Nuclear Pore Complex Biogenesis Research Articles

UBAP2L ensures homeostasis of nuclear pore complexes at the intact nuclear envelope.

Assembly of macromolecular complexes at correct cellular sites is crucial for cell function. Nuclear pore complexes (NPCs) are large cylindrical assemblies with eightfold rotational symmetry, built through hierarchical binding of nucleoporins (Nups) forming distinct subcomplexes. Here, we uncover a role of ubiquitin-associated protein 2-like (UBAP2L) in the assembly and stability of properly organized and functional NPCs at the intact nuclear envelope (NE) in human cells. UBAP2L localizes to the nuclear pores and facilitates the formation of the Y-complex, an essential scaffold component of the NPC, and its localization to the NE. UBAP2L promotes the interaction of the Y-complex with POM121 and Nup153, the critical upstream factors in a well-defined sequential order of Nups assembly onto NE during interphase. Timely localization of the cytoplasmic Nup transport factor fragile X-related protein 1 (FXR1) to the NE and its interaction with the Y-complex are likewise dependent on UBAP2L. Thus, this NPC biogenesis mechanism integrates the cytoplasmic and the nuclear NPC assembly signals and ensures efficient nuclear transport, adaptation to nutrient stress, and cellular proliferative capacity, highlighting the importance of NPC homeostasis at the intact NE.

Role of the San1 ubiquitin ligase in the heat stress-induced degradation of nonnative Nup1 in the nuclear pore complex.

The nuclear pore complex (NPC) mediates the selective exchange of macromolecules between the nucleus and the cytoplasm. Neurodegenerative diseases such as amyotrophic lateral sclerosis are characterized by mislocalization of nucleoporins (Nups), transport receptors, and Ras-related nuclear proteins into nucleoplasmic or cytosolic aggregates, underscoring the importance of precise assembly of the NPC. The assembly state of large protein complexes is strictly monitored by the protein quality control system. The ubiquitin-proteasome system may eliminate aberrant, misfolded, and/or orphan components; however, the involvement of the ubiquitin-proteasome system in the degradation of nonnative Nups in the NPC remains unclear. Here, we show that in Saccharomyces cerevisiae, although Nup1 (the FG-Nup component of the central core of the NPC) was stable, C-terminally green fluorescent protein-tagged Nup1, which had been incorporated into the NPC, was degraded by the proteasome especially under heat stress conditions. The degradation was dependent on the San1 ubiquitin ligase and Cdc48/p97, as well as its cofactor Doa1. We also demonstrate that San1 weakly but certainly contributes to the degradation of nontagged endogenous Nup1 in cells defective in NPC biogenesis by the deletion of NUP120. In addition, the overexpression of SAN1 exacerbated the growth defect phenotype of nup120Δ cells, which may be caused by excess degradation of defective Nups due to the deletion of NUP120. These biochemical and genetic data suggest that San1 is involved in the degradation of nonnative Nups generated by genetic mutation or when NPC biogenesis is impaired.

Coordinating nucleoporin condensation and nuclear pore complex assembly.

The nuclear pore complex (NPC) is among the most elaborate protein complexes in eukaryotes. While ribosomes and proteasomes are known to require dedicated assembly machinery, our understanding of NPC assembly is at a relatively early stage. Defects in NPC assembly or homeostasis are tied to movement disorders, including dystonia and amyotrophic lateral sclerosis (ALS), as well as aging, requiring a better understanding of these processes to enable therapeutic intervention. Here, we discuss recent progress in the understanding of NPC assembly and highlight how related defects in human disorders can shed light on NPC biogenesis. We propose that the condensation of phenylalanine-glycine repeat nucleoporins needs to be carefully controlled during NPC assembly to prevent aberrant condensation, aggregation, or amyloid formation.

Distinct domains in Ndc1 mediate its interaction with the Nup84 complex and the nuclear membrane.

