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Published: 13 August 2024

Docking a flexible basket onto the core of the nuclear pore complex

Nature Cell Biology volume 26, pages 1504–1519 (2024)Cite this article

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Abstract

The nuclear basket attaches to the nucleoplasmic side of the nuclear pore complex (NPC), coupling transcription to mRNA quality control and export. The basket expands the functional repertoire of a subset of NPCs in Saccharomyces cerevisiae by drawing a unique RNA/protein interactome. Yet, how the basket docks onto the NPC core remains unknown. By integrating AlphaFold-based interaction screens, electron microscopy and membrane-templated reconstitution, we uncovered a membrane-anchored tripartite junction between basket and NPC core. The basket subunit Nup60 harbours three adjacent short linear motifs, which connect Mlp1, a parallel homodimer consisting of coiled-coil segments interrupted by flexible hinges, and the Nup85 subunit of the Y-complex. We reconstituted the Y-complex•Nup60•Mlp1 assembly on a synthetic membrane and validated the protein interfaces in vivo. Here we explain how a short linear motif-based protein junction can substantially reshape NPC structure and function, advancing our understanding of compositional and conformational NPC heterogeneity.

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Main

A basket typically has a framework of interwoven components forming an open, hollow structure. The nuclear pore basket, true to its name, was proposed to share similar characteristics. Composed of proteins known as nucleoporins (Nups), the basket decorates the nucleoplasmic face of the nuclear pore complex (NPC) core scaffold. The basket forms a truncated cone, composed of eight filament-like structures as shown by ultrathin section negative stain and scanning electron microscopy (EM)^1,2,3. The height of the basket varies between species (~60–120 nm)^4,5. Whereas the NPC core forms the central conduit for nucleocytoplasmic transport, the nuclear basket adds additional layers of complexity, asymmetry and regulation to the process.

The nuclear basket has been implicated in maintaining free access to the NPC channel by excluding (hetero-) chromatin^6,7, docking ribonucleoprotein particles before export^8,9, selecting export-competent messenger RNAs and retaining faulty mRNAs¹⁰ and participating in protein quality control¹¹, as well as physically tethering and regulating genes through basket-associated adaptors^{12,13,14,15,16}. The intrinsically disordered phenylalanine–glycine (FG) regions of basket Nups also contribute to the NPC’s permeability barrier^17,18,19, and certain basket Nups provide docking sites for transport receptors^18,20. Nuclear baskets assemble only on a subset of NPCs in Saccharomyces cerevisiae. The region where the nucleolus meets the inner nuclear membrane (INM) features ‘basketless’ NPCs^7,21.

Basket-containing NPCs recruit a specific RNA and protein interactome, suggesting different transport capabilities compared with basketless NPCs²¹. Thus, the basket is a prominent example of NPC compositional heterogeneity, a poorly understood phenomenon that refers to variations in subunit stoichiometry among different NPCs within a cell or across different cell types^21,22,23,24. Basket Nups exhibit remarkable dynamics at the NPC. Whereas their protein turnover is relatively slow, the exchange rates of NPC-bound basket Nups with Nups from a free pool are high, suggesting constant swapping at the NPC^25,26,27,28. Intriguingly, basket assembly depends, directly or indirectly, on RNA polymerase II transcription and subsequent messenger ribonucleoprotein processing²¹, suggesting that gene expression itself can shape NPC structure, which further complicates mechanistic studies. Currently, we do not understand how NPCs become functionalized by the basket because we lack a biochemical and structural understanding of how the basket docks onto the core of the NPC.

Substantial progress has recently been made in visualizing the structure of the NPC’s symmetric core scaffold and the cytoplasmic filaments^{23,24,29,30,31,32,33,34}. In contrast, the basket has so far resisted a high-resolution structural characterization. An in situ cryo-electron tomography (cryo-ET) study described the basket as a pair of ‘bifurcated densities’ that associate with each Y-complex in the nuclear outer ring across an extended area²⁴. These densities were interpreted as ‘basket anchors’; however, it is currently unclear which proteins account for them and which parts of the basket are missing in the model. Another in situ cryo-ET study found ‘rod-like densities’ at the connection between the nuclear and inner ring structure and around the Y-complex vertex, which were interpreted as coiled-coil structures³⁴. Nup153 was shown to mediate membrane interactions of the Y-complex during interphase assembly³⁵ and another study proposed a ubiquitin-dependent interaction of Nup60 with the Nup84 subunit of the Y-complex³⁶. Collectively, these studies suggest that basket components are somehow docked onto the outer ring of the NPC core.

In S. cerevisiae, the nuclear basket consists of Nup1, Nup2, Nup60, the paralogous Mlp1/2 proteins and Pml39. The metazoan basket comprises only four Nups, namely, Nup153 (a likely orthologue of yeast Nup1/Nup60), Nup50 (Nup2), Tpr (Mlp1/2) and ZC3HC1 (Pml39)^37,38,39. We have previously identified the intrinsically disordered S. cerevisiae Nup60 as the basket’s flexible ‘suspension cable’⁴⁰. Nup60 harbours short linear motifs (SLiMs) as contact sites for the basket subunits Nup2 and the Mlp1 (ref. ⁴⁰), while interacting with the INM through an N-terminal amphipathic helix (AH)²⁰. How Nup60, as the basket’s suspension cable, ‘weaves’ through the framework to simultaneously connect the INM, Mlp1/2, Nup2 and possibly the NPC core, is unknown. The number of Nup60 molecules per NPC was estimated as 8 (refs. ^41,42) or 16 (refs. ^43,44). The outer ring of the NPC core, implicated in contacting the basket, is formed by the head-to-tail arrangement of Y-complexes (also known as coat Nup complex or Nup84 complex in yeast and Nup107–160 complex in metazoa), which may be present as a single ring (8 copies) or double ring (16 copies) of Y-complexes depending on the species^{34,41,45,46,47,48,49,50}. In S. cerevisiae, the ~580 kDa Y-complex consists of seven Nups: the two short arms are composed of Nup120 and the Nup85•Seh1 heterodimer, whereas the kinked stem is formed by the Nup145C•Sec13•Nup84•Nup133 heterotetramer^46,50,51,52. The short arms and the stem meet at the triskelion region, which is defined by interactions between Nup120, Nup85 and Nup145C^46,51. The Y-complex interacts with the nuclear envelope (NE) membranes via Nup120 and Nup133 (ref. ⁵³). However, the binding strength and purpose of these membrane interactions remain unclear. Of note, the majority of yeast NPCs was reported to contain a single nuclear ring comprising eight Y-complexes, whereas a subset may contain a double nuclear ring with sixteen Y-complexes²⁴. Whether and how this different stoichiometry relates to basket assembly is unknown.

To understand how the functionality of the NPC is tuned, we need to understand the molecular mechanism by which the basket docks onto the NPC core. This is important because it will explain (1) how an NPC becomes fully assembled, (2) how the basket functionalizes the core for transport and gene regulation and (3) how this creates NPC heterogeneity within a cell and across cell types. Understanding the interface between the basket and the NPC core will permit the design of specific basket separation-of-function mutants that are critical to disentangle the wealth of cellular functions of ‘basket-powered’ NPCs.

In this Article, we have reconstituted a supramolecular assembly comprising the Y-complex, the Nup60 suspension cable and critical parts of the Mlp1 filaments on a membrane. We have identified the precise docking sites of a tripartite Nup60 region on a conserved groove of Nup85 and a hinge region of Mlp1. In addition, we explain at single residue level how this junction couples Mlp1 filament docking to the Y-complex with INM tethering of the basket in vitro and in vivo. We further demonstrate that Mlp1 forms a parallel homodimer of rigid coiled coils and flexible hinges providing direct evidence for the dual nature of the basket—being both rigid and conformationally flexible. A stoichiometric basket–Y-complex assembly can form independently of external factors, yet, mRNA metabolism may stabilize it.

Results

Nup60 ‘suspension cable’ oligomerizes via its helical region

Nup60 was identified as the basket’s membrane-anchored suspension cable, containing SLiMs that interact directly with Mlp1 and Nup2 (ref. ⁴⁰). To determine how the basket docks onto the core of the NPC, we employed an artificial intelligence (AI)-based SLiM screen, extending AlphaFold-Multimer’s success in structure prediction towards identifying unknown protein–protein interactions. We segmented Nup60 into pieces of equal length (31 amino acids (aa)) covering all conserved SLiMs and determined the average interface predicted template modelling (ipTM) score for every binary combination with all Nups (Fig. 1a). Out of 264 combinations tested, two pairs stood out by their high ipTM score: the helical region (HR) of Nup60 (HR; aa119–149) with itself, and a SLiM at aa285–315 with Nup85. The HR of Nup60 is predicted to adopt a helix-turn-helix fold with two copies arranged in an antiparallel orientation, held together by a conserved hydrophobic patch that is buried at the dimer interface (Fig. 1b; see Extended Data Fig. 1a,b for model confidence). The solvent-exposed dimer surface is largely hydrophilic (Extended Data Fig. 1c–f). To verify a potential oligomerization of Nup60, we performed a two-step split-affinity purification of differentially tagged Nup60 HR constructs co-expressed in Escherichia coli. We fused the Nup60 HR (aa48–162; including an N-terminal linker) with either a His₆ or FLAG tag and appended a NusA (57 kDa) or SUMO (14 kDa) tag to create a size difference. Following a two-step affinity purification on Ni-NTA and anti-FLAG beads, we retrieved both NusA and SUMO-tagged Nup60 species (Fig. 1c), indicating an assembly of at least two Nup60 HR copies.

