Changing the guard—nuclear pore complex quality control

The integrity of the nuclear envelope depends on the function of nuclear pore complexes (NPCs), transport channels that control macromolecular traffic between the nucleus and cytosol. The central importance of NPCs suggests the existence of quality control (QC) mechanisms that oversee their assembly and function. In this perspective, we emphasize the challenges associated with NPC assembly and the need for QC mechanisms that operate at various stages of an NPC's life. This includes cytosolic preassembly QC that helps enforce key nucleoporin–nucleoporin interactions and their ultimate stoichiometry in the NPC in addition to mechanisms that monitor aberrant fusion of the inner and outer nuclear membranes. Furthermore, we discuss whether and how these QC mechanisms may operate to sense faulty mature NPCs to facilitate their repair or removal. The so far uncovered mechanisms for NPC QC provide fertile ground for future research that not only benefits a better understanding of the vital role that NPCs play in cellular physiology but also how loss of NPC function and/or these QC mechanisms might be an input to aging and disease.

Maintaining the integrity of the nuclear envelope (NE) is of key importance to the proper functioning of eukaryotic cells as it ensures nucleocytoplasmic compartmentalization. Ultimately, the integrity of the NE depends on the impermeability of its double membrane and the selective transport properties of its nuclear pore complexes (NPCs) -massive 50-100 MD conduits that facilitate macromolecular trafficking between the nucleus and cytosol.Each NPC is constructed from ~500 to 1000 individual proteins called nucleoporins (nups) that form a wellconserved eightfold radially symmetric architecture [1].Recent advances in elucidating the NPC structure have culminated in nanometer-resolution maps that illuminate the position and connectivity of most nups within the higher order assembly [2][3][4][5][6][7].These maps underscore how the overall complexity of the NPC architecture-that is, the vast number of nups and their interconnectivityarises from simplicity in its repetitive structural organization: individual nups, with a limited number of structural folds assemble into subcomplexes, and these subcomplexes form repeated modules that build concentric cytoplasmic, inner, and nuclear rings.These ring assemblies are tied together by flexible connectors that also provide flexibility to the overall structure to accommodate dilation of the NPC as observed under certain environmental and transport conditions [2, 8,9].The rings also provide anchor Abbreviations AH, amphipathic helix; ALPS, ArfGAP1 lipid packing sensor; ESCRT, endosomal sorting complexes required for transport; INM, inner nuclear membrane; NE, nuclear envelope; NPC, nuclear pore complex; NTR, nuclear transport receptor; nup, nucleoporin; NVJ, nuclear-vacuolar junction; ONM, outer nuclear membrane; PMN, piecemeal microautophagy of the nucleus; QC, quality control.
points for the cytoplasmic filaments, the nuclear basket, and the intrinsically disordered phenylalanine-glycine (FG) repeat containing nups that line the central channel.These FG-nups, together with nuclear transport receptors (NTRs), constitute the permeability barrier that facilitates selective transport between the nucleus and cytoplasm [10].Although there are high-resolution structures of the NPC scaffold ring assemblies, resolving the organization of the FG-network is challenging because it is thought to be highly dynamic-several models of the FG-network have been proposed [11][12][13][14].Although the overall structure of the NPC is conserved, the precise stoichiometry and composition of nups within NPCs differs between organisms and, in some cases, cell types [10].In addition, recent evidence suggests that multiple NPCs with distinct biochemical compositions coexist in the same nucleus [10].Although we have made great strides toward understanding the design principles of NPCs, we understand considerably less about the mechanisms that ensure that the NPC is properly assembled and, once built, how cells monitor and maintain their function.
To consider NPC quality control (QC) mechanisms, it is worthwhile to discuss the inherent challenges that cells face every time a new NPC is built.Depending on the cell type, hundreds to thousands of NPCs are assembled de novo during the interphase of actively dividing cells.Throughout each NPC biogenesis event, hundreds of proteins are brought together at a single spot on the NE, from both the nuclear and the cytoplasmic sides, where they are incorporated into stoichiometries that must be tightly controlled; the mechanisms by which this is achieved are just coming to light (reviewed in Ref. [15]).As about a third of the nups are intrinsically disordered proteins and are prone to aberrant aggregation, they likely require specific surveillance mechanisms to keep them soluble [16][17][18].Additionally, these QC and assembly events are cooccurring with membrane remodeling and local lipid metabolism that contribute to inner nuclear membrane (INM) evagination and, ultimately, the fusion of the outer nuclear membrane (ONM) and INM to form a pore.Thus, in addition to the challenges commonly associated with assembling a multiprotein complex, NPC biogenesis must occur with safeguards that prevent inappropriate perforation of the NE, which would risk a breach of the permeability barrier.On top of that, once a new NPC is formed, its function needs to be maintained to prevent the loss of NE integrity as cells grow and age.As the NPC is a long-lived structure [19][20][21] with most nups exhibiting slow turnover kinetics [21,22], whether and how NPCs can be repaired and/or replaced when they functionally decline with age and in the context of disease [19, [23][24][25], remains to be fully understood.Here, we explore the current state of understanding of the mechanisms overseeing the quality and function of NPCs, from the synthesis of individual and complexes of nups to the maintenance of mature NPCs.

