# Puzzling out nuclear pore complex assembly

Arianna Penzo, Benoit Palancade

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Arianna Penzo, Benoit Palancade. Puzzling out nuclear pore complex assembly. FEBS Letters, 2023, 10.1002/1873-3468.14713. hal-04236050

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REVIEW

# Puzzling out nuclear pore complex assembly

Arianna Penzo and Benoit Palancade

<figure><img src="image_4.png" /></figure>

Université Paris Cité, CNRS, Institut Jacques Monod, Paris, France

**Correspondence**

B. Palancade, Université Paris Cité, CNRS,
Institut Jacques Monod, F-75013 Paris,
France

Tel: +33 1 57 27 80 39

E-mail: benoit.palancade@ijm.fr

(Received 5 June 2023, revised 12 July 2023, accepted 17 July 2023)

doi:10.1002/1873-3468.14713

Edited by Birthe Fahrenkrog

Nuclear pore complexes (NPCs) are sophisticated multiprotein assemblies embedded within the nuclear envelope and controlling the exchanges of molecules between the cytoplasm and the nucleus. In this review, we summarize the mechanisms by which these elaborate complexes are built from their subunits, the nucleoporins, based on our ever-growing knowledge of NPC structural organization and on the recent identification of additional features of this process. We present the constraints faced during the production of nucleoporins, their gathering into oligomeric complexes, and the formation of NPCs within nuclear envelopes, and review the cellular strategies at play, from co-translational assembly to the enrolment of a panel of cofactors. Remarkably, the study of NPCs can inform our perception of the biogenesis of multiprotein complexes in general – and vice versa.

**Keywords:** co-translational assembly; multiprotein complex assembly; nuclear envelope; nuclear pore complex; nucleoporin

The existence of multiprotein complexes performing essential structural or catalytical functions is a hallmark of biological systems. However, the efficient and faithful production of macromolecular assemblies represents a challenge for cellular metabolism in view of their sometimes high biogenesis rates, for example, 2000 ribosomes per minute in yeast [1]; their structural complexity and defined stoichiometry—from one repeated subunit in homomers up to several dozens in heteromers; their compositional heterogeneity, that is, the existence of spatiotemporal variants for a large fraction of cellular complexes [2]; and their specific targeting within different subcellular compartments or membrane-bound organelles [3].

Among the multiprotein assemblies that specifically define eukaryotic cells are the nuclear pore complexes (NPCs), which localize at the points of fusion of the

inner and outer nuclear membranes, ranging from several dozens in the yeast nucleus [4] to a few thousand in animal cells [5], and thus delineate a transport channel through the nuclear envelope (NE), connecting the nucleus and the cytoplasm. Although NPCs were imaged since the 1950s, their composition and their structural organization were only uncovered during the last decades, thanks to a combination of genetics, biochemistry, and microscopy approaches, recently culminating in almost complete maps at near-atomic resolution (reviewed in Refs [6–9]). These gigantic multiprotein complexes are made of around 30 subunits called nucleoporins (or Nups), mostly conserved among eukaryotes and present in multiple of eight copies per complex, thus totaling ~ 500–1000 polypeptides per pore for a mass of 50–100 MDa, depending on the species. Most nucleoporins are assembled into

**Abbreviations**

AH, amphipathic helix; AL, annulate lamellae; ALPS, ArfGAP1 lipid packing sensors; EM, electron microscopy; ER, endoplasmic reticulum; FG, phenylalanine-glycine; FP, fluorescent protein; HSP, heat shock protein; ID, intrinsically disordered; INM, inner nuclear membrane; LEM, Lap2, emerin, MAN1; LINC, linker of nucleus and cytoskeleton; NE, nuclear envelope; NLS, nuclear localization sequence; NPC, nuclear pore complex; NTR, nuclear transport receptor; Nup, nucleoporin; ONM, outer nuclear membrane; PTM, post-translational modification; RBP, RNA-binding protein; RHD, reticulon homology domain; RIP, RNA immunoprecipitation; SeRP, selective ribosome profiling; SPB, spindle pole body; SUMO, small ubiquitin-like modifier; TM, transmembrane; v, vertebrate; y, yeast.
<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Simplified representation of NPC structural organization and subunit composition. Nups names are indicated for budding yeast (left) and vertebrate (right) orthologues. FG-Nups appear in bold and FG repeat domains are schematized as curvy lines. Membrane binding domains are represented as semicircles and linker Nup connectivity is illustrated for yNic96/vNup93 and yNup100-yNup116-yNup145N/vNup98. Commonly used alternative names for vertebrate Nups are as follows: Nup358: RanBP2; Nup53: Nup35; Nup210: GP210. Color code and alternative names for Nup subcomplexes are as follows; Outer ring (in blue), Nup84 complex, NUP107-160 complex, Y-complex, coat nucleoporin complex (CNC); Inner ring (in red): Inner Ring Complex (IRC); Central channel (in yellow): channel nucleoporin heterotrimer (CNT), central transport nups (CTN); Membrane ring (in orange); Cytoplasmic filaments (in green): Nup82 complex, Cytoplasmic Export Platform, Cytoplasmic Filament Nucleoporin Complex (CFNC); Nuclear basket (in purple). Depending on inner ring dilation, the diameter of the central channel varies from 425 to 585 Å in yeast (450–600 Å in vertebrates) [9,12]. Note that this schematic representation does not fully recapitulate Nups interactions or recent additional Nups localizations scored in integrative structural analyses (e.g., Nup93 and Nup205, which in addition to their localization within the inner ring, are also detected at outer rings in vertebrates [240]). For a detailed illustration of alternative NPC structural organizations in yeast or other species, the reader shall refer to earlier studies [8,12].</figcaption></figure>

stable subcomplexes, which polymerize in an eightfold symmetrical manner along the nucleocytoplasmic axis, forming a central inner ring flanked by two outer rings on each side of the NE. Multivalent linker nucleoporins connect these distinct building blocks, conferring some flexibility to the whole complex, which can undergo large-scale conformational changes in vivo, notably impacting on the dimensions of its central channel [10,11]. Specialized peripheral extensions, namely the cytoplasmic filaments and the nuclear basket, further distinguish the cytosolic and nucleoplasmic faces of NPCs, contributing to their multiple functions in nucleocytoplasmic exchanges, genome expression

and organization (Fig. 1). Although this global architecture is generally conserved across species, an increasing number of lineage-specific differences are getting reported, for example, regarding the stoichiometry of outer ring components [12–15] or the composition of cytoplasmic appendages [16,17].

