Rapid Evolution Exposes the Boundaries of Domain Structure and Function in Natively Unfolded FG Nucleoporins

Abstract

Nucleoporins with phenylalanine-glycine repeats (FG Nups) function at the nuclear pore complex (NPC) to facilitate nucleocytoplasmic transport. In Saccharomyces cerevisiae, each FG Nup contains a large natively unfolded domain that is punctuated by FG repeats. These In eukaryotes, nuclear pore complexes (NPCs) 1 regulate the movement of cellular material across the nuclear envelope by functioning as a permeability barrier and as transport machine (1). Small molecules diffuse through NPCs, but large proteins and RNAs (Ͼ40 kDa) are excluded unless they contain localization sequences that permit translocation. These targeting signals are recognized by karyopherins (Kaps), mobile receptors that interact with the NPC to facilitate nucleocytoplasmic transport (2). Multiple copies of 30 nucleoporin (Nup) proteins comprise each NPC (3, 4), and approximately half of these Nups are classified as FG Nups due to their content of phenylalanine-glycine (FG) motifs. In Saccharomyces cerevisiae, each FG Nup contains a large domain (150 -700 AA in length) composed of FG repeats spaced 10 -20 AAs apart. These FG domains function as docking sites for Kaps (5), which bind to phenylalanines in the FG motif (6, 7).The FG domains of S. cerevisiae Nups are natively unfolded (8, 9). Such "natively unfolded," "intrinsically unstructured," and "disordered" proteins or protein domains lack stable secondary structure and behave as flexible filaments (10, 11). Despite their structural disorder, these domains are often essential for protein-protein and protein-nucleic acid interactions (11). Although the role of disordered structure in FG Nup function is unclear, two hypotheses have been proposed. First the disordered structures in Nups may facilitate rapid translocation of Kap-cargo complexes through the NPC by capturing and releasing Kaps with fast association and dissociation rates. Disordered proteins can exhibit unusually rapid interaction dynamics with binding partners due to a lack of steric limitations (11). In nuclear transport, fast interactions between Kaps and Nups may be necessary for the rapid flux of Kap-cargo complexes through NPCs (12). Thus, the disordered structures of FG Nups might be optimized for highly specific, yet transient interactions with a variety of transport factors. A second hypothesis proposes that the NPC permeability barrier is a meshwork of disordered FG Nup filaments that are interconnected by weak hydrophobic interactions between FG motifs (12). In principle, such a barrier could permit small particles to pass through the interfilament space yet exclude larger molecules from entering the NPC. Large Kap-cargo complexes could gain access by interacting with multiple FG Nups.If structural disorder in FG Nups serves a critical role in NPC function and architecture, then this feature should be conserved throughout Eukaryotae. In the present study we analyzed the AA composition of FG Nups from evolutionarily distant eukaryotes and found evidence of structural disorder in nearly all FG Nups examined. We also not...

Slide-and-exchange mechanism for rapid and selective transport through the nuclear pore complex

“…In turn, each FG repeat consists of a single FG motif of various sequence flavors, such as the FSFG and GLFG motifs, and 10-30 spacer residues that separate consecutive FG motifs (15,16). The spacer residues are strongly hydrophilic (17) and rich in disorder-promoting residues, such as proline, serine, lysine, and glutamine ( Fig. 1) (18).…”

The selective permeability barrier in the nuclear pore complex

“…The overall features of these FG-Nup homologues seem to have been well conserved, AA composition, fold type, and domain structure in particular. 4,13,31 The FG-Nups in higher organisms, as those in yeast, also similarly display enriched charged and polar, or "disordered" AAs, and are depleted in hydrophobic, or "order-promoting" AAs. 13 Therefore, the structural disordered nature of FG-Nups is persistently conserved during evolution.…”

“…12 . In accordance with their name, transmembrane Nups and structural Nups are proteins with secondary structures of b-propeller and a-solenoid motifs, whereas disordered FG-Nups lack typical secondary structure and tertiary structure, displaying a very flexible nature with high net charge and low hydrophobicity as well as low compactness/nonglobularity 6,8,9,13 The NPC central channel is filled with the disordered FG-polypeptides of FG Nups, forming an effective selective permeability barrier. The barrier not only permits the passage of ions and other small molecules (< 40 kDa) via passive diffusion, but also simultaneously blocks the passage of signal-independent, large, inert molecules.…”

“…5,7,13,16 A total of 11-13 FG-Nups are described in yeast cells, and the number closely represents the total number of FG-Nups in this microorganism (Table 1, 5 ). All FG-Nups in yeast contain enriched charged and polar AAs [such as A, R, Q, E, G, K, P, S (alanine, arginine, glutamine, glutamate, glycine, lysine, proline, serine)] and reduced hydrophobic AAs [such as W, C, I, Y, V, L, N (tryptophan, cysteine, isoleucine, tyrosine, valine, leucine, asparagine)].…”

“…All FG-Nups in yeast contain enriched charged and polar AAs [such as A, R, Q, E, G, K, P, S (alanine, arginine, glutamine, glutamate, glycine, lysine, proline, serine)] and reduced hydrophobic AAs [such as W, C, I, Y, V, L, N (tryptophan, cysteine, isoleucine, tyrosine, valine, leucine, asparagine)]. 7,8,13 Disordered content varies among the subfamilies: FxFG Nups have more polar AAs than Nups with GLFG, PSFG, and xxFG repeats. 8 Interestingly, the FG-domains of the Nups located in peripheral structure (either on cytoplasmic filaments or on nuclear basket) evolved more divergently in their primary sequence structure than that of the Nups located in the central plane.…”