Cytosolic preassembly quality control: protecting against aberrant interactions
Building a NPC de novo starts with the challenge that nups are synthesized in the cytosol, where aberrant nup-nup interactions must be prevented before they reach the NE.Forming correct nup-nup contacts is not a priori assured, as it is conceivably complicated by multiple factors: (a) There is similarity and redundancy in the nucleoporin structural folds such that multiple nups could compete for the same binding partner [26], (b) several nups are part of multiple subcomplexes [1], and (c) the intrinsically disordered FGnup domains readily form condensates in vitro [27]such behavior may drive the deleterious sequestration of other nups that would deplete them from assemblycompetent nup pools.Thus, it is not difficult to imagine that there is a plethora of possible aberrant nupnup interactions that may form in the cytosol and must be mitigated (Fig. 1, left panel).

Cotranslational assembly of NPC subcomplexes
Ensuring the correct formation of NPC subcomplexes and the connectivity between these subcomplexes requires orchestration.One way this is achieved is by enforcing specific nup-nup partnerships as the nup message is being translated.This cotranslational mechanism is used to ensure key interactions in virtually all major NPC subcomplexes, including nup interactions in the inner ring, central channel, cytosolic filaments, nuclear basket, and the outer ring nups Seh1 and Nup85 [28,29].This cotranslational mechanism also helps to explain how homologous nups like the yeast connector nups Nup116, Nup100 and Nup145 each interact with their binding partners in the cytoplasmic Nup159-Nup82-Nsp1 complex, inner ring, and outer ring complexes, respectively [28].These cotranslational interactions within and between subcomplexes highlight that the modularity of the NPC structure provides an important means to protect the quality of the subcomplexes before their incorporation into the full NPC structure in the remodeling NE.

Guarding the FG-nup phase state
Cotranslational assembly not only facilitates the correct association of nups but also likely promotes their biochemical stability by shielding hydrophobic binding interfaces from the aqueous cytosol.Such a function may be particularly important in the context of the intrinsically disordered FG-nups, which are prone to forming homo-and heterotypic interactions that can lead to biomolecular condensation, but also often to aberrant and potentially pathological aggregates [23,27,[30][31][32].The latter is likely mitigated by the cotranslational binding of intrinsically disordered FGnup peptide chains by NTRs [28,33].Indeed, the yeast NTR Kap95 colocalizes with endogenous FG-nup condensates [18] and also with cytosolic nup foci in NPC assembly mutants, suggesting that it engages with the FG-nups outside the NPC [34].Recent work in mammalian cells has shown that chaperones may also fulfill a similar surveillance role as the molecular chaperone DNAJB6 specifically interacts with FG-nups in the cytosol in a way that prevents their transition into aggregates and favors their productive assembly into new NPCs [16,17].Interestingly, the ability to toggle the phase-separation and/or aggregation behavior of FG-nups may also be an inherent property of the FG-nups themselves.Specifically, biochemical assays show that Nsp1 is a modulator of FG-nup condensation, promoting a liquid-like state of FG-nup condensates and preventing aggregation [18].This activity may relate to the observation that newly synthesized Nsp1 and its binding partners appear in cytosolic condensates tied to the maintenance of a functional set of NPCs [18,35,36].It has thus been proposed that the formation of a condensate of newly synthesized FG-nups is a mechanism to biochemically stabilize the intrinsically disordered FG-nups in the time frame prior to assembly in the NPC, and, more speculatively, it may even serve as a local translation platform, in which the proper nup-nup contacts are guaranteed [18].