Nuclear pore complexes (NPCs) are embedded in the nuclear envelope and built from ∼30 different nucleoporins (Nups) in multiple copies, few are integral membrane proteins. One of these transmembrane nucleoporins, Ndc1, is thought to function in NPC assembly at the fused inner and outer nuclear membranes. Here, we show a direct interaction of Ndc1's transmembrane domain with Nup120 and Nup133, members of the pore membrane coating Y-complex. We identify an amphipathic helix in Ndc1's C-terminal domain binding highly curved liposomes. Upon overexpression, this amphipathic motif is toxic and dramatically alters the intracellular membrane organization in yeast. Ndc1's amphipathic motif functionally interacts with related motifs in the C-terminus of the nucleoporins Nup53 and Nup59, important for pore membrane binding and interconnecting NPC modules. The essential function of Ndc1 can be suppressed by deleting the amphipathic helix from Nup53. Our data indicate that nuclear membrane and presumably NPC biogenesis depends on a balanced ratio between amphipathic motifs in diverse nucleoporins.

The chaperone DNAJB6 surveils FG-nucleoporins and is required for interphase nuclear pore complex biogenesis.

Biogenesis of nuclear pore complexes (NPCs) includes the formation of the permeability barrier composed of phenylalanine-glycine-rich nucleoporins (FG-Nups) that regulate the selective passage of biomolecules across the nuclear envelope. The FG-Nups are intrinsically disordered and prone to liquid-liquid phase separation and aggregation when isolated. How FG-Nups are protected from making inappropriate interactions during NPC biogenesis is not fully understood. Here we find that DNAJB6, a molecular chaperone of the heat shock protein network, forms foci in close proximity to NPCs. The number of these foci decreases upon removal of proteins involved in the early steps of interphase NPC biogenesis. Conversely, when this process is stalled in the last steps, the number of DNAJB6-containing foci increases and these foci are identified as herniations at the nuclear envelope. Immunoelectron tomography shows that DNAJB6 localizes inside the lumen of the herniations arising at NPC biogenesis intermediates. Loss of DNAJB6 results in the accumulation of cytosolic annulate lamellae, which are structures containing partly assembled NPCs, a feature associated with disturbances in NPC biogenesis. We find that DNAJB6 binds to FG-Nups and can prevent the aggregation of the FG region of several FG-Nups in cells and in vitro. Together, our data show that the molecular chaperone DNAJB6 provides quality control during NPC biogenesis and is involved in the surveillance of native intrinsically disordered FG-Nups.

Atypical nuclear envelope condensates linked to neurological disorders reveal nucleoporin-directed chaperone activities.

DYT1 dystonia is a debilitating neurological movement disorder arising from mutation in the AAA+ ATPase TorsinA. The hallmark of Torsin dysfunction is nuclear envelope blebbing resulting from defects in nuclear pore complex biogenesis. Whether blebs actively contribute to disease manifestation is unknown. We report that FG-nucleoporins in the bleb lumen form aberrant condensates and contribute to DYT1 dystonia by provoking two proteotoxic insults. Short-lived ubiquitylated proteins that are normally rapidly degraded partition into the bleb lumen and become stabilized. In addition, blebs selectively sequester a specific HSP40-HSP70 chaperone network that is modulated by the bleb component MLF2. MLF2 suppresses the ectopic accumulation of FG-nucleoporins and modulates the selective properties and size of condensates in vitro. Our study identifies dual mechanisms of proteotoxicity in the context of condensate formation and establishes FG-nucleoporin-directed activities for a nuclear chaperone network.

An amphipathic helix in Brl1 is required for nuclear pore complex biogenesis in S. cerevisiae.

The nuclear pore complex (NPC) is the central portal for macromolecular exchange between the nucleus and cytoplasm. In all eukaryotes, NPCs assemble into an intact nuclear envelope (NE) during interphase, but the process of NPC biogenesis remains poorly characterized. Furthermore, little is known about how NPC assembly leads to the fusion of the outer and inner NE, and no factors have been identified that could trigger this event. Here, we characterize the transmembrane protein Brl1 as an NPC assembly factor required for NE fusion in budding yeast. Brl1 preferentially associates with NPC assembly intermediates and its depletion halts NPC biogenesis, leading to NE herniations that contain inner and outer ring nucleoporins but lack the cytoplasmic export platform. Furthermore, we identify an essential amphipathic helix in the luminal domain of Brl1 that mediates interactions with lipid bilayers. Mutations in this amphipathic helix lead to NPC assembly defects, and cryo-electron tomography analyses reveal multilayered herniations of the inner nuclear membrane with NPC-like structures at the neck, indicating a failure in NE fusion. Taken together, our results identify a role for Brl1 in NPC assembly and suggest a function of its amphipathic helix in mediating the fusion of the inner and outer nuclear membranes.