**Fig. 1: The Nup60 suspension cable oligomerizes via its HR.**

To characterize Nup60 oligomerization quantitatively, we performed dilution isothermal titration calorimetry (ITC) experiments with the recombinant NusA-His₆-Nup60(48–162) protein. The dilution isotherms displayed a hyperbolic profile, characteristic of a simple dimer dissociation model, whereas NusA-His₆, used as a negative control, did not exhibit such behaviour (Fig. 1d and Extended Data Fig. 1g,h). The dissociation constant (K_D) of the Nup60 HR was 76.3 ± 7.9 μM (mean ± s.d.). Additionally, we performed size exclusion chromatography coupled with multi-angle light scattering experiments. Consistent with Nup60 dimerization, we observed a concentration-dependent shift of the NusA-His₆-Nup60(48–162) peak towards higher molecular mass (Extended Data Fig. 1i). However, a low peak resolution prevented us from accurately estimating the molecular mass of the putative dimer peak. We cannot exclude the possibility that Nup60 undergoes higher-order oligomerization, beyond just forming a dimer. Conversely, there was no discernible shift in the NusA-His₆ negative control, thereby ruling out an influence of the tag on Nup60 dimerization (Extended Data Fig. 1j). Dimerization of Nup60, as explained below, is expected to affect the connectivity of the basket, its docking onto the NPC core and may stabilize the head-to-tail arrangement of Y-complexes in the nuclear ring of the NPC.

Nup60 interacts with the Nup85•Seh1 arm of the Y-complex

The second-best hit identified in our AlphaFold-based SLiM screen suggested an interaction between Nup60 and Nup85, a subunit of the Y-complex. Surprisingly, this stretch of 31 amino acids (aa285–315) is embedded in a larger region of Nup60 (aa240–318, termed Nup60 Mlp1-binding motif (MBM)) that was previously shown to recruit the basket subunit Mlp1 (ref. ⁴⁰). This finding raised the possibility that the Y-complex and the Mlp1 filaments bind to Nup60 in immediate proximity. Mlp1 was not detected in this binary screen, possibly because it interacts with Nup60 only as a coiled-coil homodimer (see further below). Moreover, the expected interaction between Nup60 and Nup2 (ref. ⁴⁰) was not detected, suggesting that AlphaFold encounters challenges in predicting SLiM-mediated interactions.

To experimentally verify a putative Y-complex–Nup60 interaction, we aimed to reconstitute it in vitro. To this end, we engineered a stable S. cerevisiae Nup60 construct, which lacks the N-terminal lipid-interacting AH (aa1–47) and the disordered FG-repeat region (aa381–504). This Nup60 construct (aa48–539ΔFG; abbreviated as Nup60*) was purified from E. coli via an N-terminal glutathione S-transferase (GST)-tag and immobilized on glutathione (GSH) beads as a bait (Fig. 2a). The Y-complex was affinity purified from an S. cerevisiae strain expressing Nup133 with a C-terminal TAP tag (Fig. 2b). When incubated with each other, the Y-complex bound Nup60* (Fig. 2c and Extended Data Fig. 2a), but did not bind to GST used as a negative control. Next, we deleted conserved regions of Nup60, which overlap or lie adjacent to the previously identified direct binding site for Mlp1. Deleting the previously mapped Mlp1 binding site on Nup60 (aa240-309) also reduced the affinity for the Y-complex (Fig. 2c). Deletion of the adjacent Nup60 region (aa200–239) similarly resulted in a partial loss of Y-complex interaction (Fig. 2c). Interestingly, deletion of both Nup60 regions (aa200–309) completely abolished the interaction with the Y-complex (Fig. 2c). This indicates that the region spanning aa200–309 of Nup60 is necessary for direct interactions with the Y-complex. We then asked whether this region of Nup60 is sufficient for binding to the Y-complex. To address this, we generated a stable recombinant GST-Nup60 construct (aa200–318) and performed binding assays with the Y-complex (Fig. 2d,e and Extended Data Fig. 2b). The Y-complex specifically interacted with this minimal Nup60 region, whereas further N- or C-terminal truncations of the Nup60 construct resulted in a loss of interaction (Fig. 2e). Thus, a region spanning aa200–318 of Nup60 is both necessary and sufficient for a direct interaction with the Y-complex.

**Fig. 2: Nup60 interacts with the Nup85•Seh1 arm of the Y-complex.**

To precisely determine the binding site of Nup60(200–318) on the heptameric, ~580-kDa Y-complex, we performed cross-linking coupled to mass spectrometry (XL–MS) experiments using disuccinimidyl suberate (DSS) as a lysine-specific cross-linker. Recombinant Nup60(200–318) cross-linked specifically to the Nup85 and Seh1 subunits of the Y-complex, which constitute one of its short arms (Fig. 2f and Extended Data Fig. 2d). Guided by these results, we tested whether a recombinant Nup85(1–564)•Seh1 heterodimer (termed Nup85*•Seh1), purified via an N-terminal His₆ tag on Seh1 (ref. ⁵⁴), directly interacts with Nup60* (Fig. 2g and Extended Data Fig. 2c). Indeed, these experiments confirmed that the region between aa200 and aa318 of Nup60 is sufficient to bind to Nup85*•Seh1. Both the regions spanning aa200–239 and aa240–318 play a role in this interaction, with the latter displaying a higher affinity for Nup85*•Seh1. In summary, we have unambiguously identified Nup85•Seh1 as a direct interaction partner of Nup60.

Mlp1 forms a homodimer of coiled coils and flexible hinges

Based on these findings, our objective was to clarify the manner in which the structurally elusive basket filaments are linked to Nup60, and consequently, the Y-complex. Despite decades of effort, the overall topology of the basket filaments remains unknown due to the difficulties of studying these large coiled-coil proteins in isolation or within the NPC context. Earlier EM studies of two separate N-terminal fragments of the human Tpr (aa1–398 and aa774–1370) had reported rectilinear particles⁵⁵. However, the manner in which these fragments are connected remained unclear.

We engineered Mlp1 to carry an N-terminal maltose-binding protein (MBP) tag (40 kDa), large enough to be visualized by EM. We succeeded in purifying a stable S. cerevisiae Mlp1(17–1,137) construct, expressed in insect cells. This construct comprises ∼60% of the full-length protein and 77% of the structured parts without the disordered C-terminus (Fig. 3a). After MBP-affinity purification on amylose beads and native elution with maltose-containing buffer, the protein exhibited a predicted size of ~174 kDa without major impurities (Fig. 3b). This protein was examined by heavy metal rotary shadowing EM. We observed filamentous particles containing multiple rod-like segments (Fig. 3c; for an overview, see Extended Data Fig. 3a). Notably, we consistently observed two globular knobs at one end of the particles, which we interpret as MBP tags (Fig. 3c), because the knobs were not present in a construct lacking MBP (Extended Data Fig. 3b). This provides direct experimental evidence that Mlp1 forms a parallel homodimer, probably consisting of rigid coiled-coil segments interrupted by flexible hinges. The number of visually distinct segments (defined as a particle segment, which can be traced by a straight line) varied from one up to six; however, most particles contained either four or three segments (Fig. 3d). The end-to-end length (defined as the sum of all segment lengths) of the four-segment particles ranged from 130–170 nm with a median length of ~150 nm (Fig. 3e). We then measured the lengths of individual segments in the largest subpopulation of particles, which contained four distinct segments (Fig. 3f). Counting from the N-terminal knobs of the MBP tag, the first, third and fourth segments displayed similar length distributions with medians between 37 and 43 nm. The second segment was noticeably shorter with a median length of only ~26 nm.

**Fig. 3: Mlp1 forms a homodimer with coiled coils and flexible hinges.**

The AlphaFold-Multimer prediction of Mlp1(17–1,137) features three extended coiled coils, each with a length of ~34–38 nm (Extended Data Fig. 4a), which closely aligns with the length of the first, third and fourth segments observed through EM. The second segment of Mlp1 is predicted to have a shorter coiled-coil structure and may correspond to the Mlp1 Nup60-binding domain (N60BD) (as explained below). AlphaFold-Multimer predictions also suggest that the overall architecture is shared between yeast Mlp1 and human Tpr (Extended Data Fig. 4a,b). In sum, we provide experimental evidence that Mlp1(17–1,137) forms a coiled-coil parallel homodimer with flexible hinge regions at distinct positions. Flexible Mlp1 hinges therefore are key features of the basket filaments, contrary to models that portray them as entirely rigid⁵⁶.

Nup60 couples Mlp1 recruitment and docking on the Y-complex

The Nup60 aa200–318 region that we identified as an interaction site with both Mlp1 and the Y-complex consists of three adjacent SLiMs (termed SLiMs A, B and C). The proximity of three SLiMs within a short stretch suggested that Nup60 organizes, and potentially co-regulates, the recruitment of Mlp1 filaments to the Y-complex. To disentangle this complexity, we generated a set of GST-Nup60(200–318) constructs with different SLiM deletions (Fig. 4a), immobilized them on GSH beads and assessed their affinity to either recombinant Nup85*•Seh1 or Mlp1 by in vitro binding assays. Deleting SLiM A reduced the interaction with Nup85*•Seh1 (Fig. 4b, upper panel; compare lane 2 with 4), and correspondingly, SLiM A alone exhibited some affinity for Nup85*•Seh1 (compare lane 2 with 3). An additional deletion of SLiM C, retaining SLiM B, completely abolished the interaction with Nup85*•Seh1 (compare lane 4 with 5). Conversely, deletion of SLiM B did not affect Nup85*•Seh1 binding (compare lane 4 with 6). The linker connecting SLiMs A and B also displayed no detectable affinity for Nup85*•Seh1 (compare lane 4 with 8). Importantly, SLiM C was sufficient to robustly interact with Nup85*·Seh1 (compare lane 4 with 10).

**Fig. 4: Nup60 couples Mlp1 filament recruitment and Mlp1 docking onto the Y-complex.**

We then tested the same set of GST-Nup60 constructs for their capacity to bind a recombinant fragment of Mlp1. We used the previously identified N60BD of Mlp1 (aa382–620, Mlp1 N60BD)⁴⁰, which was N-terminally tagged with NusA-His₆ for improved protein stability and expression. The GST-Nup60(200–318) construct exhibited robust affinity for Mlp1 N60BD (Fig. 4b, lane 2 of the lower panel). Deleting SLiM A from Nup60 largely retained the affinity for Mlp1 N60BD (compare lane 2 with 4). Correspondingly, SLiM A alone exhibited only a weak affinity for Mlp1 N60BD (compare lane 2 with 3). An additional deletion of SLiM C had no major effect on Mlp1 N60BD binding (compare lane 4 with 5). Consistently, SLiM C alone exhibited only a minor affinity for Mlp1 N60BD (lane 10). Further dissection of SLiMs B and C showed that deletion of SLiM B had a strong effect on Mlp1 N60BD binding (compare lane 4 with 6). Importantly, when testing the affinities of SLiMs A, B and C for Mlp1 N60BD in isolation, SLiM B displayed the highest affinity for Mlp1 N60BD (compare lanes 3, 9 and 10). This establishes the 18-aa-long Nup60 SLiM B as a critical contact point for the Mlp1 filaments.