Regulating nup-membrane interactions
Protecting hydrophobic interfaces from nonspecific interactions might be particularly important for nups that have membrane-binding motifs such as amphipathic helices (AH) and ArfGAP1 lipid packing sensor (ALPS)-motifs [37].The colocalization with or binding of nups to lipid droplets and endoplasmic reticulum (ER) membranes under conditions of environmental stress [38][39][40] highlights that nup-lipid interactions are not limited to the assembly site and could thus seed unwanted membrane interactions in the cytosol if not properly regulated.Intriguingly, Nup1 and Nup2 both have membrane binding capacities [37] and are locally translated at the NPC [28].Local translation of these nup messages may provide an elegant mechanism to ensure that the resulting proteins do not bind to the broader ER but are instead immediately imported through NPCs and deposited at the pore membrane or INM [41].Whether such a mechanism can be extended to other AH or ALPS containing nups remains to be established.It may not be a coincidence, however, that AH-containing nups (e.g., Nup53 and Nup153 [37,42,43]) often also engage NTRs to promote their assembly in the NPC.Thus, it is conceivable that cytosolic NTR-nup interactions serve a dual purpose of targeting nups and/or NPC subcomplexes to the nucleus while at the same time preventing unwanted cytosolic membrane interactions.

Tuning nup levels in the cytosol
Collectively, all these findings suggest that cytosolic QC mechanisms are geared toward enabling rapid and correct formation of subcomplexes and condensates to benefit NPC assembly and avoid improper interactions and aggregation of the monomers in the cytosol.In addition to the abovementioned mechanisms, tuning cellular nup levels likely provides another means to prevent aberrant cytosolic nup-nup or nup-membrane interactions.While the data supporting such regulation of nup levels are scarce, there is evidence for mechanisms that couple nup synthesis and degradation to cellular demand.Specifically, some FG-nups are under tight control of transcriptional feedback loops and proteasomal turnover that regulate their cytosolic levels, and it has been proposed that these mechanisms could indeed function to prevent unintended cytosolic interactions [44].In addition, nups can also be stored on lipid droplets [40].This might keep their concentration in the cytosol low, and in addition, spatially segregating nups on lipid droplets may prevent their interactions with other cellular structures or facilitate their turnover [39].Lastly, the local translation of Nup1 and Nup2 at the NPC strongly suggests that their cytosolic levels have to remain low at all times [28].Understanding the mechanisms by which cytosolic nup levels are tuned is an exciting area for future research endeavors.

Quality control at the nuclear envelope: protecting the NE barrier
Even in the presence of cytosolic QC mechanisms that enforce specific nup-nup interactions, it is likely that aberrant NPC assembly can occur.Such events would be predicted to result in the accumulation of stalled or dead-end assembly intermediates at the NE and/or lead to the triggering of NPC QC pathways that act at the NE.A major challenge is the dearth of tools available that allow us to definitively visualize and assess the functionality of a malformed NPC.Nonetheless, clues to how defective NPC assembly events may manifest functionally and at the ultrastructural level comes from studies of the phenotypes observed when chaperone availability is limited [16,17], during aging [24] and in nup or NPC assembly factor perturbations ( [45] and references therein).For example, NPC misassembly triggered by genetic insult results in the accumulation of evaginations of the INM, NE herniations over misassembled NPC-like structures and often clusters of misassembled NPCs [16,[45][46][47][48][49].Disentangling whether these morphologies are caused by the formation of a misassembled NPC, the triggering of a NPC QC pathway or a misfiring of such a QC pathway remain open and challenging questions to address.For instance, NE herniations are more prominent in old cells [24], but whether these become detectable because of an increase in the incidence of NPC misassembly or because cells fail to properly deal with the misassembled NPCs-or both-is not well-understood.

