Holzer G et al. (2021), The nucleoporin Nup50 activates the Ran guanine...

XB-ART-58595

EMBO J 2021 Dec 01;4023:e108788. doi: 10.15252/embj.2021108788.

Show Gene links Show Anatomy links

The nucleoporin Nup50 activates the Ran guanine nucleotide exchange factor RCC1 to promote NPC assembly at the end of mitosis.

Holzer G , De Magistris P , Gramminger C , Sachdev R , Magalska A , Schooley A , Scheufen A , Lennartz B , Tatarek-Nossol M , Lue H , Linder MI , Kutay U , Preisinger C , Moreno-Andres D , Antonin W .

???displayArticle.abstract???
During mitotic exit, thousands of nuclear pore complexes (NPCs) assemble concomitant with the nuclear envelope to build a transport-competent nucleus. Here, we show that Nup50 plays a crucial role in NPC assembly independent of its well-established function in nuclear transport. RNAi-mediated downregulation in cells or immunodepletion of Nup50 protein in Xenopus egg extracts interferes with NPC assembly. We define a conserved central region of 46 residues in Nup50 that is crucial for Nup153 and MEL28/ELYS binding, and for NPC interaction. Surprisingly, neither NPC interaction nor binding of Nup50 to importin α/β, the GTPase Ran, or chromatin is crucial for its function in the assembly process. Instead, an N-terminal fragment of Nup50 can stimulate the Ran GTPase guanine nucleotide exchange factor RCC1 and NPC assembly, indicating that Nup50 acts via the Ran system in NPC reformation at the end of mitosis. In support of this conclusion, Nup50 mutants defective in RCC1 binding and stimulation cannot replace the wild-type protein in in vitro NPC assembly assays, whereas excess RCC1 can compensate the loss of Nup50.

???displayArticle.pubmedLink??? 34725842
???displayArticle.pmcLink??? PMC8634129
???displayArticle.link??? EMBO J
???displayArticle.grants??? [+]

Species referenced: Xenopus laevis
Genes referenced: nup153 nup35 nup50 nup62 ran rcc1
GO keywords: GTPase activity [+]
???displayArticle.antibodies??? Nup50 Ab1 Nup62 Ab3

???attribute.lit??? ???displayArticles.show???