While the formation of this modular assembly involves a relatively small set of protein motifs, defining membrane-binding and protein-protein interaction interfaces (Box 1), a large fraction of the NPC mass is accounted by the presence of ID (intrinsically disordered) FG (phenylalanine-glycine) repeat domains, which establish a permeability barrier limiting the
**Box 1. Nucleoporin domains, in a nutshell.**

Among the ~30 Nups, only 3–4 contain transmembrane domains (TM) and thus traffic from the ER to the NE fol-
lowing their synthesis. Some of them (e.g., yPom34, yPom33, yNdc1 and vNdc1) are multi-pass proteins, with 2–6
TM helices and both N and C termini exposed to the nucleo-/cytoplasm, while others (e.g., yPom152, vPom121 and
vNup210) are anchored within the membrane through a unique TM helix [218]. In some cases, NLS located in their
extraluminal region contribute to their targeting to the pore membrane [153,180]. Several other Nups feature mem-
brane interaction domains such as amphipathic α-helices (AH), in which hydrophobic and polar amino acid residues
are segregated at the two opposite faces of the protein structure, or ArfGAP1 lipid packing sensors (ALPS), which
are weakly charged AH with a polar face rich in Ser/Thr residues, and reportedly acting as membrane curvature sen-
sors [198].

A handful of protein domains and protein-protein interaction motifs further contribute to the architecture of the NPC scaffold. Inner and outer ring Nups are essentially built from modular arrangements of α-helical solenoids and β-propeller domains, an architecture found in COPI and COPII vesicle coatomers with which they likely share a common ancestry [219]. α-helical solenoids are rod-shaped structures formed by packed α-helical repeats, while β-propellers are disk-shaped domains consisting of tandem repeats of β-strands arranged in blades, with surface-exposed loops often mediating protein-protein interactions [220]. Outer ring Nups (i.e., ySeh1-yNup120 and ySec13-yNup145C) offer a distinctive example of β-propeller-mediated interactions: the domain invasion motifs of Nup120 and Nup145C complement the missing blade in the incomplete Seh1 and Sec13 β-propellers, locking them into position [221–223]. Another prominent type of protein-protein interaction mode is provided by components of the central channel heterotrimer (e.g., yNsp1-yNup57-yNup49 or vNup62-vNup58-vNup54) and the cytoplasmic filaments (e.g., yNsp1-yNup159-yNup82 or vNup62-vNup214-vNup88), which intertwine their successive C-terminal α-helical coiled-coil domains into trimeric bundles [16,224–227].

A number of nucleoporins contain intrinsically disordered (ID) domains, such as those found in phenylalanine-
glycine (FG) repeat Nups. FG-Nups typically display dozens of hydrophobic FXFG, FG or GLFG motifs, inter-
rupted by characteristic charged or polar ~ 20 residues spacers. These domains can form biomolecular condensates
*in vitro*, mediate the interactions with NTRs and establish the NPC permeability barrier *in vivo* [228–232]. Remark-
ably, similar ID domains, featuring YG repeats, were recently reported to form a selective phase for protein translo-
cation through the peroxisome membrane [233,234]. In addition to their role in transport, Nups FG domains also
function in holding together Nups subcomplexes and stabilizing the NPC structure [105]. Multivalent linker Nups,
for example, yNup145N/vNup98, yNup53/vNup53 or yNic96/vNup93, also feature unstructured short linear motifs
(SLlMs), embedded within FG repeats or flexible regions, and used as interaction domains connecting distinct NPC
modules [12,103,108,235].

diffusion of small macromolecules (< ~ 40 kDa), and
dynamically associate with nuclear transport receptors
(NTRs), hence authorizing the selective translocation
of cargoes. The association of NTRs with their sub-
strates, for example, proteins harboring nuclear locali-
zation sequences (NLS), is regulated by the small
GTPase Ran, which is enriched in its GTP-bound
form within the nucleus, thus providing a positional
information that orientates the direction of transport
events [18].

Although the biogenesis of NPCs faces the same
constraints as those evoked above for multiprotein
complexes in general, their high content of ID proteins
and their insertion within the nuclear envelope raises
specific issues for the maintenance of proteome
homeostasis and cell compartmentalization. Several
studies have revealed that NPC assembly sequentially

involves the synthesis of nucleoporin subunits, their
assembly into hetero-oligomeric subcomplexes and
their recruitment at the NE [19,20] (Fig. 2). In all spe-
cies examined so far, NPCs can be formed de novo
within an intact NE during interphase, concomitantly
with nuclear growth. This mode of assembly is the
unique one in cells undergoing a closed mitosis
(e.g., *Saccharomyces cerevisiae* or *Schizosaccharomyces
pombe* yeasts), where the nuclear envelope does not
disassemble during division. In addition, in cells under-
going open mitosis (e.g., in vertebrates), NPC assem-
bly also occurs in a postmitotic pathway, alongside
with NE reformation around daughter nuclei, by recy-
cling pre-existing NPC components [21] (Fig. 2). In
this review, we will summarize our knowledge of the
mechanisms underlying the different steps of NPC bio-
genesis, as defined above, based on experimental
<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Schematic representation of NPC assembly pathways. (A) Translation-associated mechanisms regulating nucleoporin production and interactions. The role of NTRs in localized translation at NPCs and FG-Nups dynamics within cytoplasmic condensates is represented. Nup subcomplexes formed in a post-translational or co-translational manner can assemble into NPCs either from the cytoplasmic side (1), or from the nucleoplasmic side, following their entry through existing pores (2). (B) Schematic representation of *de novo* NPC assembly: new complexes form through an inside-out mechanism, with the progressive addition of submodules. Transmembrane NE/ER proteins (in gray) may assist in membrane remodeling or fusion events. The putative assembly chronology is shown for the yeast NPC; note that the reported assembly order differs between yeast and vertebrates, especially for nuclear basket modules (see text for details). (C) Schematic representation of postmitotic NPC assembly. Top panel: cell cycle-dependent Nups phosphorylation leads to NPC disassembly into stable modules; Bottom panel: NPC reassembly is seeded by Elys-mediated recruitment of Nups to chromatin, together with the engagement of pre-existing membrane fenestrations (indicated by a star *). Putative assembly intermediates are represented using the same color coding for Nup subcomplexes as in Fig. 1.</figcaption></figure>

evidence obtained from classical model systems—yeasts (y) and vertebrate (v) cells. In particular, we will highlight how recent conceptual and technological

advances have refined our understanding of the spatio-temporal coordination of these processes, drawing parallels with other multiprotein complexes.
# Starting from scratch: coordinating the synthesis of NPC subunits

