A short linear motif in scaffold Nup145C connects Y-complex with pre-assembled outer ring Nup82 complex - PubMed (original) (raw)

A short linear motif in scaffold Nup145C connects Y-complex with pre-assembled outer ring Nup82 complex

Roman Teimer et al. Nat Commun. 2017.

Abstract

Nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs), which are formed from multiple copies of ~30 different nucleoporins (Nups) and inserted into the double nuclear membrane. Many of these Nups are organized into subcomplexes, of which the Y-shaped Nup84 complex is the major constituent of the nuclear and cytoplasmic rings. The Nup82-Nup159-Nsp1 complex is another module that, however, is only assembled into the cytoplasmic ring. By means of crosslinking mass spectrometry, biochemical reconstitution, and molecular modeling, we identified a short linear motif in the unstructured N-terminal region of Chaetomium thermophilum Nup145C, a subunit of the Y-complex, that is sufficient to recruit the Nup82 complex, but only in its assembled state. This finding points to a more general mechanism that short linear motifs in structural Nups can act as sensors to cooperatively connect pre-assembled NPC modules, thereby facilitating the formation and regulation of the higher-order NPC assembly.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1

Fig. 1

XL-MS analysis of the _Ct_Nup82 complex with the central vertex of the Y-shaped _Ct_Nup84 complex. a In vitro reconstitution of the _Ct_Nup82–Y complex (_Ct_Nup82–Nup159C–Nsp1C–Nup145C–Nup85–Nup120). An immobilized Flag-_Ct_Nup159C–Nup82–Nsp1C complex was incubated with the soluble _Ct_Nup145C–Nup85–Nup120 complex (Y-vertex). Shown is the analysis of the Flag eluate by SDS-PAGE and coomassie staining. _Ct_Nup145C is highlighted in blue as it interacts directly with the _Ct_Nup82 complex. M marker, MW molecular weight. An uncropped image of the gel is shown in Supplementary Fig. 4a. b Schematic representation of the _Ct_Nup82–Y complex showing the crosslinks determined by mass spectrometry. Crosslinks are depicted as straight gray lines, intramolecular self-links as curved purple lines, and intermolecular self-links as dark red loops. DIM domain invasion motif. Additional data are shown in Supplementary Table 2 and Supplementary Data 1

Fig. 2

Fig. 2

The N-terminal domain (NTD) of _Ct_Nup145C is both necessary and sufficient for the recruitment of the _Ct_Nup82 complex. a Crystal structure of the majority of the Y-shaped _Sc_Nup84 complex (PDB ID: 4xmm). The region upstream of the _Sc_Nup145C-DIM domain is indicated by a dashed pink line (right panel) as it is missing from the crystal structure. b Schematic representation of _Sc_Nup145C and _Ct_Nup145C and the design of truncated _Ct_Nup145C-NTD and _Ct_Nup145C-CTD; see also Supplementary Fig. 2. The crosslinking hotspots found in _Ct_Nup145C-NTD (Fig. 1b) are indicated by dashed pink lines. DIM domain invasion motif. c In vitro binding assay with immobilized _Ct_Nup145C, _Ct_Nup145C-NTD, or _Ct_Nup145C-CTD and soluble _Ct_Sec13 and/or the _Ct_Nup82–Nup159C–Nsp1C complex (Nup82-com). Shown is the analysis of SDS eluates by SDS-PAGE and coomassie staining. The experiment was performed at least twice with consistent results. Asterisks indicate bands corresponding to IgG heavy and light chains; M marker, Mock purification buffer with E. coli whole cell lysate, MW molecular weight. An uncropped image of the gel is shown in Supplementary Fig. 4b