	Synopsis Nup50, part of the nuclear pore complex basket structure, is known as auxiliary factor for nuclear import. This work shows that it also plays a crucial role in nuclear pore complex (NPC) assembly at the end of mitosis by stimulating the Ran GTPase pathway. -Nup50 depletion impairs NPC assembly at the end of mitosis in frog extracts and human cells. -Nup50 function in NPC assembly is independent of its interaction with MEL28/ELYS and Nup153 or its NPC localization. -Nup50 interacts with the Ran guanine nucleotide exchange factor RCC1 to stimulate RanGTP production critical for NPC assembly.
	Figure 1. Nup50 is crucial for NPC assembly Western blot analysis of untreated, mock- and Nup50-depleted Xenopus egg extracts, with or without addition of recombinant Xenopus Nup50. 1, 2, and 4 µl of extracts were analyzed with indicated antibodies. Confocal microscopy images of fixed nuclei assembled for 120 min in mock-depleted (mock) and Nup50-depleted (ΔNup50) Xenopus egg extracts supplemented with either buffer or recombinant Nup50. In the left column, membranes were prelabeled with DiIC18 (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, red), and chromatin was stained with DAPI (4′,6-diamidin-2-phenylindol, blue). Three right columns show the immunofluorescence staining for Nup50 (green) and NPCs (mAB414, red) on the chromatin (DAPI, blue). Scale bars: 10 µm. The average percentage of closed nuclear envelopes (upper panel) and mAB414-positive nuclei (lower panel) for 100 randomly chosen chromatin substrates in each of 3 independent experiments is shown. Data points from individual experiments are indicated. HeLa cells were transfected with 20 nM control or Nup50 siRNA. 72-h post-transfection, cells were fixed with 4% PFA and stained with antibodies against Nup50 and mAB414, chromatin was labeled with DAPI. Scale bars: 50 µm. Quantitation of the mAB414 rim intensity at three different Nup50 RNAi and control oligo concentrations: 10 nM control (n = 84) or Nup50 siRNA (n = 171), 20 nM control (n = 101) or Nup50 siRNA (n = 157) and 40 nM control (n = 86) or Nup50 siRNA (n = 204). The means are indicated as diamonds; error bars show the standard deviations. P-values have been calculated from a Student t-test comparing the mean between the experimental conditions. Western blot analysis of HeLa cells treated as in (D) 72 h post transfection.
	Figure 2. Two Nup50 paralogs in mouse cells Mouse 3T3-NIH cells were transfected with 20 nM control, Nup50A, Nup50B, or a combination of Nup50A and Nup50B siRNA. After 72 h, cells were fixed and stained with mAB414 (green) and antibodies against mouse Nup50 (red). Chromatin is stained with DAPI (blue). The merge of the three channels is shown on the left column. Scale bar: 50 µm. An insert shows a zoom on a representative nucleus for each picture. Scale bar: 10 µm. Quantitation of the mAB414 rim intensity from n = 4 independent experiments done as in (A), each represented by a different color. The mean of each individual experiment and condition is represented by a diamond; the mean of all four experiments by a horizontal black line. P-values have been calculated from a paired ratio t-test comparing the mean between the experimental conditions. Error bars show the standard deviations of the means. Western blot showing the amount of Nup50A (top row) for each of the experimental condition. The actin (lower row) is shown as a loading control.
	Figure 3. Nup50 and MEL28 do not depend on each other for chromatin binding A, B. Demembranated sperm chromatin was preincubated in Xenopus egg extract, depleted for MEL28/ELYS or Nup50 (B), or control treated (mock, A). After 10 min, membranes were added to the reaction. Reactions were stopped at the indicated time points by fixation and analyzed by confocal microscopy after immunostaining with α-MEL28/ELYS, α-Nup50, and mAb414. Chromatin was stained with DAPI (blue). Scale bar: 10 μm.
	Figure 4. Nup50 is a chromatin binding nucleoporin 3 µM recombinant EGFP, EGFP-tagged MEL28/ELYS (aa 2290–2408), or EGFP-tagged Nup50 was incubated with empty or DNA-coated magnetic beads, which were chromatinized with Xenopus egg extracts (right panel) or unchromatinized DNA beads (left panel). After 3 h, the beads were re-isolated, washed, co-stained with DAPI, and analyzed by confocal microscopy. Scale bar: 10 µm.
	Figure 5. Minimal 46-aa region is required for Nup50 NPC localization HeLa cells were transfected with EGFP-Nup50 and different truncations. After 24 h, cells were shortly pretreated with 0.1% Triton X-100 in PBS, fixed with 4% PFA, and analyzed by confocal microscopy (left panel). Scale bar: 10 µm. The graph (right panel) shows the Nup50 domains (blue: importin α interaction domain, green: NPC-targeting domain, and pink: Ran-binding domain), and the truncation tested. Ability to bind NPCs is indicated on the right. Sequence alignment of Nup50 144–189, species are indicated on the left, the black boxes highlight residues tested by single-point mutation in (C) and (D), and the residue number are shown above or below the alignment. The color scheme indicates the type of amino acids according to the alignment software default setting. HeLa cells were transfected with EGFP-Nup50 comprising different point mutations in the minimal NPC-binding region and analyzed as in (A). Scale bar: 10 µm. GST fusion constructs of the Xenopus Nup50 minimal NPC binding fragment (aa 144–189) comprising no or single-point mutations were incubated with Xenopus egg extracts. Starting material as well as bound proteins were analyzed by Western blotting with indicated antibodies. The quantitation shows the average MEL28/ELYS and Nup153 bead bound signal from three independent experiments, normalized to the signal of their respective wild-type GST fusion (see Appendix Fig S1D for a gel showing the GST baits). Data points from individual experiments are indicated.
	Figure 6. A N-terminal Nup50 fragment is required for NPC assembly Confocal microscopy images of nuclei assembled for 120 min in mock-depleted, Nup50-depleted (ΔNup50), and Nup50-depleted Xenopus egg extracts supplemented with recombinant wild-type Nup50 or different mutants. Nuclei were fixed in 4% PFA and 0.5% glutaraldehyde, stained for NPCs (mAB414) and the chromatin (DAPI). Scale bar: 10 µm. Average percentage of mAB414-positive nuclei for 100 randomly chosen chromatin substrates in each of at least three independent experiments shown in (A). Data points from the individual experiments are indicated. Confocal microscopy images of nuclei assembled with N- and C-terminal Nup50 truncations and an minimal NPC binding fragment (144–189). Samples were analyzed as in (A). Average percentage of mAB414-positive nuclei for 100 randomly chosen chromatin substrates in each of at least three independent experiments shown in (C). Data points from the individual experiments are indicated.