Comparing the affinities of the three Nup60 SLiMs for Nup85*•Seh1 and Mlp1 N60BD, SLiM B binds exclusively to Mlp1 N60BD, SLiM C binds primarily to Nup85*•Seh1 and SLiM A has the capacity to interact with both Nup85*•Seh1 and Mlp1 N60BD under the conditions tested (Fig. 4c). These data define the detailed molecular architecture of the junction connecting the NPC basket and the NPC core. The interaction pattern of this tripartite junction suggests that SLiM A may sense the presence of basket filaments and Y-complex simultaneously, acting as a ‘coincidence detector’.

XL–MS confirms Nup60 interactions with Nup85 and Mlp1

For a refined understanding of the NPC basket-core connection, we modelled Nup60 interactions with either Nup85•Seh1 or Mlp1 by AlphaFold-Multimer. The Nup60 region spanning SLiMs B and C (aa260–318) is predicted to form two α-helices connected by a linker, with each SLiM containing a short α-helix (Fig. 4d). The α-helix of Nup60 SLiM C wedges between the ‘crown’ and ‘trunk’ of the Nup85 α-solenoid fold⁵⁴ (Fig. 4d). In contrast, no contacts were predicted between SLiM B and Nup85 or Seh1, consistent with our in vitro binding assays (Fig. 4b). AlphaFold-Multimer also did not predict an interaction of Nup60 SLiM A with Nup85•Seh1, possibly because their affinity is too low (Fig. 4b, lane 3). To test the validity of the model, we performed XL–MS experiments with the reconstituted Nup60(285–318)•Nup85*•Seh1 complex and mapped the DSS cross-links onto the predicted structure (Fig. 4d,e and Extended Data Figs. 5a,b and 6a–c). Supporting the structural model, the K307 residue of the Nup60 SLiM C α-helix cross-linked specifically to the Nup85 K163 residue, positioned in a groove between the crown and trunk of Nup85. The measured distance between these cross-linked lysine residues satisfies the maximal distance restraints imposed by the DSS cross-linker (<30 Å between Cα atoms). A second cross-link was detected between the Nup60 K286 residue and Nup85 K238, providing additional support for the predicted relative orientation of SLiM C with respect to Nup85 (Fig. 4d). Notably, despite the large number of surface-exposed lysine residues in Nup85*•Seh1 (29 in Nup85* and 22 in Seh1), only Nup85 K163 and K238 were cross-linked to Nup60 SLiM C, suggesting that the intrinsically disordered Nup60 protein binds to the Y-complex in a defined orientation.

We then modelled Nup60 interactions with Mlp1 following the same approach. The Mlp1 N60BD construct (aa390–620) was predicted to form two dimeric coiled-coil segments, arranged in a V-shape with an acute angle of ~45° (Fig. 4f). The apex of the V-shaped Mlp1 structure, which connects and orients the two coiled-coil segments, is decorated by short α-helices, located on opposite sides of the apex. Nup60(260–318) forms two α-helices connected by a linker similar to the Nup60•Nup85•Seh1 model (Fig. 4d,f). In contrast, SLiM B is predicted to wedge into a groove on the C-terminal coiled-coil segment of Mlp1 N60BD close to the hinge region, whereas SLiM C remains unbound (Fig. 4f). Interestingly, the Mlp2 N60BD is predicted to interact with SLiM B of Nup60 in a similar fashion (Extended Data Fig. 6h). Noting that a segment of human Tpr is predicted to adopt a structure almost identical to Mlp1 N60BD (Extended Data Fig. 4), we asked whether AlphaFold-Multimer predicts a similar interaction mode between human Nup153 and Tpr, which have been shown to interact directly in the past⁵⁷. We ran an unbiased prediction using full-length Nup153 and a Tpr(390-620) homodimer. Interestingly, AlphaFold-Multimer predicted that Nup153(305–320) forms an α-helix that interacts with the Tpr(390–620) homodimer in a fashion identical to the Nup60 SLiM B interaction with the Mlp1 N60BD (for a superposition of the predicted structures, see Extended Data Fig. 6g). The predicted interactions are consistent with previously performed in vitro binding assays demonstrating a direct interaction between recombinant Nup153(228–611) and Tpr(172–651) (ref. ⁵⁷). In a yeast two-hybrid assay, Tpr(172–651) interacted with Nup153(228-439), but neither interacted with Nup153(1–244) nor Nup153(337–611) (ref. ⁵⁷), which is in line with our structural modelling. Hence, despite low overall sequence conservation between yeast Nup60 and human Nup153, as well as between yeast Mlp1 and human Tpr, the predicted interactions are well conserved.

To further test the validity of the model, we performed XL–MS experiments with the reconstituted Nup60(267–284)•Mlp1 N60BD complex and subsequently mapped the identified DSS cross-links onto the structure (Fig. 4f,g and Extended Data Figs. 5a,c and 6d–f). The majority of identified inter-protein cross-links (14 out of 15) mapped onto the C-terminal coiled-coil segment of Mlp1 N60BD (Fig. 4g) even though both coiled-coil segments of the V-shaped Mlp1 model contain a large number of surface-exposed lysine residues (26 in the C-terminal and 12 in the N-terminal segment). The 15-residue stretch (aa559–573) within the Mlp1 N60BD forms a cross-linking hotspot, where Mlp1 K559, K563, K566, K568 and K573 are identified as being cross-linked to both K267 and K268 of the Nup60 SLiM B (Fig. 4g). In each of these instances, at least one chain of the Mlp1 N60BD dimer satisfies the DSS distance restraints. Moreover, the cross-link between K267 of Nup60 and K545 of Mlp1 N60BD is satisfied by one of the Mlp1 chains. The cross-link between K267 of Nup60 and K537 of Mlp1 N60BD, although violated by distance, can be explained by the flexibility of the SLiM B N-terminus. Only two cross-links, involving K267 and K268 of Nup60 and K590 of Mlp1 N60BD, remain unexplained. Thus, the AI-predicted interaction patterns are in excellent agreement with our reconstitution and XL–MS experiments and confirm a division of labour between SLiM B (Mlp1 contact) and SLiM C (Nup85 contact).

Mutational analyses verify the interaction surfaces

To verify the critical interfaces of the Nup60•Nup85•Mlp1 junction, we introduced rationally designed point mutations. The interaction between Nup60 SLiM C and Nup85 is predicted to involve a salt bridge between Nup60 R306 and Nup85 D216 or the adjacent E218 (note that out of the five high-confidence models generated by Alphafold-Multimer, residue R306 can accommodate two alternative salt bridges) (Fig. 5a). A second salt bridge is formed between Nup60 K309 and Nup85 E177 or E176. Hydrogen bonding between Nup60 N296 and the Nup85 E218-P219 peptide group is expected to further stabilize the interface (Fig. 5a). Notably, the NPY triad (aa296–298) of Nup60 is conserved across yeast species (Extended Data Fig. 7a). Hence, we generated mutations targeting these interactions in Nup60 and Nup85 (alanine substitutions or charge inversions) and tested the ability of recombinant GST-Nup60(285–318), spanning SLiM C, to interact with Nup85*•Seh1. The in vitro binding assays showed that alanine substitutions of Nup60 N296 and Y298 reduced the affinity for Nup85*•Seh1, while mutating P297 to alanine had a minor effect (Fig. 5b and Extended Data Fig. 7b). A double mutant, in which both Nup60 R306 and K309 were mutated to E, also had a disruptive effect (Fig. 5b). Mutating the relevant Nup85 residues also disrupted the Nup60 interaction, although to a lesser extent (Fig. 5b).

For the Nup60 SLiM B interface with Mlp1, AlphaFold predicted a salt bridge between Nup60 D281 and Mlp1 R532, and a network of hydrogen bonds involving Nup60 S270 and N271, and Mlp1 D524, E561 and E564 (Fig. 5c). To assess the relevance of these residues, we examined how mutations impacted the binding strength between recombinant GST-Nup60(267–284), spanning SLiM B, and Mlp1 N60BD. Mutating Nup60 D281 to K reduced the affinity for Mlp1, whereas mutating Nup60 S270 to D abolished binding to Mlp1 (Fig. 5d and Extended Data Fig. 7c). A double mutation, in which both S270 and N271 were mutated to A, had a similarly strong effect (Fig. 5d). Conversely, mutating Mlp1 D524 to A, Mlp1 R532 to E or mutating both E561 and E564 to A strongly reduced the affinity between Mlp1 N60BD and the Nup60(267–284) (Fig. 5d). In summary, our integrative approach, combining in vitro binding assays (Fig. 4b), XL–MS (Fig. 4d–g) and point mutagenesis (Fig. 5a–d), provides robust support for the validity of the AlphaFold models.

Reconstitution of the basket–outer ring junction in vitro

Having identified the Nup60•Mlp1 and Nup60•Nup85 binary interfaces, we set out to obtain a structural model of the entire Nup85•Nup60•Mlp1 junction. Because large multi-protein structures can pose a challenge for AlphaFold, it became necessary to truncate the proteins further. We used the crown-trunk junction (aa101–430) of Nup85 (ref. ⁵⁴), two copies of Mlp1 covering the hinge region and shortened coiled-coil segments (aa450–580), and Nup60(260–318), spanning SLiMs B and C, as before. The predicted model of the composite ternary complex recapitulates the main features of the binary interactions, yet offers additional insights (Fig. 5e). Nup60 now simultaneously interacts with Mlp1 and Nup85 via SLiM B and SLiM C, respectively. In doing so, it stitches the Y-complex and basket filaments together. The hinge region of Mlp1 lies directly opposite to Nup85, yet, the two proteins exhibit no noticeable contacts, suggesting that the Nup85•Nup60•Mlp1 junction could be intrinsically flexible. These predicted features align with a host of previous observations suggesting that the Mlp1/Tpr filaments can exhibit extensive movements on the NPC core⁵⁸. Of note, the predicted interfaces of the tripartite junction are evolutionarily conserved across yeast species, highlighting their structural relevance: the surface patch of Mlp1, which interacts with Nup60 SLiM B, displays a high degree of conservation across yeast species (Extended Data Fig. 8a–c). Similarly, multiple residues in the groove of Nup85, which accommodates the Nup60 SLiM C, are conserved (Extended Data Fig. 8c).