Constraining membrane remodeling to prevent fusion
It is sensible that the greatest threat to the cell during an aberrant NPC biogenesis event would be a loss of nucleocytoplasmic compartmentalization caused by the formation of a NE hole without an effective NPC diffusion barrier in place.Thus, we will consider possible NPC QC mechanisms that function to either prevent aberrant fusion of the INM and ONM and those that are triggered if this occurs (Fig. 1, right panel).A possible interpretation, for example, of the accumulation of INM evaginations and NE herniations over misassembled NPCs is that there are pathways that act to enforce the proper timing of INM and ONM fusion.
It is likely that this timing is encoded in the assembly order of the NPC, where one can imagine that INM and ONM fusion can only occur when a specific membrane architecture is established by the spatial organization of nups in the nascent NPC-there are likely key roles for nups with AHs and ALPS motifs [41,42,50,51] that must be properly positioned along the emerging pore membrane.This idea is supported by perturbation of several scaffold and FG-nups ( [45] and references therein) that likely alter the normal positioning and orientation of the other NPC subcomplexes, similar to what has been shown in cryo-EM structures of NPCs under herniations caused by the deletion of the linker nup Nup116 [52].Fusion timing could also be modulated by controlling local lipid metabolism that might alter membrane properties in ways that promote or inhibit fusion [53][54][55][56].Ultimately, fusion will likely depend on the recruitment of protein fusogens to the site of close apposition of the INM and ONM.The yeast Brl1 has been proposed to have this function [46,57].In support of this, overexpression of Brl1 can rescue the NE herniations observed in nup116 or gle2-mutants [53], which suggests that sufficient accumulation of Brl1 at the assembly site can stimulate fusion.Thus, regulation of Brl1 may be a key element of putative QC mechanisms that might prevent inappropriate INM-ONM fusion.

NE/NPC assembly surveillance: sensing the lack of a permeability barrier
In cases where fusion of the INM and ONM occurs but there is a failure to establish a robust permeability barrier, a NE and NPC-assembly surveillance pathway kicks in.The principal components of this pathway are the integral INM protein Heh1/LEM2 and Chm7/CHMP7, a NE-specific component of the endosomal sorting complexes required for transport (ESCRT) [58][59][60][61].Interestingly, under normal conditions, CHMP7 and LEM2 are spatially segregated on either side of the NE-LEM2 at the INM and CHMP7 in the cytosol and ER, which prevents their physical association [49,62].As this segregation depends on the functioning of the nuclear transport system, the formation of a NE hole without a diffusion barrier would lead to the local meeting of LEM2 and CHMP7, which stimulates CHMP7 polymerization [49,63] and the recruitment of additional ESCRT proteins that are thought to seal the NE hole [48,49,58,64,65].Recent work further supports that Chm7 can directly interact with highly curved, PA-rich membranes at NPC misassembly sites [48], which may be an early signal that reinforces the CHMP7-LEM2 interaction.As such, incorrectly assembled NPCs that make it through the fusion event are rapidly sealed off by this system to re-establish nuclear compartmentalization [48,49,58,65].In addition, ESCRT proteins have direct membrane deforming capacities that can drive membrane curvature and scission (reviewed in Ref. [66]), but whether they contribute to NE remodeling in normal, nondefective NPC biogenesis remains unclear.