## Providing the right amount of Nups

As a prerequisite for the formation of multiprotein assembles, the appropriate amounts of individual subunits have to be synthesized from their corresponding transcripts, ultimately reaching their respective stoichiometry and satisfying the overall cellular need for new complexes. While a constant supply of Nups is needed to cope with the assembly of NPCs, whose number gradually doubles during each cell cycle in dividing yeasts [4] or human cells [5,22], the accumulation of *orphan*, that is, nonassembled, subunits can in turn be detrimental for cellular homeostasis. In this line, overexpression of nucleoporins reportedly leads to distinct phenotypic outcomes, likely reflecting their different biochemical properties and interaction partners: (a) NE deformation or aberrant membrane structures have been observed upon accumulation of full-length or truncated membrane-binding Nups such as vNup153 [23], yNup53 [24], yNup1, yNup60 [25], or yNdc1 [26]; (b) cytoplasmic foci trapping a subset of Nups have been detected upon overproduction of a fragment of the scaffold nucleoporin yNup170 [27]; (c) increased dosage of different FG-Nups, for example, vNup62 [28] or yNup1 [29], leads to their accumulation within ectopic aggregates. In these situations, mislocalized subunits have not only the potential to interfere with NPC assembly and NE homeostasis, but could also directly impact nucleocytoplasmic transport by titrating NTRs, as suggested by the trapping of the karyopherin yKap60 within yNup1 foci [30], or the displacement of the mRNA export receptor yMex67 upon overexpression of yNup116 GLFG domain [31].

Systematic and targeted analyses have revealed general mechanisms that can tune the synthesis of multiprotein complex members. *Coordinated transcriptional regulation* can synchronize the production of subunits with cellular metabolism, as demonstrated for proteasome components or ribosomal proteins, whose cognate mRNAs are expressed under the control of dedicated transcription factors [1,32]. In addition, translational controls can lead to *proportional synthesis*, where the subunits of the same complex are made in exact proportion to their stoichiometry, a widespread mechanism in prokaryotes and eukaryotes, as revealed by ribosome footprinting [33,34]. Alternatively, degradation by the ubiquitin-proteasome system can lead to *post-translational buffering*, eliminating the subunits produced in superstoichiometric amounts and failing to assemble into mature complexes [35]. While the synchronized production of multiprotein complex members is believed to generally involve a combination of

coordinated synthesis and post-translational tuning in eukaryotic cells [36], possible mechanisms specifically avoiding the unbalanced production of Nups are now emerging.

## Expression of Nup-encoding mRNAs: matching supply to demand

Several reports support the notion that the expression of Nup-encoding mRNAs is constitutive in dividing cells yet varies during development and upon differentiation, globally correlating with NPC assembly activity. In budding yeast, *NUP* transcripts levels are stable over the cell cycle [37,38] and during replicative aging [39]. In contrast, Nups mRNAs are globally downregulated during *Caenorhabditis elegans* and *Drosophila melanogaster* development, or during cell differentiation in mammals [40,41]. Strikingly, the levels of mRNAs encoding scaffold nucleoporins are more strongly impacted in post-mitotic cells than those encoding peripheral or dynamic NPC components. This differential regulation could reflect the need to “repair” old NPCs by expressing and exchanging the Nups that are dynamically associated with mature complexes [42–45], while the scaffold itself would have to be maintained in the long term without exchanges [40,46,47], thus preserving the nuclear permeability barrier. Alternatively, certain nonscaffold Nups could fulfill tissue- or cell type-specific functions, as already exemplified [48]. In this line, pronounced variations in mRNA expression levels were observed for several peripheral, nonessential Nups in different tissues or cells lines [48–51]. Supporting the notion that transcription itself could be regulated in these different situations, expression of fluorescent reporters driven by either Nup160 or Nup153 promoters recapitulates the differential regulation of these Nups in developing worms [40].

Which *cis*-elements in Nup genes and *trans*-acting transcriptional regulators could thus scale nucleoporin production with cell division and/or ensure tissue-specific expression? In yeast, systematic mapping of transcription factor binding revealed that Nup promoters are bound by General Regulatory Factors (e.g., Abf1, Reb1) [52], whose transcriptional activity depends on nutrient-sensing signaling pathways [53]. Of note, earlier analyses of mammalian Nup promoters [49,54] had identified binding sites for Sp1, an ubiquitous transcriptional activator. Expression of Nup mRNAs may thus be constitutively directed by housekeeping transcriptional activators, and further tuned by specific regulatory elements and factors in different physiological situations. For instance, transcriptional induction of vNup210, whose promoter encompasses binding sites
for myogenin, has been proposed to rely on this muscle-
specific transcription factor during myogenic differentia-
tion [48]. Similarly, the upregulation of vNup98-96
mRNAs observed during viral infection is believed to
involve interferon-stimulated response elements within
the corresponding promoter [55]. Transcriptional con-
trols may thus ensure the flux of Nup mRNAs required
for NPC biogenesis and further contribute to build NPC
functional heterogeneity.

Post-transcriptional regulations of Nup synthesis: a NUP regulon?

Systematic analyses indicate that Nup protein levels
span a narrower range of concentrations than their
respective mRNAs, as generally reported for eukaryotic
multiprotein complexes [2]. Such a coordinated regula-
tion could follow the *post-transcriptional operon* or *regu-
lon* principle, in which functional classes of mRNAs
share *cis*-elements bound by specific RNA-binding pro-
teins (RBPs), synchronizing their fate, including transla-
tion [56]. A systematic search for such factors revealed
that a subset of Nup-encoding mRNAs is bound by
Hek2/Khd1, a hnRNP-K orthologue, which functions
as a translational repressor in budding yeast [29]. Hek2/
Khd1 activity keeps a fraction of its target mRNAs in a
translationally silent state, likely avoiding the excess
accumulation of a subset of FG-Nups and controlling
the expression of yNsp1 [29], a critical player in NPC
inheritance and FG-Nup surveillance [57–59]. Of note,
untranslated pools of Hek2/Khd1 target mRNAs are
still observed in the absence of this RBP. Furthermore,
a substantial fraction of yNUP133 mRNAs, that are not
detectably associated with Hek2, is also translationally
repressed [29]. Additional, yet-to-be identified transla-
tional regulators may thus partake in the control of
Nups synthesis. Interestingly, the translation of the
transmembrane nucleoporin yPom34 was proposed to
be specifically controlled by another hnRNP-K relative,
Scp160, in complex with the translational repressor
Eap1 [60]. Since a subset of Nup mRNAs was also
detected in interaction with hnRNP-K in human cells
[61], such translational regulations may have been con-
served during evolution.