	Figure 7. Both mouse Nup50 orthologs can replace the Xenopus protein Western blot analysis of mock- and Nup50-depleted Xenopus egg extracts (ΔNup50), with or without addition of recombinant mouse Nup50 orthologs. Samples were analyzed with Xenopus Nup50 antibodies, which do not recognize the mouse proteins, which in turn were detected with an His6 antibody. Confocal microscopy images of fixed nuclei assembled for 120 min in mock-depleted (mock) and Nup50-depleted (ΔNup50) Xenopus egg extracts supplemented with recombinant mouse Nup50 orthologs. Nuclei were stained for Nup50 (Nup50 antibody for Xenopus protein, His6 for mouse proteins) and NPCs (mAB414, red) on the chromatin (DAPI). Scale bar: 10 µm. The average percentage of mAB414-positive nuclei for 100 randomly chosen chromatin substrates in each of three independent experiments (performed as in B) is shown. Data points from individual experiments are indicated. Sequence comparison of the conserved 46-aa fragment of the human, mouse, rat, and Xenopus laevis sequences, with the residue 178 (Xenopus numbering), crucial for NPC binding in the Xenopus protein, is highlighted (black box). The color scheme indicates the type of amino acids according to the alignment software default setting. GST fusion constructs of the Xenopus Nup53 RRM domain (aa 162–267, control) full-length Xenopus Nup50, and the two mouse Nup50 orthologs were incubated with Xenopus egg extracts. Starting material as well as bound proteins were analyzed by Western blotting with indicated antibodies (see Appendix Fig S1E for a gel showing the GST-baits). The right panel shows the average MEL28/ELYS, Nup153, and importin β (Impβ) and ran bead-bound signal from two independent experiments normalized to their respective Xenopus signal. Data points from individual experiments are indicated. HeLa cells were transfected with EGFP-tagged constructs of both mouse Nup50 orthologs and the Nup50B Q177D (ortholog to the position 178 in Xenopus) mutant. After 24 h, the cells were analyzed by live cell imaging. Panel shows cells exiting mitosis (time normalized to metaphase-to-anaphase transition). The merge shows histone 2B in pink and Nup50 in green, and the black-and-white panel shows EGFP-Nup50 signal. Scale bar: 10 µm.
	Figure 8. Nup50 stimulates RCC1 activity for NPC assembly A. HEK293T cells were transfected with empty FLAG-tag vector as a control or FLAG-Nup50 N-terminal fragment (aa 1–120 of the human sequence). 24 h post-transfection, cells were lysed, FLAG-tagged proteins were immuno-isolated, and analyzed by mass spectrometry with the Volcano plot showing the identified interactors (four-fold change compared with the control and P < 0.01 in black, red if confirmed by Western blotting (B), see Dataset EV2 for full list) of Nup50 (yellow, data from three independent experiments). KPNA2 is the gene name of importin α. B. HEK293T cells were transfected with empty FLAG-tag vector or FLAG-Nup50 N-terminal fragments (aa 1–120, 29–120 and 48–120 of the human sequence), processed as in (A) and analyzed by Western blotting, with 10% of the inputs loaded. The quantification (lower panel) shows the mean signal intensity normalized on the input of at least two independent experiments. Data points from individual experiments are indicated. C. HEK293T cells were transfected with both Nup50 mouse orthologs N- and C-terminals tagged with EGFP. 24 h post-transfection, cells were lysed, EGFP-tagged proteins immuno-isolated, and analyzed by Western blotting. The quantification of the Western blot (C, lower panel) shows the mean signal intensity normalized on EGFP of three independent experiments. Data points from individual experiments are indicated. D. Xenopus sperm chromatin (6,000 sperm heads/µl were incubated with 120 µl of control), Nup50-depleted or egg extracts supplemented with excess (1X = 0.07 µM final) recombinant Xenopus Nup50 and re-purified. Total input extracts were analyzed by Western blotting against Nup50, and isolated chromatins were analyzed by Western blotting against RCC1 and histone H3 as a loading control (left panel). The quantification (right panel) shows the mean signal intensity of the RCC1 signal normalized over histone H3 from three independent experiments. Data points from individual experiments are indicated. E. 2 µM recombinant Ran, loaded with MANT-GDP was incubated with 2 mM GppNHp in buffer control, supplemented with 2 nM recombinant RCC1, 20 nM recombinant Xenopus Nup50 proteins, or RCC1 and Nup50 together. GDP-to-GppNHp exchange was monitored by the decrease in MANT fluorescence of the liberated GDP-MANT. The lower panel shows the rate constant of each experimental condition with n = 4 independent experiment per condition, bars represent the mean, and individual data points are indicated. F. Xenopus egg extracts were supplemented with 5 µM RanQ69L, RCC1, Xenopus Nup50 wild-type, and RCC1-binding mutants as well as a combination of 5 µM Nup50 and 5 µM RCC1. After 90 min, annulate lamellae were isolated by centrifugation and quantified by Western blotting with mAB414 antibody. Quantitation shows the relative Nup62 signal as a mean from three independent experiments, normalized to the buffer control. Individual data points are indicated. G. Confocal microscopy images of nuclei assembled for 120 min in mock-depleted, Nup50-depleted (ΔNup50), and Nup50-depleted Xenopus egg extracts supplemented with recombinant Xenopus wild-type Nup50 or RCC1 binding mutants. Nuclei were fixed in 4% PFA and 0.5% glutaraldehyde, stained for NPCs (mAB414) and the chromatin (DAPI). Scale bar: 10 µm. Quantitation shows the average percentage of mAB414-positive nuclei for 100 randomly chosen chromatin substrates in each of three independent experiments. Individual data points are indicated. H. Confocal microscopy images of nuclei assembled for 120 min in mock-depleted, Nup50-depleted (ΔNup50), and Nup50-depleted Xenopus egg extracts supplemented with RCC1 excess as indicated. Nuclei were fixed in 4% PFA and 0.5% glutaraldehyde and stained for NPCs (mAB414) and the chromatin (DAPI). Scale bar: 10 µm. Quantitation shows the average percentage of mAB414-positive nuclei for 100 randomly chosen chromatin substrates in each of three independent experiments. Individual data points are indicated.

References [+] :

Aksenova, Nucleoporin TPR is an integral component of the TREX-2 mRNA export pathway. 2020, Pubmed