Turnover of misassembled NPCs and NPC subcomplexes
What is the fate of misassembled NPCs or misassembled intermediates?On the one hand, if premature fusion is prevented or reversed through the activities of the NE surveillance pathway, misassembled intermediates may not pose an immediate threat to the NE barrier.However, the remaining structures may be inherently unstable and could lead to NE ruptures, as proposed for the herniations that are formed over misassembled intermediates [48].In such a scenario, misassembled or stalled intermediates warrant removal from the NE.Although again we do not possess the tools to directly detect and monitor the fate of defective NPC assembly intermediates, there is evidence that this may nonetheless occur.For example, likely defective NPC assembly intermediates are degraded by the proteasome in collaboration with the AAA ATPase Vps4 [65].Such a degradation mechanism might take advantage of nuclear basket-tethered or INM-tethered proteasomes, which might also play a role in the surveillance of soluble proteins transiting the NPC and in the QC of INM proteins [67].More recently, the nuclear-vacuolar junction (NVJ), a vacuole-ONM contact site that is required for piecemeal microautophagy of the nucleus (PMN), has also been implicated in dealing with NPC misassembly stress triggered by NPC assembly mutants or by altering lipid metabolism [68].Logical hypotheses are that PMN is required for ensuring a proper lipid balance that supports NPC assembly and/or for the clearance of defective NPC assembly structures [69].The latter model is consistent with the observation that functional NPCs are excluded from the NVJ and are not targets of PMN [70,71], whereas some nups may be degraded by this pathway [68].Interestingly, the ESCRT machinery is also required for proper NVJ formation and PMN [68,72], but whether the ESCRTs are involved in the turnover of misassembled NPC intermediates through this junction is unknown.

Triaging of NPCs postassembly
How to distinguish a functional NPC from a nonfunctional NPC?
Once NPCs are assembled, it is likely that there are QC mechanisms that directly monitor NPC function and triage faulty NPCs.Perhaps the strongest evidence for this idea is the wholesale removal of NPCs from the NE by autophagy and ESCRT-dependent mechanisms [20,52,73,74].In the case of autophagy, there is evidence that starvation or TORC1 inhibition-in a process that is distinct from other selective nuclear autophagy mechanisms [75][76][77]-leads to NPC clearance from the NE.It is likely that these NPC-phagy events represent the turnover of mature NPCs rather than NPC assembly intermediates as they rely on the recognition of Nup159, a nup that is added to a nascent NPC postfusion, by the autophagy machinery.However, whether these NPCs actually represent damaged NPCs that are deliberately targeted for degradation is unclear.In support of this idea, mutants with compromised NPC structures display higher levels of such turnover [73], raising the possibility that cells can distinguish functional from nonfunctional NPCs.Perhaps such mechanisms rely on "sensing" the structural or functional integrity of the NPC, as has been proposed [78][79][80], but the jury is still out.