The *regulon* mode of post-transcriptional regulation
represents a powerful mean to collectively adjust the
metabolism of functionally related mRNAs, by modulat-
ing the activity of their cognate RBPs [62]. Notably,
Hek2/Khd1 RNA-binding ability is repressed by
SUMOylation, a post-translational modification (PTM)
relying on the coupling of the Small Ubiquitin-like MOdi-
fier (SUMO) polypeptide to its target proteins [29]. Hek2/
Khd1 SUMOylation is reversed through the activity of

the Ulp1 SUMO-protease, which is itself stabilized
through association with mature NPCs. This PTM could
thus provide a possible feedback regulation of NPC integ-
rity through regulated RBP binding and subsequent Nup
mRNA translation. In the same line, Eap1 mediates
translational repression of the POM34 mRNA in condi-
tions of spindle pole body (SPB) duplication defects, pro-
viding cross-regulations between the biogenesis of these
two macromolecular assemblies in fungi NE [60]. Interest-
ingly, Eap1 was also reported to mediate global transla-
tional repression in response to distinct types of
membrane stress [63], although the underlying molecular
mechanisms have remained unaddressed. Whether Nup
mRNA translation is thereby responsive to changes in
NPC or NE homeostasis remains to be determined.

Beyond RBP-mediated regulations, Nup availability
and stoichiometry can also be adjusted through post-
translational mechanisms. Excess, nonassembled Nups
are subjected to degradation, likely through the
ubiquitin-proteasome system, as revealed by
the increased decay rates of FG-Nups upon derepressed
translation [29], or by the post-translational buffering of
outer ring Nups amounts in aneuploid yeasts [64].
Another mechanism is represented by the post-
translational, autoproteolytic cleavage of yNup145
(vNup98-Nup96) polypeptide [45,65,66], which gener-
ates equimolar amounts of the yNup145-N (vNup98)
and yNup145-C (vNup96) moieties. Numerous other
PTMs have been reported to target nucleoporins and
modulate their availability for NPC assembly processes.
Upon mitotic exit, dephosphorylation of several Nups
occurs concomitantly with pore reassembly, reversing
the multisite phosphorylation-induced dismantling of
NPCs occurring prior to mitosis [20,67-69]. Whether the
timing of dephosphorylation of distinct Nups further
defines their availability during mitotic exit remains
however to be explored. Similarly, phosphorylation
events specifically regulate yNup60 attachment to the
NPC during meiosis [70] or vNup153 binding to the
outer ring complex [71]. Other reported Nups PTMs, for
example, glycosylation [72] or ubiquitylation [73,74]
could also control their availability and interactions, a
matter that will require further investigation.

Building the blocks: assembling NPC
modules away from the nuclear
envelope

Channeling individual subunits toward assembly
routes

It is generally admitted than once nucleoporins are syn-
thesized, they associate into individual submodules
**Box 2.** A toolbox to investigate NPC assembly.

**Defining the chronology of NPC assembly** – The successive stages of postmitotic assembly can be characterized owing to their synchrony *in vivo*, for example, in mammalian cell lines [82,136,137], or *in vitro*, in *Xenopus* egg extracts, in which a NPC-containing nuclear envelope assembles around exogenous DNA in a process resembling nuclear reformation at the exit of mitosis [236]. The same model systems can also be used to analyze *de novo* NPC formation, which initiates shortly after mitosis at core NE regions, that is, at the vicinity of the spindle attachment site in human cell lines [85], or within nuclei assembled in *Xenopus* extracts [135,147,194]. In both experimental systems, a panel of electron microscopy approaches, from TEM (transmission EM)/FESEM (field emission scanning EM) [133,134] to high-resolution three-dimensional electron tomography [151,159], can depict and stage NPC assembly intermediates based on their structural morphology in fixed samples. In parallel, live imaging assays can determine the NE recruitment of specific Nups tagged with fluorescent proteins (FP) [135–137]. These approaches have gained into precision and accuracy with the integrative application of CRISPR-Cas9-based tagging techniques, reducing the biases linked to changes in Nups expression, high-resolution microscopy, thus tracking individual NPCs, and computational modeling, providing a quantitative assessment of Nup assembly. As an alternative to imaging approaches, which do not simultaneously monitor all Nups, metabolic labeling followed by affinity purification of assembly intermediates can provide an exhaustive depiction of the kinetics of subunit incorporation into mature NPCs, as recently achieved in yeast [86].

**Identifying the factors required for NPC biogenesis** – Systematic genetic screens assessing the localization of GFP-tagged Nups or nucleo-cytoplasmic transport have revealed instances of impaired NPC assembly in yeast mutants [204,237]. The requirements for Nups or other factors in distinct NPC assembly processes can be further defined using the abovementioned assays in yeast mutants [80,105,145], upon siRNA treatment, gene knock-out or auxin-induced depletion in mammalian cells [140,142,238], or following immunodepletion or pharmacological treatments in *Xenopus* extracts [135,143,239]. The search for interaction partners of NPCs or assembly intermediates may also identify exogenous factors required for NPC biogenesis [208].

**Characterizing NPC assembly intermediates** – Genetic perturbation of NPC biogenesis can allow to capture halted assembly intermediates, as characterized by EM approaches [105,145,152]. Newly assembling NPCs can also be distinguished using photoactivatable FPs (e.g., Dendra [80]) or Recombination-Induced Tag Exchange (RITE [148]), which results in the differential labeling of “old” and newly synthetised proteins by switching the tag on the Nup of interest *via* the transient induction of a site-specific recombinase. Assembly intermediates or Nup oligomers can be further characterized through affinity purification approaches, following pulse-chase epitope or metabolic labeling [86,87]. Finally, co-translational assembly events are detected by affinity purification of Nups followed by the analysis of the co-enriched mRNAs (RNA Immunoprecitation, RIP [30]) and ribosome-protected mRNA footprints (Selective Ribosome Profiling, SeRP [102]), allowing to identify the engaged nascent protein and the onset of subunit association with respect to the position of the translating machinery.

prior to their further incorporation at the NE (Fig. 2A). Supporting this view, stable Nup subcomplexes, for example, the yNup84 complex, the major component or the NPC outer ring, can be reconstituted *in vitro* from recombinant proteins in the absence of membranes [14,75], and several Nup modules can be isolated as stable biochemical entities from yeast or animal cell extracts [66,76–80]. Postmitotic NPC assembly directly takes advantage of such pre-existing building blocks, released during NE breakdown, as supported by the persisting association of nucleoporins within subcomplexes in mitotic extracts [68,81–84] and by the

quantitative analysis of Nups recruitment from the cytoplasm to the NE upon mitotic exit [85]. Interphasic assembly similarly utilizes pre-assembled modules, as shown by the determination of Nup incorporation rates into subcomplexes or mature NPCs following pulse-chase labeling in yeast (Box 2) [86,87].

