Available online at www.sciencedirect.com ScienceDirect The nuclear pore complex core scaffold and permeability barrier: variations of a common theme Ryo Hayama, Michael P Rout and Javier Fernandez-Martinez The study of the nuclear pore complex (NPC) is a fascinating that fenestrates the NE and forms a channel of approxi- endeavor, as it not only implies uncovering the ‘engineering mately 40 nm wide; it is an eightfold symmetrical assem- marvel’ of its architecture and function, but also provides a key bly, formed by more than 500 copies of 30 different window into a significant evolutionary event: the origin of the proteins called nucleoporins (nups) [1] that have been eukaryotic cell. The combined efforts of many groups in the shown to be arranged in conserved, biochemically stable field, with the help of novel methodologies and new model subcomplexes acting as the NPC’s building blocks [2–4]. organisms, are facilitating a much deeper understanding of this These building blocks coat the NE membrane with a complex assembly. Here we cover recent advances on the highly modular symmetrical scaffold, formed by eight characterization of the structure of the NPC scaffold and of the protomers called spokes, connecting radially to form biophysical mechanisms that define the permeability barrier. several concentric rings [2,3]: the outer ring—formed We identify common architectural and functional principles by a head-to-tail arrangement of a Y-shaped complex between those two NPC compartments, expanding the [5–7]; the inner ring; and the membrane ring. Attached previous protocoatomer hypothesis to suggest possible to these rings are two asymmetrically localized modules, evolutionary origins for the FG nucleoporins and the NPC the cytoplasmic Nup82 complex and the nuclear basket. permeability barrier. From the scaffold, a special type of nups, called FG nups for their high content in phenylalanine–glycine repeats, Address project their intrinsically disordered FG repeat containing Laboratory of Cellular and Structural Biology, The Rockefeller University, domains into the central channel [3] (Figure 1). An New York, NY 10065, USA estimated 6 MDa worth of FG disordered domains fill Corresponding author: Fernandez-Martinez, Javier the NPC central channel to form the permeability barrier ([email protected]) that confers selectivity and specificity to the NPC. Such a ‘cloud’ of FG domains prevents passive diffusion of most macromolecules through the NPC, while at the same time Current Opinion in Cell Biology 2017, 46:110–118 provides docking sites for specific transport factors (TFs), This review comes from a themed issue on Cell nucleus mainly belonging to the karyopherin family of TFs [8]; Edited by Aaron F. Straight and Roland Foisner after binding to their cargo, TFs transiently interact with For a complete overview see the Issue and the Editorial the FG regions and rapidly transit through the central channel in a matter of milliseconds [9]. Available online 15th June 2017 http://dx.doi.org/10.1016/j.ceb.2017.05.003 Most components of the NPC core scaffold have been 0955-0674/ã 2017 Elsevier Ltd. All rights reserved. shown to be structurally and evolutionarily related to vesicle coating complexes [10–13], which led to the proposal of the protocoatomer hypothesis, that suggests a common evolutionary origin for NPCs and coated vesicles in an early membrane-curving module (the proto-coatomer) [10]. However, tracing the evolutionary Introduction: the nuclear pore complex and origin of other parts of the NPC, including the nuclear transport permeability barrier, have been more challenging than The hallmark organelle of the eukaryotic cell is the that of the scaffold. Here, we give an overview of the nucleus, a compartment delimited by a double membrane recent advances in our understanding of: (i) the detailed termed the nuclear envelope (NE). The nucleus confers architecture of the NPC scaffold; and (ii) the biophysical the primary compartmentalization within the eukaryotic characteristics of intrinsically disordered FG domains and cell, segregating the DNA, and associated processes in their molecular behaviors. Finally, we integrate this new the nucleoplasm, from the cytoplasm; however, compart- knowledge into a hypothesis for the evolutionary origin of mentalization comes with a cost, as it requires systems the FG nups/karyopherins and the NPC permeability that ensure proper communication and exchange of barrier. macromolecules between different cellular compart- ments. In the case of the nucleus, the nuclear pore complex (NPC) ensures efficient trafficking between Evolving view of the NPC structure nucleus and cytoplasm. The NPC is a massive protein The structural characterization of the NPC has been an assembly, of 