Fate of defective NPCs
If a mature NPC that becomes defective is detected, what could be its fate (Fig. 2)?Conceivably, structurally aberrant NPCs may be repaired.Such an idea could be supported by the evidence of exchange of individual nups within the NPC [20,21], which may provide a means to keep the NPC functional over time.It may even be possible to stimulate nup exchange in the context of damaged or missing nups but the underlying mechanism to detect such damage is elusive-for example, structural aberrancies in one NPC subcomplex leads to more turnover of members of the same subcomplex [21].
How the NPC permeability barrier itself is maintained is even less well-understood, but two theoretical scenarios of compromised barrier function are possible.First, NPCs may get clogged.A few clogged NPCs out of hundreds (or thousands) are unlikely to threaten bulk transport across the NE but they may nonetheless be a risk for general protein homeostasis as they may entrap other proteins.Whether there are mechanisms to detect clogged NPCs is not known.Second, NPCs may become leaky over time.For leaky NPCs, it makes considerable sense that they pose a risk to NE compartmentalization and that the CHMP7-LEM2 surveillance system can detect those, similar to its ability to detect and seal over membrane ruptures [81,82].Future research needs to establish whether and how structural aberrations, clogging, and leakiness indeed specifically trigger NPC-phagy that could facilitate the removal of such structures from the NE.
Quality control of mature and assembling NPCs: two sides of the same coin?
Quality control of mature NPCs is likely important given the long-lived nature of NPCs.However, whether the mechanisms at play during assembly are the same as those responsible for the QC of mature NPCs remains unanswered.Furthermore, a question arises: How does the QC system discern between an assembling NPC that has not yet attained its functional state, warranting preservation, and one that was once mature but has now become dysfunctional, necessitating degradation?To address these questions, the development of experimental tools capable of distinguishing between assembly intermediates, mature functional NPCs, and damaged NPCs would be beneficial.
An illustrative example of the complications in interpreting whether QC mechanisms deal with misassembled NPCs and damaged NPCs in the same way comes from the interpretation of clustered NPCs.A consistent observation, made over several decades, reports that NPCs that are missing key components are often found confined to specific areas of the NE [16,46,52].It remains unclear, however, whether the clustering reflects the relocation of mature NPCs that were originally spaced out over the NE (Fig. 3  Scenario 1), or a failure of clusters of assembling NPCs to move apart (Fig. 3, Scenario 2).
Related to Scenario 1, it has been proposed, for example, that clustering of faulty NPCs in one place could facilitate their recognition and clearance by autophagy [73].Similarly, there is evidence to support that clustered defective or damage-containing NPCs are part of a spatial QC mechanism that restricts these structures to mother nuclei to prevent their inheritance to progeny [65,83].In the Scenario 2, NPCs would assemble in defined regions in the NE; once fully mature, they can populate the rest of the NE.Such a scenario could explain why structures resembling early assembly intermediates are found close to each other [34,47,84].We hypothesize that by restricting NPC assembly to defined NE domains, it may also facilitate the coupling of biogenesis and QC mechanisms.
What could drive the clustering of NPCs in the NE?At present, the recruitment of specific QC proteins that actively mediate NPC clustering cannot be ruled out.However, there is more support for the alternative that NPC clustering may be energetically favorable and must therefore be actively prevented by yet to be defined mechanisms [80].As such, one could speculate that a failure of the assembly intermediate or damaged NPC to interact with specific proteins or anchoring structures would promote its clustering with other (mis)assembling NPCs.These anchoring proteins could conceivably be part of the NPC structure itself, as deletions of various nups from the outer ring [85][86][87][88], membrane ring [89], and nuclear basket [90] drive alterations in the positioning of NPCs.Alternatively, interactions with other nuclear structures such as chromatin [91,92], integral NE membrane proteins, and/or the nuclear lamina in higher eukaryotes [80,90,[93][94][95][96] could be a means to regulate the distribution of NPCs over the NE.
In conclusion, the need for better tools to follow specifically NPC assembly intermediates or damaged NPCs will undoubtedly spark many exciting discoveries as to how QC of NPCs is a relay of several surveillance mechanisms.A better understanding of NPC assembly and NPC QC go hand in hand and may in the long run help us understand the relationship between NPC function and how it impacts aging and disease.

Fig. 1 .
Fig.1.Cytosolic preassembly QC and QC at the assembly site.Schematic representation of the mechanisms protecting against aberrant interactions between nups in the time frame between their synthesis and incorporation into NPCs (left) and the mechanisms that either prevent premature fusion of the inner and outer membranes or protect against aberrant fusion (right).Different subcomplexes of the NPC are indicated in the schematic on the left: inner rings, outer rings, central channel, nuclear basket, and cytoplasmic filaments.

Fig. 2 .
Fig. 2. Conceivable functional and structural NPC decline of mature NPCs and possible mechanisms for QC to deal with such faulty structures (indicated in yellow).

References1Fig. 3 .
Fig. 3. Spatial organization of NPCs over the NE.Cartoon depicts clustering of NPCs versus spreading out of NPCs over the NE with two scenarios: clustering as outcome of QC mechanisms (Scenario 1) or clustering as default during biogenesis (Scenario 2). ,