To reconstitute a supramolecular assembly comprising the Nup60 suspension cable, Y-complex and part of the Mlp1 filaments, we devised a step-wise approach. First, the affinity-purified Y-complex was incubated with recombinant Nup60(200–318) spanning SLiMs A, B and C (generated by TEV protease cleavage of GST-TEV-Nup60(200–318)). The Y-complex was captured on nanobody-coated anti-FLAG beads using a FLAG-tagged nanobody (VHH-SAN8-SNAP-FLAG), which binds Nup84 (ref. ⁵⁹). Next, NusA-His₆-Mlp1 N60BD was added, and after washing, the interacting proteins were eluted using a FLAG peptide. As expected, Nup60(200–318) alone bound to the Y-complex (Figs. 2e and 5f). Importantly, Mlp1 N60BD co-purified with the Y-complex at near-stoichiometric amounts in the presence of Nup60(200–318) (Fig. 5f). In contrast, Mlp1 N60BD did not co-purify with the Y-complex when GST was used as a negative control. This shows that docking the Mlp1 basket filaments to the Y-complex critically depends on Nup60, which organizes a tripartite protein junction via adjacent SLiMs.

Nup60 co-recruits Mlp1 and Y-complex to synthetic membrane

Traditionally, Nup protein–protein interactions were studied in solution. However, the behaviour of membrane-anchored Nups may differ substantially⁶⁰. Membrane anchoring can affect local protein concentration, orientation and accessibility towards interacting partners. Thus, membranes can act as platforms for higher-order self-assembly, but may also restrict interactions. Moreover, the lateral diffusion of membrane-tethered Nups could be restricted to specific regions within the membrane, for example, curved membranes⁵³ or laterally segregated lipid domains⁶¹, leading to anisotropic lateral diffusion. We therefore interrogated the assembly of Nup60, Mlp1 and the Y-complex on a chemically defined membrane in vitro, using a previously established ‘visual biochemistry’ approach⁴⁰ involving multicolour detection of recombinant proteins on giant unilamellar vesicles (GUVs; 10–20 μm in diameter).

We employed a set of recombinant Nup60 constructs C-terminally tagged with monomeric green fluorescent protein (mGFP) as well as recombinant Mlp1 N60BD N-terminally tagged with NusA-His₆ and C-terminally tagged with mCherry-FLAG (Fig. 6a,b). The Y-complex was tandem-affinity purified from an S. cerevisiae strain expressing Nup133 with a C-terminal SNAP-TAP tag and Nup120 with a C-terminal 6xFLAG tag (Fig. 6b). The Y-complex was fluorescently labelled with Alexa Fluor 647 via the SNAP tag. We chose to fluorescently label Nup133 because it is positioned at one end of the Y-complex, away from Nup85 (Fig. 2b) and tends to dissociate more readily than other subunits⁴⁶. Thus, the labelled Nup133 would serve as a sensitive indicator demonstrating that Nup60 recruits an intact Y-complex through Nup85. We first tested whether recombinant Nup60 (aa27–318ΔHR; abbreviated as Nup60**) can bind to GUVs and recruit both Mlp1 N60BD and the Y-complex. The Nup60** construct contains all relevant SLiMs, but lacks the N-terminus, HR and FG repeats for improved protein stability. Indeed, co-incubation of Nup60**, Mlp1 N60BD and the Y-complex with GUVs resulted in the enrichment of all proteins on the membrane (Fig. 6c). Both Y-complex and Mlp1 fluorescence intensities positively correlated with Nup60 fluorescence intensity (Fig. 6d,e). Membrane recruitment was specifically mediated by the N-terminal AH of Nup60, as the introduction of an I36R mutation, which disrupts the hydrophobic face of the AH, abolished the recruitment of all three proteins (Fig. 6c).

**Fig. 6: Nup60 SLiMs co-recruit basket filaments and Y-complexes to a synthetic membrane.**

We then tested how deleting the Nup60 SLiMs, which mediate interactions with Mlp1 N60BD and the Y-complex in vitro, affects membrane recruitment. Deletion of either SLiM A or SLiM C of Nup60** decreased the co-recruitment of the Y-complex compared with wild-type Nup60** (Fig. 6c), in agreement with the in vitro binding assays (Fig. 2c). To gain quantitative insights into the relative recruitment efficiencies of different Nup60 constructs and to account for the slight variation in the levels of membrane-bound Nup60 across different constructs, we fitted linear regression models to these data and extracted the slope values (m) as population-wide estimates for the relative recruitment efficiency (Fig. 6d,e). This analysis revealed that deletion of either SLiM A or SLiM C decreased Y-complex recruitment ~4–5-fold compared with wild-type Nup60** (Fig. 6d). Interestingly, the recruitment of Mlp1 N60BD increased upon deletion of either Nup60 SLiM A or SLiM C, suggesting that Y-complex binding attenuates Mlp1 N60BD recruitment under the conditions tested (Fig. 6e). Whether this represents a physiologically relevant weakness of the protein junction remains to be tested. Double deletion of both SLiMs A and C reduced the membrane recruitment efficiency of the Y-complex by a factor of ~50 (Fig. 6d). As predicted, deletion of SLiM B abolished the recruitment of Mlp1 N60BD, but did not affect recruitment of the Y-complex (Fig. 6d,e), consistent with our in vitro binding assays (Fig. 4b).

These results confirm that the membrane can act as a platform for the higher-order, SLiM-dependent assembly of the Y-complex-Nup60-basket filament junction. They also suggest that the membrane may attenuate the simultaneous interaction of Mlp1 and the Y-complex with Nup60, potentially creating a built-in instability of the interface between the NPC core and the basket. This may account for the high exchange rate between basket-bound and free nucleoplasmic pools of Mlp1/2 proteins²⁸.

Nup60 SLiMs organize the Mlp1-outer ring junction in cells

Having determined how Nup60 couples Mlp1 filaments with the Y-complex, we sought to verify these findings in vivo. To this end, we examined the localization of both Nup60, C-terminally tagged with mCherry, and Mlp1, C-terminally tagged with mGFP. Both Nups were expressed from plasmids under their endogenous promoters in a nup60∆mlp1∆ strain and visualized by live cell fluorescence microscopy. Wild-type Nup60 and Mlp1 displayed a punctate NE staining that is characteristic of NPCs (Fig. 7a and Extended Data Fig. 9a). Nup60 is distributed throughout the NE, whereas Mlp1 is absent in the NE region adjacent to the nucleolus^7,21. This implies a discrepancy in the quantities of Nup60 and Mlp1 at the NE, with all NPCs containing Nup60, but only a portion containing Mlp1. We determined the NE enrichment factor of both Nups by quantifying the ratio between NE and nucleoplasm (N) fluorescence intensities. Due to some cells losing plasmids, only cells expressing both proteins were taken into account. Notably, upon deletion of SLiM B, Mlp1 mislocalized into the nucleoplasm and formed prominent foci in ~90% of cells, which may reflect an aggregated state of Mlp1 (Fig. 7a,b). By comparison, the localization of Nup60 itself was largely unaffected (Fig. 7a), probably because Nup60 remains tethered to the NE via multiple interactions—to the INM via its AH and to the NPC core via the HR and SLiMs A and C. Collectively, these data confirm the importance of SLiM B for the positioning of the Mlp1 basket filaments in vivo and in vitro.

**Fig. 7: Nup60 SLiMs A, B and C organize the basket filament–outer ring junction in cells.**

Next, we explored under which conditions Nup60 will detach from the NPC. Deleting the HR within the full-length Nup60 enriched Nup60 in the nucleoplasm, but some Nup60 was retained at the NE (Fig. 7a,b), suggesting that Nup60 oligomerization is not strictly required for NPC localization. The positioning of Mlp1 remained mostly unchanged in this mutant. To test for the role of the Nup60–Y-complex connection, we deleted SLiM A and SLiM C, alone or in combination, in a Nup60∆HR background. While deletion of either SLiM A or SLiM C slightly increased the mislocalization of Nup60 (Fig. 7b), the deletion of both SLiMs strongly mislocalized Nup60 and consequently Mlp1, which formed foci in ~70% of cells (Fig. 7b). Thus, our data indicate a multivalent tethering mechanism, where Nup60 localization is governed by a lipid anchor (AH) and at least two protein–protein interactions with the NPC core (SLiMs A and C), possibly reinforced by oligomerization (HR). In contrast, Mlp1 localization primarily relies on the structurally distinct Nup60 SLiM B, whose efficiency might be tuned by the occupancy of the adjacent SLiMs A and C. Our in vivo findings align well with the in vitro biochemical reconstitution of the basket–NPC outer ring junction, providing strong support for our model of how the basket is docked to the core of the NPC.

Discussion

A basket-like structure on the nuclear side of the NPC was first observed in the early 1990s^1,2,3,62. However, despite decades of research, key aspects of the basket’s structure remain unclear, positioning the basket as a final frontier in the structural elucidation of the NPC. Through an integrated approach, including biochemical reconstitution in solution and on synthetic membranes, high-resolution connectivity mapping and AI-guided structure–function interrogation, we have now identified the elusive docking mechanism of basket filaments onto the NPC core. Additionally, we have uncovered a basis for the intrinsic flexibility of basket filaments. These results provide the foundation for understanding at the molecular level how the nuclear side of the NPC is functionalized.