Yet, the assembly of individual subunits into heterooligomeric complexes is expected to face several general constraints. For interactions to take place, polypeptides have to find each other in the crowded cellular milieu, a theoretically inefficient process [88]. However, oligomer formation must be kinetically
efficient, that is, rapid enough to outcompete the deg-
radation of non-assembled subunits, as initially shown
for ribosomal proteins [89], and hierarchically ordered,
as revealed by the existence of preferred assembly
sequences under evolutionary selection [90]. The bio-
genesis of NPC submodules is further complexified by
the prevalence of ID domains in nucleoporins (Box 1),
possibly prone to rapid degradation if not assembled,
and the existence of multiple competing partners for
several Nups, as exemplified by yNsp1 (vNup62),
which establishes coiled-coil-mediated interactions with
either yNup82 (vNup88) within cytoplasmic filaments
or yNup49-yNup57 (vNup58-vNup54) within the cen-
tral transport channel [91] (Fig. 1). Some nucleoporins
also belong to distinct cellular complexes, that is, the
COPII coatomer complex for Sec13 [77], the SEA/
GATOR complex for Sec13 and Seh1 [92] or the SPB
for yNdc1 [93], a possibly incomplete picture in view
of the numerous off-pore functions of nucleoporins,
likely involving additional partners [81,94–97]. Mecha-
nisms governing the association of individual Nups
within subcomplexes, and thus possibly solving this
jigsaw, have recently been brought to light.

When NPC assembly begins during translation

Co-translational assembly has recently emerged as a
widely used mechanism to couple the synthesis of mul-
tiprotein complex subunits with their association into
oligomeric intermediates, as notably revealed through
systematic studies [98–100]. On the one hand, a fully
folded subunit can interact with its nascent protein
partner during the translation of the latter, a process
referred to as *sequential* or *co-posttranslational assem-
bly*. Alternatively, two nascent polypeptides can
interact together when their respective translating ribo-
somes are brought to vicinity, leading to *simultaneous*
or *co-cotranslational assembly* [3,101]. Exploring these
possibilities, systematic investigation of co-
translational interactions between yeast nucleoporins
and Nup-encoding mRNAs *via* RNA immunoprecipi-
tation approaches (Box 2) recently uncovered a dozen
of distinct *sequential* assembly events, within or among
distinct NPC modules [30,102] (Fig. 2A). Co-
translational assembly was detected for nucleoporin
pairs within virtually all NPC subcomplexes, that is,
the inner ring complex (yNup53 → yNup170), the
outer ring complex (ySeh1 → yNup85), the central
transport Nup trimer (yNsp1 → yNup57), the cyto-
plasmic filaments (yGle2 → yNup116; yNup116 →
yNup82; yNup159 → yNup82) and the nuclear basket
(yNup60 → yNup2). In addition, linker Nups,
which connect sub-modules in the final NPC

structure [12,103–108], were also found to associate in
a *co-posttranslational* manner with their partners, as
exemplified by the binary interactions involving
yNup145N, yNup100 and yNic96, whose nascent poly-
peptides are bound by yNup157, yNup192 and the
yNsp1-yNup49-yNup57 trimer, respectively [30,102].
Co-translational events thus support the formation of
“velcroable” modules, that is, pre-assembled heterote-
tramers that join the NPC scaffold to form cytoplas-
mic filaments or the central channel, defining as such
the hierarchy of the assembly process. In the same line,
the early recruitment of yNup157 onto *NUP145*
mRNA may spatially co-localize components of the
inner ring (yNup157) and outer ring (yNup145C) with
the yNup145N linker, favoring the gathering of differ-
ent NPC sub-modules. Of note, the only partial over-
lap between the co-translational interactions detected
in these studies suggests that their occurrence and/or
detection may depend on experimental and physiologi-
cal conditions, necessitating additional systematic
investigations to unravel their prevalence in NPC
assembly. In addition, in view of the conservation of
co-translational processes, as shown for proteasome
assembly [109], it will be insightful to investigate
whether such events also contribute to NPC biogenesis
in other species, notably in reported cases of species-
specific structural variations [15,17,107].

What would be the functional impact of co-
translational assembly on the efficiency and accuracy
of NPC biogenesis? For most of the co-translational
events described above, the protein–protein interfaces
that were found to form during translation are experi-
mentally proven to be critical for NPC integrity or cell
fitness [106,108,110]. However, this is not sufficient to
conclude about the importance of co-translational
assembly since experimental manipulation of protein–
protein interaction domains similarly impacts post-
translational assembly. Determining whether polypep-
tide composition, codon usage or ribosome-nascent
chain complexes binding partners [3,111] impact
translation elongation kinetics and possibly promote
co-translational assembly could instead provide experi-
mental tools to specifically interfere with this process.
Nevertheless, the previously discussed benefits of co-
translational assembly [112–114] could also apply to
the NPC (Fig. 3). First, all reported Nup co-
translational interactions are nonreciprocal (*sequential
assembly*) and involve protein domains which in most
cases map at the N ter of the recognized nascent poly-
peptides and at the C ter of the engaged, fully folded
partners (Fig. 3A). In line with similar observations
for other complexes [115–117], this genetically encoded
arrangement of interaction domains within primary
<figure><img src="image_7.png" /><figcaption>(A) ordered assembly</figcaption></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

Gle2-Nup116-Nup82

<figure><img src="image_10.png" /><figcaption>(B) protection from degradation / aggregation</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>(C) co-translational folding</figcaption></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /><figcaption>(D) choice between competing pathways</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>(E) localized translation at NPCs</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>**Fig. 3.** Possible outcomes of co-translational assembly. (A) Ordered assembly for oligomers such as Gle2-Nup116-Nup82 [30,102]; (B) Protection from degradation or aggregation for ID proteins, as suggested for FG-Nups [30]; (C) Co-translational folding, as proposed for the Seh1-Nup85 interaction [102]; (D) Prioritization of competing assembly pathways [102]. (E) Localized translation at NPCs upon co-translational recruitment of NTRs onto nascent NLS in Nups [30,241].</figcaption></figure>

sequences would thus impose an ordered assembly through co-translational events. In addition, the NPC co-translational interaction map shows a high representation of Nups harboring ID or SLIM domains (Box 1) [30]. Their association with binding partners prior to translation termination could counteract their

premature degradation, aggregation or engagement in nonproductive interactions, as previously reported for other complexes [99,109] (Fig. 3B). Furthermore, co-translational associations could assist in the folding of nascent polypeptides, as described [118] and exemplified by the recruitment of ySeh1 onto nascent yNup85,
possibly locking the conformation of a flexible domain in the latter [102] (Fig. 3C). Finally, co-translational assembly seems to occur for only some of the pathways that could engage Nups belonging to distinct complexes. For instance, ySeh1 interacts with nascent yNup85, but associates post-translationally with its partner ySea4 in the SEA complex [102] (Fig. 3D). Of note, the lack of detection of co-translational interactions may not be sufficient to infer their absence, notably in light of the high sensitivity of subcomplex preservation to lysis and purification conditions [79,92]. However, it is tempting to speculate that - translation-dependent regulatory mechanisms could thus differentially prioritize competing assembly pathways.