Fig. 3

Fig. 3

Interaction between the _Ct_Nup82 complex and the _Ct_Y-vertex. In vitro binding assay with the immobilized _Ct_Nup82 complex (Flag-_Ct_Nup159C–Nup82–Nsp1C) and different soluble prey proteins of the _Ct_Y-shaped complex. Shown is the analysis of SDS eluates by SDS-PAGE and coomassie staining. The experiment was performed at least twice with consistent results. The asterisk indicates bands corresponding to the anti-Flag light chain; M marker, Mock purification buffer with E. coli whole cell lysate, MW molecular weight, Y-vertex co-purified _Ct_Nup145C–Nup85–Nup120 complex, Y-vertex-CTD co-purified _Ct_Nup145C-CTD–Nup85–Nup120 complex. Uncropped images of the gels are shown in Supplementary Fig. 4c, d

Fig. 4

Fig. 4

Identification of a short linear motif (SLiM) in _Ct_Nup145C-NTD that recruits the _Ct_Nup82 complex. a Schematic representation of _Sc_Nup145C and _Ct_Nup145C and the design of truncated _Ct_Nup145C-A and _Ct_Nup145C-B motifs; see also Supplementary Fig. 2. The crosslinking hotspots found in _Ct_Nup145C-NTD (Fig. 1b) are indicated by dashed pink lines. DIM domain invasion motif. b In vitro binding assay with Flag-immobilized _Ct_Nup145C-A, _Ct_Nup145C-B, and _Ct_Nup82 complex (Flag-_Ct_Nup159C–Nup82–Nsp1C) and soluble GST-_Ct_Nup145C-A, GST-_Ct_Nup145C-B, _Ct_Nup82 complex, and/or _Ct_Nup145N-ΔFG. Shown is the analysis of SDS eluates by SDS-PAGE and coomassie staining. The image section marked by the dashed box was subjected to image processing to increase the visibility of the small bait peptides _Ct_Nup145C-A-Flag and _Ct_Nup145C-B-Flag. c In vitro binding assay with Flag-immobilized _Ct_Nsp1C, _Ct_Nup159C, _Ct_Nup82, and _Ct_Nup82 complex (Flag-_Ct_Nup159C–Nup82–Nsp1C) and soluble GST-_Ct_Nup145C-B or _Ct_Nup145C. The experiments were performed at least twice with consistent results. The asterisk indicates bands corresponding to the anti-Flag light chain; M marker, Mock purification buffer with E. coli whole cell lysate, MW molecular weight. Uncropped gel images are shown in Supplementary Fig. 4e, f

Fig. 5

Fig. 5

XL-MS suggests that _Ct_Nup145C binding to the _Ct_Nup82 complex resembles _Ct_Nic96 binding to the _Ct_Nsp1 complex. The model of the _Ct_Nup82 complex a was built based on the _Ct_Nsp1 complex (b, PDB ID: 5cws). The _Ct_Nup145C-82CIM motif (residues 215–270), which could not be reliably modeled at the atomic level, is shown as a low-resolution density. Note that although sequence similarity suggests that the _Ct_Nup82 complex has an architecture similar to the _Ct_Nsp1 complex; the exact orientation of the coiled-coil domains might be different (curved double arrows). Residues crosslinked to _Ct_Nup145C-82CIM are shown as red spheres and the crosslinked residues of _Ct_Nup145C are marked as K242, K258, and K266. Crosslinks between _Ct_Nup82, _Ct_Nsp1, and _Ct_Nup159 are indicated as sticks, crosslinks to _Ct_Nup145C as dashed lines. Crosslinks that satisfy the distance threshold of 30 Å are colored blue, and red otherwise. The two violated (red) crosslinks (_Ct_Nup159 Lys1249-_Ct_Nup82 Lys710 and _Ct_Nsp1 Lys649-_Ct_Nup82 Lys769) have relatively low ld-scores of 25.3 and 28.46, respectively. They thus might be false positives, in line with the calibrated FDR of 5% for the entire XL-MS data set. Alternatively, flexibility or alternative arrangements of coiled-coil domains might account for the distance violations

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