Multivalent network connecting the NPC core and basket

The critical junction between the core scaffold of the NPC and the nuclear basket consists of three adjacent, functionally specialized SLiMs of Nup60, exhibiting affinities to both Nup85 and Mlp1. We consider three potential connection states of Mlp1, each characterized by the Nup60 SLiMs being utilized: Firstly, Mlp1 can independently bind to Nup60 via SLiM B, without involving Nup85. Due to Nup60 interacting with the membrane through its AH, this scenario could lead to an INM-bound Mlp1 filament diffusing on the inner nuclear membrane before interacting with the NPC core, for example, during NPC assembly. In the second state, the Nup60-tethered Mlp1 filament would be anchored to Nup85 through the Nup60 SLiM C. In the third state, Nup60 SLiM A would simultaneously bind Mlp1 and the Y-complex, acting as a coincidence detector. The molecular details of these interactions remain to be identified in future higher-order reconstitutions of the NPC. The cumulative strength of the basket–NPC core interaction would be governed by the avidity among the three identified SLiMs (Fig. 7c).

The nature of these interactions prompts the question of why a seemingly straightforward task—tethering Mlp1 to Nup85—is addressed in such a complex manner. We propose three reasons: Firstly, the remarkably interwoven NPC core–basket junction is conformationally flexible, which explains the ability of basket filaments to undergo axial and radial deformations¹. Secondly, as an architectural principle, the built-in redundancy of the multivalent NPC core–basket junction may accommodate for disruptions caused by constrictions/dilations of the NPC core⁶³. Loss of one SLiM interaction would not entirely break the junction. Thus, the multivalent network may act as a stress-absorber, keeping the basket components in place. Building on our work, future studies can investigate how the SLiM network is utilized and regulated to enable or inhibit basket assembly and to tune basket assembly in response to changes in nuclear RNA metabolism.

Domain architecture and flexibility of basket filaments

The putative nuclear basket densities, as recently revealed by a cryo-ET reconstruction of the S. cerevisiae NPC, extend ~20–30 nm into the nucleoplasm when measured from the nuclear outer ring²⁴. However, these densities probably represent only a smaller portion of the entire basket, which is difficult to capture by cryo-ET given its flexibility. Improved biochemistry and EM allowed us to examine 77% of the likely structured parts of Mlp1(1–1450). Our data indicate that Mlp1 forms a parallel dimer characterized by extended coiled coils interspersed with pliable linkers. We experimentally determined an end-to-end length of 130–170 nm for a construct, which consists of all except one of the extended coiled-coil regions. It is possible that Mlp1 adopts a more compact configuration in cells, where the coiled-coil segments might intertwine, potentially assisted by other Nups or by attachment to the NPC core. This hypothesis requires further investigations.

Importantly, we identify the N60BD of Mlp1 as a key structural element and interaction hub, unresolved in prior studies. This Mlp1 domain may adopt a V-shaped structure, introducing a kink in the Mlp1 filament, thereby orienting the N- and C-terminal coiled-coil segments that emanate from it. Assuming that the Nup85•Nup60•Mlp1 junction forms the NPC core-proximal part of the basket, the apex of the Mlp1 N60BD (Fig. 4f) could function as the structural apex of the entire basket (Fig. 7c). The long C-terminal segment of Mlp1, following the N60BD, may constitute the primary body of Mlp1 filaments, probably featuring three long coiled-coil segments connected by linkers. Notably, the AlphaFold-predicted V-shaped structure of the Mlp1 N60BD and its hypothetical equivalent in human Tpr exhibit remarkably similar folds (Extended Data Fig. 6g), despite low sequence identity (18%). This indicates an evolutionarily conserved structure–function relationship for this critical element from yeast to human. The overall organization of coiled-coil segments also seems conserved between yeast Mlp1 and human Tpr (Extended Data Fig. 4).

Our data suggest a high degree of intrinsic flexibility facilitated by linkers between the coiled coils. This could explain the observed flexibility of basket filaments, documented back in 1998⁵⁸, when axial compression, extension and radial dilation of the basket was first observed. Our study elucidates the dual nature of the nuclear basket, exhibiting both rigidity in the coiled-coil segments and flexibility in the hinge regions. Drawing parallels with man-made structures such as suspension bridges and space frames, similar engineering principles may apply to Mlp/Tpr coiled-coil proteins, optimizing basket performance and durability under diverse conditions. Linkers between the coiled coils presumably enable the nuclear basket to accommodate cargoes of various sizes⁵⁸. Moreover, the flexible suspension of the filaments on the Y-complex seems adapted to respond to each dilation or constriction of the NPC core, which involves a rearrangement of the Y-complex rings⁶³. On the other hand, the rigid parts may act as spacers that keep basket-associated export adaptors and heterochromatin^6,21 at a certain distance, preventing the entry point into the NPC’s transport channel from getting clogged.

The NPC core–basket filament interactions in a membrane context

Why is it crucial to also anchor the basket–NPC core junction on the pore membrane? It is conceivable that the ALPS motifs of Nup133 and Nup120 lack adequate membrane affinity for the de novo recruitment of the Y-complex to the NE membrane^35,53. Notably, we identify a third membrane attachment point for the Y-complex, specifically the tethering of the Nup85•Seh1 arm to the membrane through the Nup60 AH. Consequently, there are at least three distinct membrane attachment sites for the Y-complex. Of note, earlier pulse labelling experiments suggested that the head part of the Y-complex (Nup85•Seh1•Nup120) and Nup60 are incorporated into the maturing NPCs simultaneously⁶⁴. This intuitively points to a lock-and-key mechanism for NPC assembly, where basket filament assembly on a membrane is intricately linked to the assembly and integrity of the NPC core.

In fact, dimerization (or higher-order oligomerization) of Nup60 through its HR is expected to create another layer of membrane-tethered, multivalent interactions. Nup60 dimerization would cross-link two Y-complexes of the nuclear ring (Fig. 7c). So far, the circularization of Y-complexes has been attributed to head-to-tail interactions between the N-terminal extension of Nup133 and Nup120 of a neighbouring Y-complex⁴⁵. Our data suggest that these interactions and Y-complex ring formation could be reinforced by Nup60 connecting the Nup85 arms of neighbouring Y-complexes. As a consequence, both the circularization and membrane attachment of the NPC’s nuclear ring is promoted. As reported earlier, besides self-interaction, the Nup60 HR may also bind to as-yet-unidentified components of the NPC core⁴⁰. Multiple lines of evidence suggest that multivalent interactions of membrane proteins can apply compressive stress to membrane surfaces, which can change membrane curvature⁶⁰. How the membrane-tethered NPC core–basket filament junction and other lipid-interacting Nups (for example Nup1) could bend the pore membrane is an interesting avenue for future studies.

Functional implications of basket heterogeneity in cells

Two distinct NPC core scaffold variants have been identified in S. cerevisiae: NPCs featuring a single nuclear ring and a recently discovered isoform with a double nuclear ring^23,24. Assuming that all Nup85 binding sites in a double nuclear ring NPC can interact with Nup60•Mlp1, this isoform could accommodate up to 16 copies of Nup60, recruiting up to 16 dimeric Mlp1 filaments (that is, 32 Mlp1 monomers). As Nup60 directly interacts with Nup2 (ref. ⁴⁰), the Nup2 stoichiometry would also double. Consequently, basket filament stoichiometry in cells could vary from 0 (‘basketless’ NPC adjacent to the nucleolus) to 8 (single nuclear ring) to 16 (double nuclear ring), resulting in structural heterogeneity on the nucleoplasmic face of the NPC. This may explain discrepancies in basket subunit stoichiometry reported in different studies^{41,42,43,44,65}. In principle, a double ring could template a double basket, and vice versa, suggesting a potential reinforcement of the double ring architecture by the basket. This has implications for basket-associated nuclear transport adaptors and enzymes^13,21. An intriguing possibility is that doubling the basket filament number, adaptor and enzyme stoichiometry could enhance the export competency of specific NPCs, consequently boosting NPC functionality locally.

In conclusion, our findings shed light on the once enigmatic process of constructing a basket on the nuclear face of the NPC, providing a comprehensive molecular understanding of a missing link of NPC structural biochemistry. Building upon this foundation, future studies can investigate the involvement of the basket in NPC and pore membrane biogenesis, explore the origins and implications of NPC asymmetry, and embark on the question of how NPC heterogeneity encodes cell identity and function.

Methods

The study complies with all relevant ethical regulations.

The AlphaFold-Multimer screen for protein–protein interactions

The AlphaFold-Multimer deep learning model was used to predict the structures of all possible binary protein complexes between S. cerevisiae Nup60 SLiMs and all other Nups (in total, 264 unique pairwise combinations). Nup60 query SLiMs were defined as stretches of 31 consecutive amino acid residues containing at least 10 highly conserved residues across yeast orthologues. The computational results were obtained on the Cloud Infrastructure Platform high-performance computing cluster. For every prediction, the mean ipTM score of models ranked one to five (confidence estimate of protein–protein interface) was calculated and plotted in the heatmap using Python data visualization library seaborn (v0.11.2). AlphaFold3 (ref. ⁶⁷) models of Nup60(260–318)•Nup85•Seh1, Nup60(260–318)•dimeric Mlp1(390–620) and Nup85(101–430)•Nup60(260–318)•dimeric Mlp1(450–580) recapitulated the positioning of polypeptide chains predicted by AlphaFold-Multimer.

Reagents, plasmids and yeast strains

Reagents, plasmids and yeast strains used in this study are listed in Supplementary Tables 1–3, respectively. All plasmids were verified by either Sanger sequencing (PCR-based molecular cloning approaches) or diagnostic restriction digest (subcloning). Yeast genes were chromosomally tagged by a standard one-step PCR-based technique⁶⁸. Microbiological techniques followed standard procedures.

Recombinant protein expression in E. coli

Recombinant proteins were expressed in E. coli BL21 CodonPlus (DE3) RIL cells. E. coli cultures were grown in lysogeny broth (Miller) medium supplemented with expression vector-compatible antibiotics (140 r.p.m., 37 °C) to optical density at 600 nm of 0.30–0.40, transferred to 130 r.p.m. 23 °C, induced with 0.5 mM IPTG (final concentration) at optical density at 600 nm of 0.70–0.80 for 3 h before harvesting. Pellets were frozen in liquid nitrogen and stored at −20 °C.