## Cytoplasmic condensates: places where NPC biogenesis gets started?

Even if co-translational assembly could chaperone Nups or facilitate their hierarchical assembly, it is not expected to favor *per se* their encounter within the cytoplasm. Although some Nups exhibit self-assembling properties when simultaneously overexpressed in bacteria [75], their association at physiological concentrations may require them to meet at a restricted subcellular location. In this line, asymmetrical mRNA localization and site-specific translation have recently been suspected to facilitate multiprotein complex biogenesis, based on the observation of translationally active granules, in some cases encompassing functionally related mRNAs, from yeast to human cells [3,119–121]. Such membrane-less compartments are proposed to form upon phase separation and could enhance oligomer assembly by (a) multiplexing mRNAs encoding subunits of a same complex prior to translation, as put forward in the *transperon* model [122], (b) promoting the recruitment of fully folded proteins at the vicinity of their nascent partners by virtue of *matchmakers* RBPs [123,124], or (c) protecting nascent chains and paused ribosomes from quality control mechanisms, as in *assemblysomes* [109]. While Nup-encoding mRNAs are generally not translated at the NE, with a few exceptions (see below, [30,125]), distinct nucleoporin foci have been observed in various model systems [57–59,125,126].

An example of Nup-containing cytoplasmic granules is provided by ER-bound yNsp1 foci, which are specifically detected in the daughter cells of budding yeasts [57]. These structures contain a cytoplasmic pool of newly synthesized Nups, that is, not previously assembled at existing pores (as assessed by RITE, Box 2), including yNsp1 and its direct interactors from both the central transport channel (yNup49, yNup57) and the

cytoplasmic filaments (yNup82), together with the NTR yKap95 [57,59]. Their phase separation is driven by the FG domain of yNsp1, which is sufficient to rescue NPC stoichiometry and transport defects arising upon yNsp1 depletion *in vivo*, and can modulate FG-Nups phase state and solubility *in vitro* [59], akin to vKap95/important-β [127]. These granules could thus maintain isolated FG-Nups in an assembly competent state, preventing promiscuous interactions prior to their incorporation into NPCs. Strikingly, these condensates enrich pairs of Nups that assemble co-translationally (i.e., yNsp1-yNup57 and yNup116-yNup82 [30,102]), and are targeted to the cell bud *via* the actomyosin cytoskeleton [57], as shown for yeast translation factories [119], suggesting that they could couple Nup synthesis, assembly and transport. Similarly, cytoplasmic condensates encompassing several distinct FG and scaffold Nups were also observed in human cells deficient for fragile X-related (FXR) proteins, which are RBPs involved in translational processes [126], or in worm embryos [128].

Nups condensates were actually demonstrated to fuel NPC assembly in the particular context of the *D. melanogaster* oocyte. In this species, pore assembly is achieved during the rapid early embryonic cell cycles through the *en bloc* insertion of pre-existing NPC scaffolds embedded in ER membranes stacks and referred to as *annulate lamellae* (AL) [129]. The prior generation of maternal stockpiles of AL-NPCs in turn involves compositionally heterogenous nucleoporin condensates: precursor Nup-containing granules form in the oocyte, while large Nup358 assemblies are generated in the cytoplasm of adjacent nurse cells [125]. Strikingly, a subset of Nup mRNAs were found to associate to these granules in a translation-dependent manner, suggesting that they could couple Nup synthesis and assembly. Microtubule-dependent transport would then foster granule interactions, nucleating the formation of AL-NPCs [125]. Although it is still unclear whether this mechanism can be generalized, notably in view of the frequent detection of AL in germ and cancer cells [130,131] or upon Nup altered dosage [46,132], the oocyte granules share several features with some of the Nup foci mentioned above: (a) their liquid–liquid phase separation in the cytoplasm, guided by FG-Nups ID domains; (b) their dynamic interplay with the nuclear transport machinery, likely controlling Nup interactions; and (c) their transport through the cytoskeleton toward sites of NPC biogenesis. Whether such granules generally represent genuine assembly intermediates targeted to sites of interphasic NPC assembly (Fig. 2A), and/or aberrant condensates formed upon accumulation of cytosolic precursors, remains however an opened question (discussed in Ref. [128]).
# Assembling NPCs at the nuclear envelope: right time, right place

## Multiple choreographies for a same finale

Fully assembled NPCs are specifically located at pores piercing the double lipid bilayer of the NE, a unique situation in living systems, necessarily requiring dedicated targeting and membrane remodeling events. A large panel of experimental strategies (Box 2) has unveiled the mechanisms that thereby underlie the interphasic, *de novo* and postmitotic NPC assembly pathways, in yeast and vertebrate cells. On the one hand, the chronology of NPC assembly has been determined through either imaging approaches, tracking the recruitment of fluorescently tagged Nups and detecting ultrastructural assembly intermediates *via* electron microscopy [82,85,133–137], or labeling strategies, coupled to affinity purification of subcomplexes [86,87]. On the other hand, loss-of-function experiments have analyzed the requirement of distinct Nups for NPC formation *in vivo*, mainly in yeasts, nematodes or mammalian cells, or *in vitro*, using *Xenopus* egg extracts (see for example [80,135,138–147]). While these strategies have generally revealed a good agreement between the stage of recruitment of Nups and their requirement for NPC assembly in given experimental systems, they uncovered marked differences between NPC biogenesis pathways.

Interphase assembly had been initially hypothesized to involve the splitting of existing pores, as subsequently observed for other macromolecular complexes or organelles [148], and in line with the detection of possible intermediates exceeding the eightfold symmetry [149], or the fusion of Nup-containing vesicles within intact nuclear envelopes [150]. It is now generally accepted that during interphase, NPCs form instead *de novo*, without recycling pre-existing NPC material [135], and that their assembly requires discrete fusion events between the INM and the ONM to create the pore conduits [151]. The “inside-out” assembly process sequentially involves the recruitment of NPC building blocks at the inner face of the NE, the evaporation of the INM, and its fusion with the ONM, culminating with the final maturation of the complex (Fig. 2B). This scenario is supported by the temporal staging of assembly intermediates visualized through a panel of microscopy approaches in mammalian cells [85,151], and the detection of NE evaginations or NPC-like structures at the INM in a subset of yeast nucleoporin mutants [105,145,152]. While the overall process is similarly slow in both yeast and mammals (~1 h to completion), possibly rate-limited by the

membrane fusion stage, the order of Nup recruitment seems to differ between these model systems. In vertebrates, interphase NPC assembly initiates with the recruitment of transmembrane nucleoporins (i.e., vPom121) at the INM, followed by nuclear basket components (vNup153; vTpr), prior to outer ring, then inner ring Nups [85,147,153]. In contrast, in yeast cells, transmembrane and inner ring Nups are recruited at an early stage of pore assembly, and the nuclear basket components, especially vTpr orthologues yMlp1-2, assemble much later [86]. Although these different chronologies were reported through distinct approaches, i.e., microscopy [85] vs. metabolic labeling [86], they are generally consistent with nucleoporin requirements for interphase assembly in the two models (for example, vPom121, vNup153 and outer ring Nups in vertebrates [135,146,147,153], or inner ring Nups in yeast [80,138,145,154]). Of note, how the NE diffusion barrier is maintained during membrane fusion events in these different *de novo* choreographies still remains unclear.