50–100 MDa depending on the organism, especially challenging endeavor, due to the sheer size of Current Opinion in Cell Biology 2017, 46:110–118 www.sciencedirect.com The nuclear pore complex core scaffold and permeability barrier Hayama, Rout and Fernandez-Martinez 111 Figure 1 Nup82 complex Outer ring Cytoplasm (Nup84 complex) ONM Inner ring Membrane ring complex INM Nucleus CCentralentral NNuclearucu basket cchannel Coatomereerr nupsnups FG regionreegioio FG motif SLiM-IDRR TF sosolenoidolenen SLiM Inner ring nup TF solenoid Hydrophobic (karyopherin) Pocket Folded anchoring region Current Opinion in Cell Biology The nuclear pore complex architecture. Upper part, schematic illustrating the major features of the NPC organization. Lower part, schematic of linker nucleoporins yNup145N/yNup 100/yNup116/hNup98, showing their interaction with the NPC scaffold through their disordered connectors (based on Ref. [20 ]) and interaction with transport factors through their FG repeat regions. the assembly, its intrinsic flexibility, the dynamic nature into new hybrid models covering a substantial portion of of some of its components, and the fact that 1/3 of its the NPC symmetric core [17 ,18 ,22 ]. These analyses mass is predicted to be disordered. Nevertheless, recent showed that the outer rings of the vertebrate NPC are groundbreaking advances in the structural analysis of the formed by a reticulated arrangement of two concentric NPC are revealing exciting new details about this beau- rings of 8 Y-complexes (32 copies total). The monomeric tifully complex assembly. One of these breakthroughs complexes run in a head-to-tail fashion and seem to came thanks to technical advances in cryo electron connect both through direct contacts and through bridges microscopy [14], that allowed Martin Beck and colleagues formed by other nups, like Nup358 [17 ,22 ]; in yeast, ˚ to generate a first 30 A resolution cryo-electron tomogra- though, the arrangement is simpler [3]. The Y-complexes phy map of the human NPC [15]. The resolution of this contact the NE membrane directly through membrane map, and that of further refinements achieving resolutions interacting motifs located in the beta-propeller tips of ˚ around the 20 A mark [16 ,17 ,18 ], along with bio- Nup133 and Nup120–Nup160 [32,33]. Similar membrane chemical reconstitutions [19,20 ,21,22 ] and cross-link- binding motifs are used to contact the NE by other nups, ing and mass spectrometry data [15,17 ], allowed the like vNup155 [18 ], yNup60, yNup1 [34 ] and Nup53 fitting of available crystal structures [23 ,24,25 ,26–31] [35,36], suggesting the presence of a network of contacts www.sciencedirect.com Current Opinion in Cell Biology 2017, 46:110–118 112 Cell nucleus across the surface of the NPC facing the pore membrane facing the central channel, and not away from it as has that probably helps stabilize and shape the NPC; an been traditionally considered [43]. important future goal will be to fully map these contacts to understand how the NPC is anchored in the NE. The An important paradigm shift in our understanding of the outer and inner rings of the vertebrate NPC are architecture of the NPC came from deciphering the connected through vertically arranged copies of vNup155. structural role of non-FG unstructured domains in nups. The core of the inner ring is proposed to be formed by For many years, the function of these domains was not vNup93, vNup155, vNup205 or vNup188, and the well understood, if not even considered an ‘inconvenient’ trimeric vNup62 complex [23 ,25 ]. In each spoke, they feature that needed to be removed to obtain constructs appear organized in four compositionally similar modules, suitable for structural analyses. This view has radically including one copy of each protein. The modules are changed thanks to groundbreaking studies by Ed Hurt’s arranged along an oblique two-fold rotational symmetry group. They showed that the thermophilic fungus Chae- axis dividing the spoke into a nuclear and a cytoplasmic tomium thermophilum (ct) nups [19] ctNup53, ctNic96, half. On each half, the two modules run parallel in an ctNup145N and their Saccahromyces cerevisae homologous approximate head-to-tail fashion, and the trimeric coiled- nups (yNup53, yNic96 and yNup145N, yNup116 and coil bundles of vNup62–vNup54–vNup45 project yNup100 respectively) contain conserved arrays of short laterally into the central channel, placing their associated linear motifs (SLiMs [44]) in their intrinsically disordered FG domains roughly around the equator of the NPC. It regions (IDRs) that mediate interactions with multiple would also seem that a number of these features are inner ring components [19,20 ] (Figure 1). These SLiM- similar in the yeast NPC [3]. However, we should