Expression of MBP-3C-Mlp1(17–1,137)-His₆ in insect cells

MBP-3C-Mlp1(17–1,137)-His₆ was produced using baculovirus expression vector system. To generate V0 baculovirus stock, EMBacY bacmid harbouring the open reading frame of MBP-3C-Mlp1(17–1,137)-His₆ was transfected into Sf9 insect cells (Expression Systems). The recombinant fusion protein was expressed in Hi5 insect cells (Expression Systems) at 27 °C. The insect biomass was collected 3 days after proliferation arrest and flash frozen in liquid nitrogen.

Protein purification

Protein purification procedures, including affinity resins used and buffer solution compositions, are summarized in Supplementary Table 4. All steps were performed at 4 °C using pre-chilled buffers and containers. Briefly, frozen pellets of biomass were thawed, resuspended in lysis buffer and lysed using either Avestin Emulsiflex C3 high-pressure homogenizer (for E. coli/insect pellets) or Fritsch Pulverisette 6 planetary ball mill (for S. cerevisiae pellets). The lysate was cleared by high-speed centrifugation (at 48,000g for 20 min). Cleared lysate was incubated with corresponding beads for 2 h at 4 °C on tube rotator. After incubation, beads were collected by centrifugation and batch washed in 2 ml microcentrifuge tubes using wash buffer (either 3× or 5× 1 ml, for 1 min each with 30 s at 500g centrifugation steps between washes). Beads were then transferred to 1 ml Mobicol columns (MoBiTec) and eluted by adding elution buffer and incubating for 30–90 min at 4 °C. All protein solutions were cleared of aggregates by high-speed centrifugation (10 min at 16,000g) before downstream use. Protein concentration was measured using Bio-Rad protein assay based on the Bradford dye-binding method using bovine serum albumin (20 mg ml⁻¹, NEB) solutions for calibration.

Nup60 oligomerization assay

NusA-His₆-Nup60(48–162) and SUMO-Nup60(48–162)-FLAG as well as binary combinations with solubility/affinity tags were co-expressed in E. coli BL21 CodonPlus (DE3) RIL cells following the procedure described above in ‘Recombinant protein expression in E. coli’ section. Pellets of biomass were then subjected to two-step affinity purification as summarized in Supplementary Table 4 and briefly described above in ‘Protein purification’ section.

ITC of Nup60 HR

NusA-His₆-Nup60(48–162) and NusA-His₆ (negative control) were purified as specified in Supplementary Table 4. After 16 h dialysis in HIS-PB (300 mM NaCl, 50 mM Tris pH 7.5, 50 mM imidazole and 1.5 mM MgCl₂) samples were diluted to final concentration of 170 μM and measured in dilution (dissociation) ITC setup on MicroCal PEAQ-ITC calorimeter (Malvern). Baseline correction, thermogram integration and fitting of simple dimer dissociation model was performed using MicroCal PEAQ-ITC Analysis Software.

Size exclusion chromatography coupled with multi-angle light scattering of Nup60 HR

NusA-His₆-Nup60(48–162) and NusA-His₆ (negative control) were purified as specified in Supplementary Table 4. After 16 h dialysis in HIS-PB (300 mM NaCl, 50 mM Tris pH 7.5, 50 mM imidazole, 1.5 mM MgCl₂) samples were diluted to a final concentration in range of 100–200 μM and loaded through a 50 μl injection loop. The samples were analysed at constant flow rate of 0.5 ml min⁻¹ on Superdex 200 Increase 10/300 GL column (GE Healthcare) connected to ӒKTA pure 25 chromatography system coupled with miniDAWN MALS detector (Wyatt Technology). The analysis was performed using ASTRA 8.2.0 software package (Wyatt Technology).

In vitro binding assays

In every binding assay setup, purified GST-Nup60 fusion proteins were used as baits (GST alone as negative control). Binding assay reactions contained 25 μl of Glutathione Sepharose 4B (Cytiva) beads (50 μl of 1:1 bead slurry), 0.25–0.75 nmol (depending on the experiment) of either GST or GST-Nup60 fusion proteins and 1–1.3× molar excess of prey proteins (Y-complex, Nup85*•Seh1 or Mlp1 N60BD) at the final volume of 100–250 μl. Binding reactions with the Y-complex were set using PB-100 buffer solution (100 mM NaCl, 50 mM Tris pH 7.5, 1.5 mM MgCl₂ and 0.5 mM dithiothreitol (DTT)), whereas binding reactions with either Nup85*•Seh1 or Mlp1 N60BD were set using PB-200 buffer solution (200 mM NaCl, 50 mM Tris pH 7.5, 1.5 mM MgCl₂ and 0.5 mM DTT). After 1 h incubation at 4 °C on tube rotator, beads were batch washed with either PB-100 (Y-complex, 2 × 1 ml for 1 min each) or PB-200 (Nup85*•Seh1 or Mlp1 N60BD, 5 × 1 ml for 2 min each) buffer solutions. Proteins were eluted by adding 50 μl GSH-EB (100 mM NaCl, 50 mM Tris pH 7.5, 1.5 mM MgCl₂, 0.5 mM DTT and 20 mM reduced GSH) and incubated for 30 min at 4 °C on tube rotator. Native eluates were mixed with 4× sample buffer (SB), boiled for 5 min at 95 °C and analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and Coomassie staining.

Chemical cross-linking of reconstituted protein complexes

Reconstituted complexes were prepared as described above in ‘In vitro binding assays’ section using HEPES-containing elution buffer instead of Tris. Protein complexes were chemically cross-linked with either isotopically coded disuccinimidyl suberate (1:1 molar mix of DSS-H12/D12, Creative Molecules) in case of the Y-complex or isotopically unlabelled DSS (Thermo Scientific) in case of Nup85*•Seh1 and Mlp1 N60BD for 30 min at 30 °C at 1,000 r.p.m. on a thermomixer. Reaction was quenched by adding 100 mM ammonium bicarbonate (final concentration) and incubating for 10 min at 30 °C at 1,000 r.p.m. Cross-linked samples were mixed with 4× SB, boiled for 5 min at 95 °C and analysed by SDS−PAGE and Coomassie staining.

Sample preparation for MS analysis

The Coomassie-stained gel band was destained with a mixture of acetonitrile (VWR) and 50 mM ammonium bicarbonate (Sigma). The proteins were reduced using 10 mM dithiothreitol (Roche) and alkylated with 50 mM iodoacetamide (Sigma). Trypsin (Promega; Trypsin Gold, Mass Spectrometry Grade) was used for proteolytic cleavage. Digestion was carried out at 37 °C overnight. Formic acid (Fisher chemical) was used to stop the digestion and peptides were extracted using a mixture of acetonitrile and formic acid. Extracts were speedvacced until dryness and resuspended in 0.1% trifluoroacetic acid (Thermo Scientific) 2% acetonitrile.

Liquid chromatography MS analysis

Peptides were separated on an ultimate 3000 nano-flow chromatography system (Thermo Fisher), using a pre-column for sample loading (Acclaim PepMap C18, 2 cm × 0.1 mm, 5 μm, Thermo Fisher) and a C18 analytical column (Acclaim PepMap C18, 50 cm × 0.75 mm, 2 μm, Thermo Fisher), applying a segmented linear gradient from 2% to 35% and finally 80% solvent B (80% acetonitrile, 0.1% formic acid; solvent A 0.1% formic acid) at a flow rate of 230 nl min⁻¹ over 120 min.

Eluting peptides were analysed on an Exploris 480 Orbitrap mass spectrometer (Thermo Fisher) coupled with a high-field asymmetric waveform ion mobility spectrometry Pro ion-source (Thermo Fisher) using coated emitter tips (PepSep, MSWil) with the following settings: The mass spectrometer was operated in data-dependent acquisition mode with the compensation voltage set to −40, −55 and −70. The cycle time was set to 1 s. The survey scans were obtained in a mass range of 375–1600 m/z, at a resolution of 120,000 at 200 m/z, and a normalized AGC target at 100%. The selected ions were isolated with a width of 1.2 m/z, fragmented in the HCD cell at 27%, 30% and 33% collision energy, and the spectra recorded for maximum 200 ms at a normalized AGC target of 200% and a resolution of 30,000. Peptides with a charge of +3 to +8 were included for fragmentation, the peptide match feature was set to preferred, the exclude isotope feature was enabledand selected precursors were dynamically excluded from repeated sampling for 30 s.

Data analysis of MS data

Raw data were converted per compensation voltage into raw files in Freestyle and searched using the MaxQuant software package (2.0.3.0)⁶⁹ against the target sequences, the UniProt E.coli and yeast reference proteome (www.uniprot.org), as well as a database of most common contaminants. The search was performed with standard identification settings, including full trypsin specificity allowing a maximum of two missed cleavages. Carbamidomethylation of cysteine residues was set as fixed, and oxidation of methionine and acetylation of protein N-termini were set as variable modifications. All other settings were left at default. Results were filtered at a false discovery rate of 1% at protein and peptide spectrum match (PSM) level.

pLink search

To identify cross-linked peptides, the spectra were searched using pLink software (version 2.3.9)⁷⁰ against the sequences of the top ten non-contaminant proteins from the MaxQuant search sorted by intensity-based absolute quantitation values. DSS and DSS-D12 were selected as the cross-linking chemistry. Carbamidomethyl on cysteine was set as fixed, oxidation of methionine and protein N-terminal acetylation as variable modifications. Enzyme specificity was selected according to the protease used for digestion. Search results were filtered for 1% false discovery rate on the PSM level limiting the precursor mass deviation to 10 ppm. To remove low-quality PSM an additional e-value cut-off of <0.001 was applied. Cross-link maps were generated in xiNET⁷¹.