Structure of cytoplasmic ring of nuclear pore complex by integrative cryo-EM and AlphaFold

INTRODUCTION The nuclear pore complex (NPC) is the molecular conduit in the nuclear membrane of eukaryotic cells that regulates import and export of biomolecules between the nucleus and the cytosol, with vertebrate NPCs ~110 to 125 MDa in molecular mass and ~120 nm in diameter. NPCs are organized into four main rings: the cytoplasmic ring (CR) at the cytosolic side, the inner ring and the luminal ring on the plane of the nuclear membrane, and the nuclear ring facing the nucleus. Each ring possesses an approximate eightfold symmetry and is composed of multiple copies of different nucleoporins. NPCs have been implicated in numerous biological processes, and their dysfunctions are associated with a growing number of serious human diseases. However, despite pioneering studies from many groups over the past two decades, we still lack a full understanding of NPCs' organization, dynamics, and complexity. RATIONALE We used the Xenopus laevis oocyte as a model system for the structural characterization because each oocyte possesses a large number of NPC particles that can be visualized on native nuclear membranes without the aid of detergent extraction. We used single-particle cryo-electron microscopy (cryo-EM) analysis on data collected at different stage tilt angles for three-dimensional reconstruction and structure prediction with AlphaFold for model building. RESULTS We reconstructed the CR map of X. laevis NPC at 6.9 and 6.7 Å resolutions for the full CR protomer and a core region, respectively, and predicted the structures of the individual nucleoporins using AlphaFold because no high-resolution models of X. laevis Nups were available. For any ambiguous subunit interactions, we also predicted complex structures, which further guided model fitting of the CR protomer. We placed the nucleoporin or complex structures into the CR density to obtain an almost full CR atomic model, composed of the inner and outer Y-complexes, two copies of Nup205, two copies of the Nup214-Nup88-Nup62 complex, one Nup155, and five copies of Nup358. In particular, we predicted the largest protein in the NPC, Nup358, as having an S-shaped globular domain, a coiled-coil domain, and a largely disordered C-terminal region containing phenylalanine-glycine (FG) repeats previously shown to form a gel-like condensate phase for selective cargo passage. Four of the Nup358 copies clamp around the inner and outer Y-complexes to stabilize the CR, and the fifth Nup358 situates in the center of the cluster of clamps. AlphaFold also predicted a homo-oligomeric, likely specifically pentameric, coiled-coil structure of Nup358 that may provide the avidity for Nup358 recruitment to the NPC and for lowering the threshold for Nup358 condensation in NPC biogenesis. CONCLUSION Our studies offer an example of integrative cryo-EM and structure prediction as a general approach for attaining more precise models of megadalton protein complexes from medium-resolution density maps. The more accurate and almost complete model of the CR presented here expands our understanding of the molecular interactions in the NPC and represents a substantial step forward toward the molecular architecture of a full NPC, with implications for NPC function, biogenesis, and regulation. [Figure: see text].

Visualizing Nuclear Pore Complex Assembly In Situ in Human Cells at Nanometer Resolution by Correlating Live Imaging with Electron Microscopy.

In eukaryotic cells that undergo open mitosis, nuclear pore complex assembly proceeds via two distinct pathways: postmitotic and interphase assembly. Studying both assembly processes is challenging because postmitotic assembly is fast, interphase assembly is rare and sporadic, and assembly intermediates in both pathways are very small with a diameter below 100nm. Here, we present a protocol for studying nuclear pore complex biogenesis in situ in cultured human cells in a spatiotemporally resolved and quantitative manner by combining live imaging with three-dimensional electron microscopy. The method described here can alsobe applied for studying other cell cycle-associated events with high spatiotemporal resolution.

