene expression requires selective transport through this double membrane. This function is provided by nuclear pore complexes (NPCs), large membrane-spanning protein complexes that perforate the nuclear envelope (Fahrenkrog and Aebi, 2003; Fernandez-Martinez and Rout, 2012; Floch et al, 2014; Gorlich and Kutay, 1999; Grossman et al, 2012; Macara, 2001). Small molecules up to 20–40 kDa (i.e., up to roughly 5 nm in diameter) can diffuse freely through the NPC, the passage of larger macromolecules is impeded unless they are bound to nuclear transport receptors (NTRs) (Gorlich and Kutay, 1999; Keminer and Peters, 1999; Mohr et al, 2009; Yang and Musser, 2006)

Methods

Results

Discussion

Conclusion