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J Cell Sci
2018 Jan 04;1311:. doi: 10.1242/jcs.208538.
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A self-inhibitory interaction within Nup155 and membrane binding are required for nuclear pore complex formation.
De Magistris P
,
Tatarek-Nossol M
,
Dewor M
,
Antonin W
.
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Nuclear pore complexes (NPCs) are gateways through the nuclear envelope. How they form into a structure containing three rings and integrate into the nuclear envelope remains a challenging paradigm for coordinated assembly of macro-complexes. In vertebrates, the cytoplasmic and nucleoplasmic rings of NPCs are mostly formed by multiple copies of the Nup107-Nup160 complex, whereas the central, or inner ring is composed of Nup53, Nup93, Nup155 and the two paralogues Nup188 and Nup205. Inner ring assembly is only partially understood. Using in vitro nuclear assembly reactions, we show that direct pore membrane binding of Nup155 is crucial for NPC formation. Replacing full-length Nup155 with its N-terminal β-propeller allows assembly of the outer ring components to the NPC backbone that also contains Nup53. However, further assembly, especially recruitment of the Nup93 and Nup62 complexes, is blocked. Self-interaction between the N- and C-terminal domains of Nup155 has an auto-inhibitory function that prevents interaction between the N-terminus of Nup155 and the C-terminal region of Nup53. Nup93 can overcome this block by binding to Nup53, thereby promoting formation of the inner ring and the NPC.
Fig.1. Direct membrane binding of Nup155 is crucial for NPC assembly. (A) Western blot analysis of mock-treated and Nup155-depleted Xenopus egg extracts, with or without addition of Nup155 addback and mutants. (B) Confocal microscopy images of fixed nuclei assembled for 120 min in mock-depleted (mock) and Nup155-depleted (δNup155) Xenopus egg extracts supplemented with either buffer, wild-type Nup155 mRNA, or mRNA encoding the membrane binding mutants L258D and δ258–267, as well as the mutant Nup155(R385H), which causes cardiac disease. Membranes were pre-labelled with DiIC18 (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, red in overlay) and chromatin was stained with DAPI (4′,6-diamidino-2-phenylindol, blue in overlay). The bottom panels show immunofluorescence staining for Nup155 (green) and NPCs (mAB414, red) on the chromatin (DAPI, blue in overlay). Scale bars: 5 µm. (C) The average percentage of closed nuclear envelopes for 100 randomly chosen chromatin substrates in each of three independent experiments. Data points from individual experiments are indicated.
Fig.2. The Nup155 β-propeller region is sufficient for formation of a closed nuclear envelope. (A) Confocal microscopy images of nuclei assembled for 120 min in mock-depleted, Nup155-depleted (δNup155), and Nup155-depleted Xenopus egg extracts supplemented with 100 nM of the Nup155(1–589) fragment. Membranes have been pre-labelled with DiIC18 (red in overlay) and chromatin was stained with DAPI (blue in overlay). The bottom panels show the immunofluorescence analysis for Nup155 (green) and NPCs (mAB414, red) on the chromatin (DAPI, blue in overlay). Scale bar: 10 µm. (B) Average percentage of closed nuclear envelopes for 100 randomly chosen chromatin substrates in each of three independent experiments. Data points from the three individual experiments are indicated.
Fig.3. Nup155 supports assembly of the NPC structural backbone. (A) Nuclei assembled in mock, Nup155-depleted extracts (δNup155) or Nup155-depleted extracts supplemented with Nup155 1–589 for 120 min, fixed and stained with the respective antibodies. Scale bar: 10 μm. (B) Western blot analysis of mock and Nup155-depleted Xenopus egg extracts from the experiment in A. (C) Nuclei were assembled as in A. After 60 min an EGFP-tagged importin-α/β-dependent nuclear import substrate was added in a final concentration of 300 nM. Nuclei were fixed after a further 70 min and analyzed by confocal microcopy. Quantification shows the average of two independent experiments with 100 chromatin substrates analyzed per condition. Data points from the two individual experiments are indicated.
Fig.4. The C-terminal domain of Nup155 weakens the Nup53–Nup155 interaction. (A) GST fusion constructs of the Xenopus Nup53 RRM domain (aa 162–267, control) or a Nup155 N-terminal fragment (aa 1–589) were incubated with the SUMO-tagged Nup53 RRM domain (aa 162–267, control) or two C-terminal Nup155 fragments (aa 589–1388 and aa 504–1388) at a final concentration of 0.5 µM of each protein, pulled down and analyzed by western blotting. The right panel shows the average of the SUMO–Nup155 preys bound to GST–control or GST–Nup155(1–589) baits from three independent experiments. Data points from individual experiments are indicated. (B) 0.5 µM GST fusion constructs of the Xenopus Nup53(RRM) (control) or a C-terminal fragment (aa 162–320) were incubated with 0.5 µM SUMO–Nup53(RRM) (control) or SUMO–Nup155(1–589). Where indicated, SUMO–Nup155(504–1388) was added in a twofold (1 µM) or sixfold (3 µM) excess over SUMO–Nup155(1–589). The right panel shows the average of the SUMO–Nup155(1–589) prey bound to GST–Nup53(162–320) bait in the absence or presence of a twofold and sixfold excess of SUMO–Nup155(504–1388) from three independent experiments. Data points from individual experiments are indicated. Eluates and 2% of the input were analyzed by western blotting using an antibody against the 6×His tag.
Fig.5. Nup93 can override the inhibitory effect of the Nup155 C-terminus. Western blot analysis of 0.5 µM GST fusion constructs of the Xenopus Nup53 RRM domain (aa 162–267, control) or an Nup53 fragment including the Nup93 and Nup155 binding regions (aa 1–312) incubated with 0.5 µM SUMO–Nup53(RRM) (control) or SUMO–Nup155(1–589). Where indicated, SUMO-tagged C-terminal fragments of either Nup155(504–1388) or Nup93(608–820) were added in sixfold excess (3 µM) over SUMO–Nup155(1–589). The right panel shows the average of the SUMO–Nup155(1–589) prey bound to GST–Nup53(1–312) bait in the absence or presence of SUMO–Nup155(504–1388) and SUMO–Nup93(608–820) from three independent experiments. Data points from individual experiments are indicated. Eluates and 2% of the input were analyzed by western blotting using an antibody against the 6×His tag.
Fig.6. Nup155 is recruited to the pore via Nup53 and requires Nup93 to become competent for NPC assembly. Model for inner pore ring assembly. Nup53 binds to the nascent pore via its pore membrane interaction and contributes to curvature stabilization. Nup155 is recruited in loco via its N-terminal domain. At this step, Nup155 is found in a self-inhibitory conformation, because of the interaction of its N- and C-terminal moieties. Binding of Nup93 to Nup53 stabilizes the Nup155–Nup53 interaction by overcoming the auto-inhibitory effect of the C-terminal of Nup155 on the complex. Nup93 then recruits the Nup62 complex, leading to assembly of transport-competent NPCs. The direct and mutually exclusive Nup93 interaction partners Nup188 and Nup205 are not shown.
