Disorder in the nuclear pore complex: The FG repeat regions of nucleoporins are natively unfolded

Abstract

Nuclear transport proceeds through nuclear pore complexes (NPCs) that are embedded in the nuclear envelope of eukaryotic cells. The Saccharomyces cerevisiae NPC is comprised of 30 nucleoporins (Nups), 13 of which contain phenylalanine-glycine repeats (FG Nups) that bind karyopherins and facilitate the transport of karyopherin-cargo complexes. Here, we characterize the structural properties of S. cerevisiae FG Nups by using biophysical methods and predictive amino acid sequence analyses. We find that FG Nups, particularly the large FG repeat regions, exhibit structural characteristics typical of ''natively unfolded'' proteins (highly flexible proteins that lack ordered secondary structure). Furthermore, we use protease sensitivity assays to demonstrate that most FG Nups are disordered in situ within the NPCs of purified yeast nuclei. The conclusion that FG Nups constitute a family of natively unfolded proteins supports the hypothesis that the FG repeat regions of Nups form a meshwork of random coils at the NPC through which nuclear transport proceeds.T he nuclear pore complex (NPC) spans the nuclear envelope of eukaryotes and serves as the conduit for all nucleocytoplasmic transport (1). In Saccharomyces cerevisiae, the NPC is composed of multiple copies of 30 different nucleoporins (Nups) that form an octagonal pore structure of 60 MDa measuring 95 nm in diameter and 35 nm in height (2). The FG family of Nups contain extensive regions of FG repeats (typically 200-700 aa in length) and facilitate the passage of karyopherin-cargo complexes through the NPC by binding directly to karyopherins (Kaps͞importins͞exportins͞ transportins) (3, 4), the soluble receptors that recognize nucleocytoplasmic transport signals within cargo (5).Although the mechanism of Kap translocation through NPCs is poorly understood, Kap-FG Nup interactions are central to the process. It is therefore likely that the structures of FG Nups play important roles in directing the movement of Kap-cargo complexes as they traverse the NPC; however, little is known about the structure of the individual FG Nups within the NPC. The FG repeat region of the mammalian Nup p62 was initially hypothesized to adopt a ␤-conformation (6). More recent models of NPC organization have favored disordered conformations for the FG Nups (7, 8), despite a lack of conclusive evidence. In support of these models, Bayliss et al. (9) observed that a fragment of the Nsp1p FG repeat region (a yeast homolog of p62) lacks ␣-helical structure and may be disordered. Also, our recent biophysical analyses of the fulllength S. cerevisiae FG Nup Nup2p characterized it as a natively unfolded protein (10). Natively unfolded proteins, such as Nup2p, lack significant ␣-helical and ␤-sheet secondary structure and exhibit high flexibility under physiological conditions. They also exhibit low overall hydrophobicity, high net charge, and low compactness͞nonglobularity (11, 12). Here, we perform structural analyses of additional FG Nups to test whether unfolded structures are a conserved feat...

Integrative structure and functional anatomy of a nuclear pore complex

“…Each of the FG repeat domains was represented as a flexible string of beads; a bead had a radius of 6 Å and encompassed 20 residues to achieve a compromise between computational efficiency and accuracy 118,146–149 . Consecutive beads were restrained by a bond with an equilibrium length of 18 Å and a constant force of 1.0 kcal/mol/Å, approximating the spring-like nature of flexible polymers 150 in general and FG repeat domains 118,147–149,151–153 in particular. The freely diffusing molecules included 1,600 NTRs and 1,600 inert macromolecules (0.33 mM each), each consisting of eight subgroups of 200 macromolecules, ranging in radius from 4 to 28 Å in 2 Å increments (10 to 75 kDa, assuming constant protein density of 1.38 g/cm 2 ).…”

Arx1 Is a Nuclear Export Receptor for the 60S Ribosomal Subunit in Yeast

“…The inner channel of the nuclear pore complex is largely composed of FG-repeat-containing nucleoporins that create a hydrophobic meshwork, posing a permeability barrier that selectively controls the translocation of macromolecules (Ribbeck and Gorlich, 2001;Denning et al, 2003). Translocation of hydrophilic cargo through this hydrophobic channel of the NPC requires a mechanism for partitioning the cargo into such an environment.…”

“…Thus, the ribosomal subunits might also employ multiple receptors. In addition, the channel of the NPC is comprised of disordered FG repeat-containing nucleoporins that form a mesh through weak hydrophobic interactions (Ribbeck and Gorlich, 2001;Denning et al, 2003;Patel et al, 2007). The highly electronegative ribosomal subunits likely require additional proteins to present hydrophobic surfaces that can partition into this milieu.…”

Nup214-Nup88 Nucleoporin Subcomplex Is Required for CRM1-mediated 60 S Preribosomal Nuclear Export

“…In fact, the FG-repeat region of Nup214 is able to cross the NPC by itself (56) and is predicted to be unstructured (57) and long enough to cross the NPC from a cytoplasmic anchoring point. In addition, overexpression of Nup214 results in a presence of this nucleoporin at both sides of the NPC (58).…”