© 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016) 129, 4439-4447 doi:10.1242/jcs.194753 COMMENTARY Perforating the nuclear boundary – how nuclear pore complexes assemble Marion Weberruss and Wolfram Antonin* ABSTRACT (Alber et al., 2007; Ori et al., 2013). Functionally, nucleoporins can be The nucleus is enclosed by the nuclear envelope, a double roughly divided into three groups. First, transmembrane nucleoporins membrane which creates a selective barrier between the cytoplasm anchor the NPC in the pore membrane. In metazoa, three and the nuclear interior. Its barrier and transport characteristics are transmembrane nucleoporins have been identified: POM121, determined by nuclear pore complexes (NPCs) that are embedded GP210 (also known as NUP210) and NDC1. Members of the within the nuclear envelope, and control molecular exchange second group of nucleoporins belong to the symmetric structural between the cytoplasm and nucleoplasm. In this Commentary, we scaffold of the NPC. Finally, largely unstructured nucleoporins discuss the biogenesis of these huge protein assemblies from containing a high number of phenylalanine-glycine (FG) repeats form approximately one thousand individual proteins. We will summarize the permeability barrier that is essential for nucleocytoplasmic current knowledge about distinct assembly modes in animal cells that transport. are characteristic for different cell cycle phases and their regulation. The NPC structural scaffold is formed by a stack of three rings (Fig. 1): the nucleoplasmic and cytoplasmic rings, and the inner ring KEY WORDS: Annulate lamellae, Nuclear envelope, Nuclear pore (for a review, see Grossman et al., 2012). This arrangement and the complex, Nuclear transport nucleoporins creating these structures are similarly found in yeast (Hoelz et al., 2011; Stuwe et al., 2015; Lin et al., 2016). However, Introduction here, we will primarily focus our discussion on vertebrate NPCs. Both Envelopment of the genetic material by the nuclear envelope is a the cytoplasmic and the nucleoplasmic rings are predominantly hallmark of eukaryotic cells. By spatially and temporally separating formed by multiple copies of an evolutionarily highly conserved nuclear transcription and RNA processing, as well as cytosolic complex, the Nup107–Nup160 complex which is, due to its overall translation, the nuclear envelope allows eukaryotes to achieve a level of shape, also referred to as the Y-complex. In vertebrates, the regulation in gene expression that is unprecedented in prokaryotes. Y-complex consists of ten nucleoporins, some of which show However, the separation of the nuclear genome from the cytosolic structural similarities to vesicle coats (Devos et al., 2004; Mans et al., protein synthesis machinery comes at a price. It requires transport gates 2004; Brohawn et al., 2008). This complex is thought to stabilize that guarantee selective passage of proteins, RNAs, RNA–protein the highly curved pore membrane. The precise arrangement of complexes and metabolites. This is achieved by nuclear pore Y-complex molecules within the cytoplasmic and nucleoplasmic complexes (NPCs), which act as the gatekeepers of the nuclear rings is a subject of active research (for a review, see Hoelz et al., envelope. In contrast to other transport gates, such as ion channels, 2016). Recent electron microscopic tomographic reconstructions of metabolite translocators or transporters for polypeptides, which span NPCs suggest that both the cytoplasmic and the nucleoplasmic rings the respective membrane to form an aqueous channel within a each consist of two concentric rings of eight Y-complexes that hydrophobic lipid bilayer, NPCs breach the barrier of the nuclear are arranged in a head-to-tail manner, resulting in 32 copies of the envelope differently: they deform the two membranes of the nuclear Y-complex per NPC (Bui et al., 2013; von Appen et al., 2015). envelope to create pores with a diameter of 100 nm into which these Embedded between the nucleoplasmic and cytoplasmic rings is complexes are inserted. Accordingly, in most cells NPCs are the largest the inner ring of the structural scaffold. This inner ring is mainly protein assemblies known, with a total mass of 125 MDa in vertebrates. formed by Nup93 complexes, which consist of Nup93, Nup155, Here, we will discuss how these huge complexes assemble and Nup53 (also referred to as Nup35) and the orthologs Nup205 or integrate into the double-membrane structure of the nuclear envelope at Nup188 (for a review, see Vollmer and Antonin, 2014). New different phases of the cell cycle, with a focus on animal cells. insights into the inner ring architecture were recently achieved by docking