Postmitotic NPC assembly shows distinct molecular requirements and kinetic features, since it occurs rather synchronously, alongside with NE formation upon mitotic exit, and in a faster manner (few minutes), as compared to the interphase process. These specificities likely stem from the dephosphorylation and immediate availability of the required Nup subcomplexes, inherited from previous prophase NPC disassembly events [68,81,82,84] (Fig. 2C, top panel), possibly in the form of higher order octameric assemblies bound to ER membranes [155]. It has been debated whether their assembly occurs concomitantly with the membrane fusion events by which ER-derived sheets, tubules or vesicles expand and/or stack over the daughter nuclei, sealing novel nuclear envelopes, or follows their formation, thereby requiring INM-ONM fusion as for interphasic assembly [19,20,156]. However, both postmitotic and *de novo* pathways are simultaneously at play shortly after mitosis in mammalian cells, where they spatially segregate in distinct NE regions surrounding the daughter nuclei [85,151], possibly accounting for the observation of fully sealed nuclear envelopes on which NPC further assemble [157]. Both pathways also occur successively upon nuclear reconstitution in *Xenopus* extracts [135], where putative membrane fusion intermediates were detected ([158] and references therein). Recent studies rather indicate that the postmitotic pathway mostly occurs within pre-existing membrane fenestration, which recruit precursors assembled at the surface of decondensing chromosomes and further dilate upon NPC maturation [85,159] (Fig. 2C, bottom panel). On the chromatin
side, assembly is initiated through DNA and/or nucle-
osome recognition by the nucleoporin Elys, together
with the recruitment of outer ring Nups [132,160–163].
At this stage, NPC formation likely involves further
interactions between these NPC seeds at the chromatin
surface and holes within the reforming NE, possibly
associated with transmembrane [164,165] and/or other
scaffold Nups, for example, inner ring components
[155]. Along with hole dilation, FG-Nups association
concomitantly restores transport competence, while
peripheral components join later, building the nuclear
baskets and cytoplasmic filaments [85,136]. In agree-
ment with this choreography, postmitotic assembly
requires Elys [132,146,162,166], outer ring components
[141,142,166,167] and to some extent, inner ring/trans-
membrane Nups [168–172].

Nuclear pore complex assembly at the NE can thus
follow distinct assembly routes, that is, the ones
described above, or those utilizing *annulate lamellae*,
observed in *Drosophila* embryos [129] and possibly
other cell types [173]. The existence of different path-
ways raises the question of whether they further vary
according to the physiological status of the cell, or
across the phylogenetic tree, in line with species-
specific flavors in the structure of NPCs or the mode
of nuclear division, that is, closed, open and beyond
(discussed in [20]). Supporting these notions, NPC
assembly has been reported to progressively complex-
ify during early embryonic cell cycles in Zebrafish,
enabling compositional and morphological changes
associated to the timely acquisition of full transport
competence prior to zygotic gene activation [166]. In
another report, assembly of the nuclear basket has
been shown to depend on the transcriptional and
mRNA processing activities of yeast cells [174]. Multi-
ple developmental and regulatory signals are thus
likely to impinge on NPC assembly routes.

Spatially restricting NPC assembly at the nuclear envelope

While the various biogenesis pathways described above
exhibit distinct requirements for specific nucleoporins
or Nup subcomplexes, they all rely on the Ran-GTP
gradient as a spatial cue, specifying NPC assembly
sites at the INM during interphase or at the chromatin
surface upon mitotic exit. The critical role of Ran and
NTRs for NPC formation was simultaneously discov-
ered through genetic screens in budding yeast [150]
and biochemical manipulations in *Xenopus* extracts
[143,175,176]. The proposed mechanism relies on
inhibitory interactions between nucleoporins and
NTRs that are released by Ran-GTP, which is

enriched in the nucleus or at the vicinity of mitotic
chromosomes through the localized action of the Ran
guanine nucleotide exchange factor RCC1. During
interphase, NTR-mediated transport and Ran-GTP-
dependent dissociation thus ensure the delivery in the
nucleus or at NPCs of several NLS-containing nucleo-
porins, including the inner ring component yNup53
[177,178], the transmembrane Nups vPom121 [153,179]
and yPom33 [180], as well as the nuclear basket sub-
units vNup153 [147], yNup1, yNup60 [25] and yNup2
[42,181]. Similarly, during postmitotic assembly, Ran-
GTP mediates the localized dissociation of NTRs
bound to various nucleoporins, including FG-Nups
[143,182], vElys and outer ring components [143,182–
184]. Mechanistically, Ran-GTP-mediated dissociation
can promote NPC assembly by exposing specific Nup
interactions domains, fostering their association with
(a) chromatin, as observed for Elys and outer ring
Nups seeding at sites of postmitotic assembly
[182,183], (b) the INM, as exemplified by vNup153
whose membrane-interacting helix is occluded by
transportin binding [147], or (c) specific interacting
partners within the NPC, as shown for yNup53 whose
NLS overlaps yNup170-binding motif [178]. NTRs
also contribute to the localized translation of NUP1
and NUP2 mRNAs at NPCs, through the recognition
of their interaction domains in nascent yNup1 and
yNup2 polypeptides (Fig. 3E) [30]. Of note, several
Nups do not reportedly interact with NTRs, raising
the question of the mechanism ensuring their localized
delivery at NPCs, at the INM or at the chromatin
vicinity. Remarkably, large scaffold Nups, i.e.,
Nup188 or Nup192, exhibit structural similarities with
NTRs, suggesting that they could get autonomously
imported through NPCs [12,185].

Although the Ran-GTP gradient and NTRs thus
appear pivotal to specify the localization of NPC
assembly events with respect to the genetic material, it
is unclear whether additional regulatory mechanisms
define their spacing and distribution at the surface of
the chromatin, or within the NE. Furthermore, while
NPC formation at mitotic exit seems to be synchro-
nized by cell cycle regulators, notably controlling Nup
phosphorylation levels, the temporal cues defining the
rates of interphasic assembly, which are notably higher
in the G1 phase of the cell cycle [85], are poorly
understood.

NPC biogenesis: autonomous or assisted?