XiSearch search

To identify cross-linked peptides, the spectra were searched using XiSearch software (1.7.6.7)⁷² against the sequences of the top ten non-contaminant proteins from the MaxQuant search sorted by intensity-based absolute quantitation. DSS and DSS-D12 were selected as the cross-linking chemistry. Carbamidomethyl on cysteine was set as fixed, and oxidation of methionine and protein N-terminal acetylation were set as variable modifications. Enzyme specificity was selected according to the protease used for digestion. Search results were filtered in XiFDR with 5% false discovery rate for residue pairs limiting the precursor mass deviation to 15 ppm. Cross-link maps were generated in xiVIEW (http://xiview.org).

Reconstitution of the basket–NPC core junction

VHH-SAN8 nanobody (VHH-SAN8-SNAP-FLAG) specific for Nup84 subunit of the Y-complex⁵⁹ was purified as specified in Supplementary Table 4. Nanobody was immobilized on beads by incubating 25 μg of purified VHH-SAN8 nanobody with 250 μl of anti-FLAG beads (Anti-FLAG M2 affinity gel, Cytiva) in a volume of 600 μl for 1 h at 4 °C on a tube rotator. To wash away a free nanobody, slurry was batch washed with PB (100 mM NaCl, 50 mM Tris pH 7.5, 1.5 mM MgCl₂ and 0.5 mM DTT) (3× 1 ml for 1 min each). After final wash, beads were resuspended in 1,000 μl and aliquoted 100 μl per reaction (each aliquot contained ~25 μl of nanobody-coated beads). Purified Y-complex (Nup133-SNAP-CBP) and equimolar amounts of either GST or Nup60(200-318)-StrepII (that is, a mix of Nup60(200-318)-StrepII and GST, generated by TEV protease cleavage of GST-TEV-Nup60(200-318)-StrepII) were then added to the beads and incubated for 1 h at 4 °C on a tube rotator. Purified Mlp1 N60BD was then added and incubated for 30 min more at 4 °C on a tube rotator. After incubation, beads were batch washed with PB (3 × 1 ml for 1 min each) and eluted with 50 μl of FEB (100 mM NaCl, 50 mM Tris pH 7.5, 1.5 mM MgCl₂ and 0.5 mg ml⁻¹ FLAG peptide). Eluates were mixed with 4 × SB, boiled for 5 min at 95 °C and analysed by SDS–PAGE.

Rotary shadowing EM of Mlp1 samples

Protein samples were first diluted to a concentration of ~0.1 mg ml⁻¹ in maltose elution buffer containing 100 mM NaCl, 50 mM Tris (pH 7.5), 1.5 mM MgCl₂, 0.5 mM DTT and 30 mM maltose. Before freezing, samples were diluted 1:1 in Mabuchi spraying buffer, containing 200 mM ammonium acetate and 60% (v/v) glycerol, with the pH adjusted to 7.4. To obtain a clean surface, mica chips (quality V3; Agar Scientific) were freshly cleaved; the diluted samples were sprayed onto them, and immediately transferred into a BAL-TEC MED020 high vacuum evaporator (BAL-TEC, Liechtenstein), equipped with electron guns. To ensure a uniform coating from all sides, the mica chips were placed on a rotating table. After reaching a vacuum of 2 × 10⁻⁵ mbar, the samples were coated with 0.7 nm platinum (BALTIC) at an angle of 4–5°, followed by 7 nm carbon (Balzers) at 90°. The obtained replicas were floated off from the mica chips, picked up on 400 mesh Cu/Pd grids (Agar Scientific) and inspected in an FEI T20 G2 transmission electron microscope operated at 200 kV. Images were acquired using an FEI Eagle 4k charge-coupled device camera (both TEM and camera: formerly FEI, now Thermo Fisher Scientific). Additionally, grids were inspected in an FEI Morgagni 268D TEM (formerly FEI, now Thermo Fisher Scientific) operated at a high tension of 80 kV. Digital images were acquired using a Megaview III CCD camera (Olympus-SIS).

Lipids

1-Palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), sodium salt of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-2-dioleoyl-sn-glycerol (DAG) and brain ammonium salt of l-α-phosphatidylinositol-4,5-bisphosphate (PIP₂) were purchased from Avanti Polar Lipids. The lipid stock solutions were stored in glass containers layered with argon at −80 °C. Lipid-containing solutions were handled using glass microliter syringes (Hamilton).

GUV binding assay and image analysis

GUVs were prepared by PVA-assisted method⁷³. A mixture of lipids corresponding to 45 mol% POPE, 30 mol% POPS, 20 mol% POPC, 4 mol% PIP2 and 1 mol% DAG was used for the ER-like lipid composition. GUVs were pre-incubated with purified proteins (at final concentrations of 100 nM) for 10 min before imaging with a 60 × 1.42 NA Plan-Apochromat oil-immersion objective using DeltaVision Elite microscope (GE Healthcare) equipped with a cooled CCD camera (CoolSNAP HQ2; Photometrics). All images were taken within an imaging window of 10 min. The imaging system was controlled with softWoRx 6.5.2 software. In-built image restoration by deconvolution (ratio conservative method) was applied for all images collected. Background-corrected fluorescence intensity along the rim of the GUVs was measured manually in FIJI. Linear regression models were fitted using the least squares method.

Live cell fluorescence microscopy of yeast cells

S. cerevisiae Δnup60 Δmlp1 strain was transformed with pRS315_MLP1p_MLP1-yeGFP_NUP1t and pRS316_NUP60p_NUP60(1-539)-mCherry_NUP1t-derived constructs and selected on solid SDC-LEU-URA medium. For live cell imaging, yeast cells were grown in SDC-LEU-URA medium at 30 °C to midlog phase and collected by centrifugation. Cells were mounted on agarose pads⁷⁴ and visualized with a 60× 1.42 NA Plan-Apochromat oil-immersion objective using DeltaVision Elite microscope (GE Healthcare) equipped with a cooled CCD camera (CoolSNAP HQ2) (Photometrics). The imaging system was controlled with softWoRx 6.5.2 software. In-built image restoration by deconvolution (ratio conservative method) was applied to all images acquired.

Quantification of live cell fluorescence images

To quantify the degree of protein (either Nup60-mCherry or Mlp1-mGFP) enrichment at the NE compared with the N, nuclear rim regions (bands of 1.29 μm in width, corresponding to 12 pixels) were manually traced and straightened in FIJI. Consistent clockwise tracing ensured that after the straightening the nuclear and cytoplasmic sides of the nuclear envelope were consistently oriented across all selections. Only cells expressing both Nup60-mCherry and Mlp1-mGFP were included in the analysis. For each cell, the straightened selection of the nuclear rim was analysed in a row-wise fashion, that is, for each row of pixels the nuclear intensity I(N) and NE intensity I(NE) values were extracted. I(N) was defined as the intensity of a pixel on the nuclear side, which is distalmost from the NE in the given selection (on average, ~600 nm away from the peak fluorescence intensity corresponding to the NE). I(NE) was defined as the maximum intensity value in the row of pixels. The enrichment factor I(NE)/I(N) was defined as the ratio between median I(NE) and I(N) for each cell.

To quantify the nuclear puncta of Mlp1-mGFP, 50 cells from every condition were visually inspected (z-stack) and cells harbouring nuclear foci were counted. Only cells expressing both Nup60-mCherry and Mlp1-mGFP were included in the analysis.

Statistics and reproducibility

No statistical method was used to predetermine sample size. When analysing live cell fluorescence imaging data (Fig. 7a,b), only cells that expressed both the Nup60-mCherry and Mlp1-mGFP plasmids were included (that is, cells expressing only one plasmid due to plasmid loss were excluded). To characterize the effect of Nup60 mutations on the localization of Mlp1, it was necessary for both proteins to be present in the cells. This exclusion criterion was determined before the experiment. To control for any time-dependent effects during in vitro binding assays, binding experiments were repeated with random sample processing order. Blinding was not implemented during experiments and outcome assessments because the same investigator performed and analysed all the experiments. Experiments were performed the following number of times: twice (Figs. 3c–f, 4b,d–g and 5b,d,f and Extended Data Figs. 1i,j, 2d, 3a,b, 5a–c, 7b,c and 9a), three times (Figs. 1d, 2c,e,g, 3b, 6b–e and 7a,b and Extended Data Fig. 2a–c) and six times (Fig. 1c). The detailed summary of multiple comparison tests (two-sided Dunnett’s T3 test, α = 0.05) for Figs. 6c and 7b is provided in Source data.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository⁷⁵ under accession number PXD052015. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank M. Hartl, D. Anrather, W. Chen and N. Hartl (Max Perutz Labs Mass Spectrometry Facility) for performing mass spectrometry measurements and analyses, the Electron Microscopy Facility at Vienna BioCenter Core Facilities (VBCF) for assistance with rotary shadowing EM and A. Zile for initial experiments with Mlp1. The expression of Mlp1 in insect cells was performed by the Protein Technologies Facility of the VBCF. AlphaFold-Multimer predictions were obtained using the Vienna Biocenter CLIP cluster. A.K. was funded by ERC-COG (772032; NPC BUILD) and the NOMIS Foundation.

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Authors and Affiliations

Max Perutz Labs, Vienna Biocenter Campus, University of Vienna and Medical University of Vienna, Vienna, Austria
Edvinas Stankunas & Alwin Köhler
Vienna BioCenter PhD Program, Doctoral School of the University of Vienna and Medical University of Vienna, Vienna, Austria
Edvinas Stankunas

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Edvinas Stankunas
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Alwin Köhler
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Contributions

A.K. and E.S. conceived the study. E.S. performed experiments. A.K. and E.S. analysed all data and wrote the paper.

Corresponding author

Correspondence to Alwin Köhler.