Reticulon-like REEP4 at the inner nuclear membrane promotes nuclear pore complex formation.

Nuclear pore complexes (NPCs) are channels within the nuclear envelope that mediate nucleocytoplasmic transport. NPCs form within the closed nuclear envelope during interphase or assemble concomitantly with nuclear envelope reformation in late stages of mitosis. Both interphase and mitotic NPC biogenesis require coordination of protein complex assembly and membrane deformation. During early stages of mitotic NPC assembly, a seed for new NPCs is established on chromatin, yet the factors connecting the NPC seed to the membrane of the forming nuclear envelope are unknown. Here, we report that the reticulon homology domain protein REEP4 not only localizes to high-curvature membrane of the cytoplasmic endoplasmic reticulum but is also recruited to the inner nuclear membrane by the NPC biogenesis factor ELYS. This ELYS-recruited pool of REEP4 promotes NPC assembly and appears to be particularly important for NPC formation during mitosis. These findings suggest a role for REEP4 in coordinating nuclear envelope reformation with mitotic NPC biogenesis.

A short perinuclear amphipathic α-helix in Apq12 promotes nuclear pore complex biogenesis.

The integral membrane protein Apq12 is an important nuclear envelope (NE)/endoplasmic reticulum (ER) modulator that cooperates with the nuclear pore complex (NPC) biogenesis factors Brl1 and Brr6. How Apq12 executes these functions is unknown. Here, we identified a short amphipathic α-helix (AαH) in Apq12 that links the two transmembrane domains in the perinuclear space and has liposome-binding properties. Cells expressing an APQ12 (apq12-ah) version in which AαH is disrupted show NPC biogenesis and NE integrity defects, without impacting Apq12-ah topology or NE/ER localization. Overexpression of APQ12 but not apq12-ah triggers striking over-proliferation of the outer nuclear membrane (ONM)/ER and promotes accumulation of phosphatidic acid (PA) at the NE. Apq12 and Apq12-ah both associate with NPC biogenesis intermediates and removal of AαH increases both Brl1 levels and the interaction between Brl1 and Brr6. We conclude that the short amphipathic α-helix of Apq12 regulates the function of Brl1 and Brr6 and promotes PA accumulation at the NE possibly during NPC biogenesis.

Torsin and NEP1R1-CTDNEP1 phosphatase affect interphase nuclear pore complex insertion by lipid-dependent and lipid-independent mechanisms.

The interphase nuclear envelope (NE) is extensively remodeled during nuclear pore complex (NPC) insertion. How this remodeling occurs and why it requires Torsin ATPases, which also regulate lipid metabolism, remains poorly understood. Here, we show that Drosophila Torsin (dTorsin) affects lipid metabolism via the NEP1R1‐CTDNEP1 phosphatase and the Lipin phosphatidic acid (PA) phosphatase. This includes that Torsins remove NEP1R1‐CTDNEP1 from the NE in fly and mouse cells, leading to subsequent Lipin exclusion from the nucleus. NEP1R1‐CTDNEP1 downregulation also restores nuclear pore membrane fusion in post‐mitotic dTorsinKO fat body cells. However, dTorsin‐associated nuclear pore defects do not correlate with lipidomic abnormalities and are not resolved by silencing of Lipin. Further testing confirmed that membrane fusion continues in cells with hyperactivated Lipin. It also led to the surprising finding that excessive PA metabolism inhibits recruitment of the inner ring complex Nup35 subunit, resulting in elongated channel‐like structures in place of mature nuclear pores. We conclude that the NEP1R1‐CTDNEP1 phosphatase affects interphase NPC biogenesis by lipid‐dependent and lipid‐independent mechanisms, explaining some of the pleiotropic effects of Torsins.