of crystal structures of individual nucleoporins or NPC architecture respective subcomplexes into the electron tomogram of the Despite their huge size, NPCs are formed by only about thirty human NPC (Kosinski et al., 2016). Similar to the Y-complex, different proteins, nucleoporins or Nups, that are – due to the eightfold the Nup93 complex forms four eight-membered stacked rings symmetry of NPCs – present in eight, sixteen, 32 or more copies resulting in 32 copies of the complex per NPC. The NPC inner ring represents the link between the pore membrane and the permeability barrier formed by FG-repeat nucleoporins: Nup93 positions the Friedrich Miescher Laboratory of the Max Planck Society, Spemannstraße 39, Nup62 complex, which forms a large part of the central transport Tübingen 72076, Germany. channel of the NPC (Sachdev et al., 2012; Chug et al., 2015). *Author for correspondence ([email protected]) Attached to this generally symmetric NPC structure are the nuclear basket and cytoplasmic filaments, asymmetric extensions that W.A., 0000-0003-4669-379X extend into their respective compartments. This is an Open Access article distributed under the terms of the Creative Commons Attribution NPCs are embedded into the nuclear envelope at sites where inner License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. and outer nuclear membranes are fused. Membrane association is Journal of Cell Science 4439 COMMENTARY Journal of Cell Science (2016) 129, 4439-4447 doi:10.1242/jcs.194753 Nup160 Nup85 Fig. 1. General organization principle of nuclear pore Nup358 Nup133 Nup37 Nup214 complexes. Simplified schematic representation of the Nup107 Sec13 Cytoplasmic filaments different structural elements of NPCs with assignment of Nup96 Seh1 the respective nucleoporins. The cytoplasmic and Nup43 nucleoplasmic rings are shown in green, each mostly POM121 formed by 16 copies of the Y-complex, arranged in two Cytoplasmic ring GP210 eight-membered rings. The inner ring, predominantly NDC1 formed by 32 copies of the Nup93 complex is shown in Nup93 red. Transmembrane nucleoporins are depicted in violet, Transmembrane nucleoporin Nup205 and/or Nup188 and the cytoplasmic filaments and nuclear basket Inner ring Nup53 Central channel structure are in orange. Attached to the inner ring are Nup155 Nup62 complexes (depicted in blue), which form a Nup62 cohesive meshwork within the central channel through Nup58 Nucleoplasmic ring their FG-repeat domains. Not indicated is the position of Nup54 Nup98, a FG-repeat-containing nucleoporin important for the transport and exclusion function of NPCs; its Nup160 Nup85 position in the NPC is less defined, but it might be part of Nuclear basket Nup133 Nup37 Nup107 Sec13 the inner ring. Similarly, Aladin (also known as AAAS), Nup96 Seh1 Gle1, Rae1 and Npl1 (also known as hCG1 and NUPL2) Nup153 Nup43 MEL28 have been omitted. Nup50 Tpr mediated by each of the three scaffold rings. In the case of the Facilitated translocation requires nuclear transport receptors (NTRs, cytoplasmic and nucleoplasmic rings, the Y-complex members also called karyopherins), which shuttle between the cytoplasm and Nup160 and Nup133 contain amphipathic helixes, which can the nuclear interior, binding cargo on one side of the nuclear envelope facilitate membrane binding (Drin et al., 2007; Doucet et al., 2010; and delivering it to the other (Fig. 2). Thereby, NTRs mediate cargo Kim et al., 2014; von Appen et al., 2015). For the inner ring, Nup53 translocation through the permeability barrier of NPCs. Regarding the and Nup155, both components of the Nup93-complex, can directly direction of transport, nuclear transport receptors can be classified as interact with the pore membrane (Vollmer et al., 2012; von Appen importins or exportins, although this categorization is not absolute as et al., 2015) and additionally interact with the transmembrane some NTRs mediate transport in both directions. Importins recognize nucleoporins NDC1 and POM121 (Mansfeld et al., 2006; Mitchell cargo proteins bearing nuclear localization signals (NLSs) and enable et al., 2010; Eisenhardt et al., 2014). their passage from the cytoplasm into the nucleus. In contrast, exportins bind cargo with nuclear export signals (NESs) in the Nucleocytoplasmic transport nucleus and translocate them into the cytoplasm. NPCs function as selective gates through the nuclear envelope and The passage of importins and exportins through NPCs occurs in allow the passage of molecules in two modes: passive diffusion, both direction and the same would, in principle, also be true for which is only effective for molecules smaller than 5 nm, and importin–cargo and exportin–cargo complexes. Directionality