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Extended data

Extended Data Fig. 1 AlphaFold-Multimer model of Nup60 HR dimer.

a, Superposition of rank 1-5 AlphaFold-Multimer models of Nup60(110-160) dimer. b, AlphaFold-Multimer models of Nup60(110-160) dimer shown in cartoon representation coloured by per-residue confidence score (pLDDT) (blue: high confidence, orange: low confidence). c, AlphaFold-Multimer models of Nup60(110-160) dimer shown in surface representation coloured by sequence conservation. d, AlphaFold-Multimer models of Nup60(110-160) dimer shown in surface representation coloured by Coulombic electrostatic potential. e, AlphaFold-Multimer models of Nup60(110-160) dimer shown in surface representation coloured by hydrophobicity. f, AlphaFold-Multimer model of a Nup60(110-160) dimer shown in cartoon (backbone) and ball-and-stick (sidechains) representation. Hydrophobic amino acid residues forming the buried dimeric core are labelled. g, Dilution ITC thermograms of NusA-His₆-Nup60(48-162) sample, corresponding to ITC isotherms provided in Fig. 1d. h, Dilution ITC thermograms of NusA-His₆ sample, corresponding to ITC isotherms provided in Fig. 1d. i, UV absorbance (280 nm) chromatograms of NusA-His₆-Nup60(48-162) size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS), performed in an injection concentration range of 100-200 μM. Arrows indicate monomer, putative dimer and void volume peaks. The molecular mass of the monomer peak (76 kDa, black trace) was measured at an injection concentration of 100 μM. j, UV absorbance (280 nm) chromatograms of NusA-His₆ SEC-MALS runs in an injection concentration range of 100-200 μM. Arrow indicates monomer peak. The molecular mass of the monomer peak (57 kDa, black trace) was measured at an injection concentration of 100 μM. Source numerical data are available in source data.

Source data

Extended Data Fig. 2 Input gels for in vitro binding assays.

a, SDS-PAGE gel showing native eluates of Y-complex and GST-Nup60* constructs used for the in vitro binding assays presented in Fig. 2c. b, SDS-PAGE gel showing native eluates of Y-complex and GST-Nup60 constructs used for the in vitro binding assays presented in Fig. 2e. c, SDS-PAGE gel showing native eluates of Nup85(1-564)•His₆-Seh1 and GST-Nup60 constructs used for the in vitro binding assays presented in Fig. 2g. d, SDS-PAGE gel showing reconstituted complex between Y-complex and GST-Nup60(200-318) (left lane) used as an input for DSS cross-linking (right lanes). Yellow box indicates a band of gel that was submitted for XL-MS analysis. Unprocessed gels are available in source data.

Source data

Extended Data Fig. 3 Gallery of Mlp1 rotary shadowing electron micrographs.

a, Selection of MBP-3C-Mlp1(17-1,137)-His₆ particles visible in rotary shadowing electron microscopy images acquired using FEI T20 G2 transmission electron microscope. Particles are manually grouped (boxes of different colours) by the number of visually distinct rectilinear segments (yellow traces). Scale bar, 40 nm. b, Overview images of MBP-3C-Mlp1(17-1,137)-His₆ (uncleaved, left) and Mlp1(17-1,137)-His₆ (cleaved with 3C protease, right) samples. Images were acquired using FEI Morgagni 268D transmission electron microscope. Scale bar, 200 nm.

Extended Data Fig. 4 Threading-based AlphaFold-Multimer modelling of S. cerevisiae Mlp1 and H. sapiens Tpr.

a, AlphaFold-Multimer models of Mlp1 dimers with overlapping amino acid residue boundaries (right) and corresponding domain organization cartoons (left). The boxed area (indicated by dashed line) highlights the V-shaped N60BD of Mlp1, which is structurally similar to the V-shaped Nup153BD of Tpr in b. The AlphaFold-predicted orientation of the remaining coiled-coil segments in relation to these V-shaped domains varies and is likely devoid of structural significance. b, AlphaFold-Multimer models of Tpr dimers with overlapping amino acid residue boundaries (right) and corresponding domain organization cartoons (left).

Extended Data Fig. 5 Chemical cross-linking of Nup60 complexes with Nup85•Seh1 and Mlp1.

a, DSS cross-linking of reconstituted Nup60(285-318)•Nup85(1-564)•His₆-Seh1 and Nup60(267-284)•NusA-His₆-Mlp1(382-620) complexes. Yellow boxes indicate bands of gel submitted for XL-MS analysis. b, Mapping of identified inter-protein DSS cross-links onto cartoon representations of Nup60(285-318) and Nup85(1-564). c, Mapping of identified inter-protein DSS cross-links onto cartoon representations of Nup60(267-284) and Mlp1(382-620). Unprocessed gels are available in source data.

Source data

Extended Data Fig. 6 AlphaFold-Multimer models of Nup60 in complex with Nup85•Seh1 and Mlp1 N60BD.

a, AlphaFold-Multimer model of Nup60(260-318)•Nup85•Seh1 shown in cartoon representation coloured by per-residue confidence score (pLDDT) (blue: high confidence, orange: low confidence). For clarity, the unstructured N-terminal extension of Nup85 (aa1-46) and internal loop of Seh1 (aa248-291) are not shown. b, Predicted interface between Nup60 (cartoon representation, dark green) and Nup85 (surface representation, coloured by Coulombic electrostatic potential). c, Predicted interface between Nup60 (surface representation, coloured by Coulombic electrostatic potential) and Nup85 (cartoon representation, light blue). d, AlphaFold-Multimer model of Nup60(260-318)•Mlp1(390-620) dimer shown in cartoon representation coloured by per-residue confidence score (pLDDT) (blue: high confidence, orange: low confidence). e, Predicted interface between Nup60 (cartoon representation, dark green) and Mlp1 N60BD dimer (surface representation, coloured by Coulombic electrostatic potential). f, Predicted interface between Nup60 (surface representation, coloured by Coulombic electrostatic potential) and Mlp1 N60BD dimer (cartoon representation, magenta). g, Superposition of Nup60(260-318)•Mlp1(390-620) dimer and Nup153(1-1475)•Tpr(390-620) dimer AlphaFold-Multimer models. h, Superposition of Nup60(260-318)•Mlp1(390-620) dimer and Nup60(260-318)•Mlp2(390-620) dimer AlphaFold-Multimer models suggests a similar interaction mode between Nup60 and Mlp1 and Nup60 and Mlp2.

Extended Data Fig. 7 Input gels for mutational analysis.

a, Multiple-sequence alignment (MSA) of Nup60 orthologs across yeast species. Amino acid residue numbering corresponds to S. cerevisiae sequence. b, SDS-PAGE gel showing native eluates of GST-Nup60(285-318) and Nup85(1-564)•His₆-Seh1 constructs used for the in vitro binding assays presented in Fig. 5b. c, SDS-PAGE gel showing native eluates of GST-Nup60(267-284) and NusA-His₆-Mlp1(382-620) constructs used for the in vitro binding assays presented in Fig. 5d. Unprocessed gels are available in source data.

Source data

Extended Data Fig. 8 AlphaFold-Multimer model of interface between NPC core and nuclear basket.

a, AlphaFold-Multimer model of Nup85(101-430)•Nup60(260-318)•Mlp1(450-580) dimer complex shown in cartoon representation and coloured by polypeptide chain. b, AlphaFold-Multimer model of Nup85(101-430)•Nup60(260-318)•Mlp1(450-580) dimer complex shown in cartoon representation and coloured by per-residue confidence score (pLDDT) (blue: high confidence, orange: low confidence). c, AlphaFold-Multimer model of Nup85(101-430)•Nup60(260-318)•Mlp1(450-580) dimer complex shown in surface representation and coloured by sequence conservation.

Extended Data Fig. 9 Immunoblot analysis of Nup60 and Mlp1 expression levels.

a, Western blot analysis of Nup60-mCherry and Mlp1-mGFP expression levels from live-cell fluorescence imaging experiment presented in Fig. 7a. Pgk1 was used as loading control. Unprocessed blots are available in source data.

Source data

Supplementary information

Reporting Summary

Supplementary tables

Supplementary Table 1: Reagents used in the study. Supplementary Table 2: Plasmids used in the study. Supplementary Table 3: Yeast strains used in the study. Supplementary Table 4: Summary of protein purification procedures. Supplementary Table 5: Nomenclature of yeast and human Nups.

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Statistical source data.

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Unprocessed gels and western blots.

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Stankunas, E., Köhler, A. Docking a flexible basket onto the core of the nuclear pore complex. Nat Cell Biol 26, 1504–1519 (2024). https://doi.org/10.1038/s41556-024-01484-x

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Received: 02 February 2024
Accepted: 17 July 2024
Published: 13 August 2024
Issue Date: September 2024
DOI: https://doi.org/10.1038/s41556-024-01484-x

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Abstract

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Main

Results

Nup60 ‘suspension cable’ oligomerizes via its helical region

Nup60 interacts with the Nup85•Seh1 arm of the Y-complex

Mlp1 forms a homodimer of coiled coils and flexible hinges

Nup60 couples Mlp1 recruitment and docking on the Y-complex

XL–MS confirms Nup60 interactions with Nup85 and Mlp1

Mutational analyses verify the interaction surfaces

Reconstitution of the basket–outer ring junction in vitro

Nup60 co-recruits Mlp1 and Y-complex to synthetic membrane

Nup60 SLiMs organize the Mlp1-outer ring junction in cells

Discussion

Multivalent network connecting the NPC core and basket

Domain architecture and flexibility of basket filaments

The NPC core–basket filament interactions in a membrane context

Functional implications of basket heterogeneity in cells

Methods

The AlphaFold-Multimer screen for protein–protein interactions

Reagents, plasmids and yeast strains

Recombinant protein expression in E. coli

Expression of MBP-3C-Mlp1(17–1,137)-His6 in insect cells

Protein purification

Nup60 oligomerization assay

ITC of Nup60 HR

Size exclusion chromatography coupled with multi-angle light scattering of Nup60 HR

In vitro binding assays

Chemical cross-linking of reconstituted protein complexes

Sample preparation for MS analysis

Liquid chromatography MS analysis

Data analysis of MS data

pLink search

XiSearch search

Reconstitution of the basket–NPC core junction

Rotary shadowing EM of Mlp1 samples

Lipids

GUV binding assay and image analysis

Live cell fluorescence microscopy of yeast cells

Quantification of live cell fluorescence images

Statistics and reproducibility

Reporting summary

Data availability

References

Acknowledgements

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Competing interests

Peer review

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Additional information

Extended data

Supplementary information

Source data

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Expression of MBP-3C-Mlp1(17–1,137)-His₆ in insect cells