Co-translational assembly and localized translation of nucleoporins in nuclear pore complex biogenesis

mRNA translation is coupled to multiprotein complex assembly in the cytoplasm or to protein delivery into intracellular compartments. Here, by combining systematic RNA immunoprecipitation and single-molecule RNA imaging in yeast, we have provided a complete depiction of the co-translational events involved in the biogenesis of a large multiprotein assembly, the nuclear pore complex (NPC). We report that binary interactions between NPC subunits can be established during translation, in the cytoplasm. Strikingly, the nucleoporins Nup1/Nup2, together with a number of nuclear proteins, are instead translated at nuclear pores, through a mechanism involving interactions between their nascent N-termini and nuclear transport receptors. Uncoupling this co-translational recruitment further triggers the formation of cytoplasmic foci of unassembled polypeptides. Altogether, our data reveal that distinct, spatially segregated modes of co-translational interactions foster the ordered assembly of NPC subunits and that localized translation can ensure the proper delivery of proteins to the pore and the nucleus.

A mutation in budding yeast BRR6 affecting nuclear envelope insertion of the spindle pole body.

BRR6 and BRL1 are two paralogs that encode transmembrane proteins of the nuclear envelope (NE) involved in membrane fluidity and nuclear pore complex biogenesis in organisms that undergo a closed mitosis. We show that mutation of a conserved cysteine in the intralumenal domain of Saccharomyces cerevisiae Brr6p results in a novel temperature sensitive allele, brr6-Y100H, that arrests growth due to defects in spindle formation. Analysis of brr6-Y100H cells by electron tomography and Brr6p localization by super-resolution imaging supports the idea that Brr6p is involved in insertion of the newly duplicated spindle pole body into the NE.

Maturation Kinetics of a Multiprotein Complex Revealed by Metabolic Labeling

All proteins interact with other cellular components to fulfill their function. While tremendous progress has been made in the identification of protein complexes, their assembly and dynamics remain difficult to characterize. Here, we present a high-throughput strategy to analyze the native assembly kinetics of protein complexes. We apply our approach to characterize the co-assembly for 320 pairs of nucleoporins (NUPs) constituting the ≈50 MDa nuclear pore complex (NPC) in yeast. Some NUPs co-assemble fast via rapid exchange whereas others require lengthy maturation steps. This reveals a hierarchical principle of NPC biogenesis where individual subcomplexes form on a minute timescale and then co-assemble from center to periphery in a ∼1 h-long maturation process. Intriguingly, the NUP Mlp1 stands out as joining very late and associating preferentially with aged NPCs. Our approach is readily applicable beyond the NPC, making it possible to analyze the intracellular dynamics of a variety of multiprotein assemblies.

Nuclear envelope-vacuole contacts mitigate nuclear pore complex assembly stress.

The intricacy of nuclear pore complex (NPC) biogenesis imposes risks of failure that can cause defects in nuclear transport and nuclear envelope (NE) morphology; however, cellular mechanisms used to alleviate NPC assembly stress are not well defined. In the budding yeast Saccharomyces cerevisiae, we demonstrate that NVJ1- and MDM1-enriched NE-vacuole contacts increase when NPC assembly is compromised in several nup mutants, including nup116ΔGLFG cells. These interorganelle nucleus-vacuole junctions (NVJs) cooperate with lipid droplets to maintain viability and enhance NPC formation in assembly mutants. Additionally, NVJs function with ATG1 to remodel the NE and promote vacuole-dependent degradation of specific nucleoporins in nup116ΔGLFG cells. Importantly, NVJs significantly improve the physiology of NPC assembly mutants, despite having only negligible effects when NPC biogenesis is unperturbed. These results therefore define how NE-vacuole interorganelle contacts coordinate responses to mitigate deleterious cellular effects caused by disrupted NPC assembly.

In-cell architecture of the nuclear pore and snapshots of its turnover.