Nuclear pore complexes have long been considered as
capable of self-assembly without the need for cofac-
tors, with the notable exception of the Ran pathway.
This is in striking contrast with several other multiprotein machineries, which require a number of exogenous, specific assembly factors, scaling with their compositional complexity: for instance, the assembly of the 33 proteasome subunits requires a dozen assembly factors [186] while the formation of the ribosome, which encompasses 80 ribosomal proteins and four rRNAs, requires ~200 cofactors and ~80 snoRNAs [187]. Biogenesis of the membrane-embedded mitochondrial oxidative phosphorylation system, a higher order assembly of multiprotein complexes gathering ~90 distinct subunits, requires almost as many cofactors [188]. Such activities are known to chaperone individual subunits, stabilize oligomeric intermediates, promote covalent modifications, compartmentalize assembly events or, in the last example, facilitate insertion within membranes. In this respect, an increasing number of players have been assigned a function in the complex membrane remodeling events associated with NPC biogenesis, or in ID Nups chaperoning.

While NPC insertion within the NE relies on a limited set of integral membrane Nups (Box 1), which diversely contribute to Nups recruitment and NPC assembly *in vivo* [146,164,171,189–192], other factors have been proposed to induce or stabilize the high level of local membrane curvature associated with pore conduits. Members of the reticulon homology domain (RHD) family, which encompasses membrane-bending proteins involved in shaping the curvature of ER cisternae and tubules [193], were found to localize to the INM or at the vicinity of NPCs, from yeasts to vertebrates [194–196]. Loss-of-function experiments further revealed that distinct RHD proteins contribute to interphase and postmitotic assembly [194,196,197], their recruitment possibly involving direct interactions with nucleoporins [189,196]. Strikingly, several yeast and vertebrate Nups [24,25,146,147,180,198,199] also exhibit membrane-binding domains such as amphipathic helixes (AH, Box 1), which are in some cases critical for interphase NPC assembly [146,147,199]. According to proposed mechanisms, Nups AH, e.g., Nup133 ALPS [146], could facilitate protein targeting to NPCs, since some of these helixes preferentially bind to membranes with a high curvature degree, similar to the pore situation [147,180,198]. Such motifs could further partake in the recruitment of soluble Nups partners, as exemplified by vNup153, whose AH-mediated interaction with the INM contributes to outer ring association [147], or promote the binding to other membrane-tethered Nups, since the reduction of the dimensions explored by membrane-bound proteins generally favors their encounter [200]. Finally, some of these domains could contribute to stabilize or

induce membrane curvature at sites of NPC biogenesis, akin to reticulons, as supported by their *in vitro* membrane deformation activity [25,199]. Lack of stabilization of the pore membrane has also been proposed to account for the typical NPC clustering phenotype observed *in vivo* in outer ring mutants ([201] and references therein). Of note, Nups alone are unlikely to be sufficient to induce INM-ONM fusion, an event possibly involving distinct NE/ER or INM transmembrane proteins reportedly contributing to interphase NPC biogenesis. Among them, the yeast LEM protein Heh1 and the human LINC complex member Sun1 were proposed to form lumenal bridges with transmembrane Nups, possibly enabling INM-ONM juxtaposition [202,203]. Another group of fungi-specific transmembrane NE/ER proteins, namely Brl1-Brr6-Apq12, was instead shown to associate with early NPC intermediates, prior to the recruitment of cytoplasmic modules, and to partake in fusion events through lumen-exposed AHs, whose perturbation triggers assembly defects and INM-associated evaginations [204–209].

In human, inactivation of the NE-associated torsin AAA+ ATPase similarly halts interphase NPC biogenesis, leading to INM herniations [210]. Strikingly, these NE blebs are enriched for several chaperones of the HSP70-HSP40 network, including the DNAJB6 cochaperone [211,212]. DNAJB6 also associates with the NE during normal interphase growth, interacts with FG-Nups *in vivo* and prevents their aggregation *in vitro*, likely contributing to NPC interphase assembly by surveilling these ID proteins at the NE. While in yeast ID Nups chaperoning may similarly require HSPs, as supported by the detection of ySsb/HSP70 binding onto nascent FG domains in systematic analyses [213], it is striking that it also involves the NPC component yNsp1 and the NTR yKap95 [59], in line with other reported chaperone activities of karyopherins [214,215]. Overall, the various membrane remodeling and ID chaperoning events associated with NPC formation thus involves both Nups/NTRs themselves and sub-stoichiometrically engaged exogenous cofactors.

## Concluding remarks

The increasingly precise description of the NPC assembly chronology, fueled by technological breakthroughs, has shed new light on our understanding of the formation of these elaborate complexes. The existence of distinct pathways, as observed in distant species [85,86], or within a same cell [85] or organism [125,129], echoes the recently-unveiled structural heterogeneity of NPCs [12,15,17,107] and suggests that further investigation
may reveal alternative routes to their assembly. The reported compositional plasticity of NPCs, which can accommodate the loss of several scaffold Nups, from yeast to mammals [216,217], further suggests that these pathways are robust and tolerant to variations. However, faulty assembly can occur, requiring removal of dead-end intermediates through quality control processes, and possibly underlying a number of human pathologies associated with Nups deregulations (discussed in accompanying reviews of this series).

It is perhaps not surprising that the Nup domains that are critical to interconnect NPC modules in the mature assembly, as revealed in the more recent structural maps, are also pivotal at different stages of the biogenesis process, as exemplified by the engagement of linker Nup domains in co-translational interactions [30,102] and their targetability by mitotic phosphorylation/dephosphorylation events [67]. Similarly, the FG-Nups appear to be under tight control at every step of their metabolism, from their expression [29] to their co-translational assembly [30,102], subcellular localization in condensates [59,125,126] and chaperone-mediated surveillance [211,212].

Possibly more striking is the fact that NPC components, together with the nuclear transport machinery, carry virtually all the activities required to support the assembly process, from membrane remodeling to ID chaperoning. In this line, although the list of exogenous assembly factors keeps expanding, NPC biogenesis appears as a rather autonomous process in comparison with other multiprotein complexes. Whether these properties contributed to its evolutionary trajectory represents an exciting question for the future.

## Acknowledgments

We are very grateful to Anna Babour, Vincent Pacini and Lionel Pintard for critical reading of the manuscript. We apologize to colleagues whose work could not be directly cited because of space limitation. Work in B.P. lab is supported by Agence Nationale pour la Recherche (ANR-18-CE12-0003, ANR-21-CE12-0029, ANR-21-CE12-0040), Fondation ARC pour la recherche contre le Cancer (projet ARC), Ligue Nationale contre le Cancer (comité de Paris) and the Université Paris Cité IdEx (ANR-18-IDEX-0001). A.P. was supported by fellowships from the BioSPC PhD program (Université Paris Cité), the Fondation pour la Recherche Médicale (grant number FDT202 106013057) and the EUR G.E.N.E. (ANR-17-EURE-0013).

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