Nuclear pore complexes (NPCs) fuse the inner and outer membranes of the nuclear envelope. They comprise hundreds of nucleoporins (Nups) that assemble into multiple subcomplexes and form large central channels for nucleocytoplasmic exchange1,2. How this architecture facilitates messenger RNA export, NPC biogenesis and turnover remains poorly understood. Here we combine in situ structural biology and integrative modelling with correlative light and electron microscopy and molecular perturbation to structurally analyse NPCs in intact Saccharomyces cerevisiae cells within the context of nuclear envelope remodelling. We find an in situ conformation and configuration of the Nup subcomplexes that was unexpected from the results of previous in vitro analyses. The configuration of the Nup159 complex appears critical to spatially accommodate its function as an mRNA export platform, and as a mediator of NPC turnover. The omega-shaped nuclear envelope herniae that accumulate in nup116Δ cells3 conceal partially assembled NPCs lacking multiple subcomplexes, including the Nup159 complex. Under conditions of starvation, herniae of a second type are formed that cytoplasmically expose NPCs. These results point to a model of NPC turnover in which NPC-containing vesicles bud off from the nuclear envelope before degradation by the autophagy machinery. Our study emphasizes the importance of investigating the structure-function relationship of macromolecular complexes in their cellular context.

Lipid and protein dynamics that shape nuclear envelope identity.

The nuclear envelope (NE) is continuous with the endoplasmic reticulum (ER), yet the NE carries out many functions distinct from those of bulk ER. This functional specialization depends on a unique protein composition that defines NE identity and must be both established and actively maintained. The NE undergoes extensive remodeling in interphase and mitosis, so mechanisms that seal NE holes and protect its unique composition are critical for maintaining its functions. New evidence shows that closure of NE holes relies on regulated de novo lipid synthesis, providing a link between lipid metabolism and generating and maintaining NE identity. Here, we review regulation of the lipid bilayers of the NE and suggest ways to generate lipid asymmetry across the NE despite its direct continuity with the ER. We also discuss the elusive mechanism of membrane fusion during nuclear pore complex (NPC) biogenesis. We propose a model in which NPC biogenesis is carefully controlled to ensure that a permeability barrier has been established before membrane fusion, thereby avoiding a major threat to compartmentalization.

Maturation Kinetics of a Multiprotein Complex Revealed by Metabolic Labeling

All proteins interact with other cellular components to fulfill their function. While tremendous progress has been made in the identification of protein complexes, their assembly and dynamics remain difficult to characterize. Here we present a high-throughput strategy to analyze the native assembly kinetics of protein complexes. We apply our approach to characterize the co-assembly for 320 pairs of nucleoporins (NUPs) constituting the ~ 50 MDa nuclear pore complex (NPC) in yeast. Some NUPs co-assemble fast via rapid exchange whereas others require lengthy maturation steps. This reveals a hierarchical principle of NPC biogenesis where individual subcomplexes form on a minute-time-scale and then co-assemble from center to periphery in a ~1-hour-long maturation process. Intriguingly, the NUP Mlp1 stands out as joining very late and associating preferentially with aged NPCs. Our approach is readily adaptable beyond the NPC, making it possible to analyze intracellular dynamics of a variety of multiprotein assemblies.

The Role of Torsin AAA+ Proteins in Preserving Nuclear Envelope Integrity and Safeguarding Against Disease.

Torsin ATPases are members of the AAA+ (ATPases associated with various cellular activities) superfamily of proteins, which participate in essential cellular processes. While AAA+ proteins are ubiquitously expressed and demonstrate distinct subcellular localizations, Torsins are the only AAA+ to reside within the nuclear envelope (NE) and endoplasmic reticulum (ER) network. Moreover, due to the absence of integral catalytic features, Torsins require the NE- and ER-specific regulatory cofactors, lamina-associated polypeptide 1 (LAP1) and luminal domain like LAP1 (LULL1), to efficiently trigger their atypical mode of ATP hydrolysis. Despite their implication in an ever-growing list of diverse processes, the specific contributions of Torsin/cofactor assemblies in maintaining normal cellular physiology remain largely enigmatic. Resolving gaps in the functional and mechanistic principles of Torsins and their cofactors are of considerable medical importance, as aberrant Torsin behavior is the principal cause of the movement disorder DYT1 early-onset dystonia. In this review, we examine recent findings regarding the phenotypic consequences of compromised Torsin and cofactor activities. In particular, we focus on the molecular features underlying NE defects and the contributions of Torsins to nuclear pore complex biogenesis, as well as the growing implications of Torsins in cellular lipid metabolism. Additionally, we discuss how understanding Torsins may facilitate the study of essential but poorly understood processes at the NE and ER, and aid in the development of therapeutic strategies for dystonia.