NLS-Mediated NPC Functions of the Nucleoporin Pom121

FEBS Letters 584 (2010) 3292–3298

journal homepage: www.FEBSLetters.org

NLS-mediated NPC functions of the nucleoporin Pom121

Sevil Yavuz, Rachel Santarella-Mellwig, Birgit Koch, Andreas Jaedicke, Iain W. Mattaj *,1, Wolfram Antonin **,1

European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany article info abstract

Article history: RanGTP mediates nuclear import and mitotic spindle assembly by dissociating import receptors Received 23 February 2010 from nuclear localization signal (NLS) bearing proteins. We investigated the interplay between Revised 31 May 2010 import receptors and the transmembrane nucleoporin Pom121. We found that Pom121 interacts Accepted 2 July 2010 with importin a/b and a group of nucleoporins in an NLS-dependent manner. In vivo, replacement Available online 14 July 2010 of Pom121 with an NLS mutant version resulted in defective nuclear transport, induction of aber- Edited by Ulrike Kuttay rant cytoplasmic membrane stacks and decreased cell viability. We propose that the NLS sites of Pom121 affect its function in NPC assembly both by inﬂuencing nucleoporin interactions and pore membrane structure. Keywords: Pom121 Nucleoporin Structured summary: Importin MINT-7951230: pom121 (uniprotkb:Q5EWX9) physically interacts (MI:0914) with nup155 (uniprotkb:O75694), Nup133 (uniprotkb:Q8WUM0) and Importin beta (uniprotkb:Q14974) by pull down (MI:0096) MINT-7951210: pom121 (uniprotkb:Q5EWX9) physically interacts (MI:0915) with Importin alpha (uniprotkb:P52170) and Importin beta (uniprotkb:P52297) by pull down (MI:0096) MINT-7951183: pom121 (uniprotkb:Q5EWX9) physically interacts (MI:0914) with nup155 (uniprotkb:Q7ZWL0), nup160 (uniprotkb:P83722), nup205 (uniprotkb:Q642R6), nup93 (uniprotkb:Q7ZX96), Importin beta (uniprotkb:P52297) and nup62 (uniprotkb:Q91349) by pull down (MI:0096) MINT-7951416: pom121 (uniprotkb:Q5EWX9) physically interacts (MI:0914) with nup155 (uniprotkb:Q7ZWL0), nup93 (uniprotkb:Q7ZX96) and Importin beta (uniprotkb:P52297) by pull down (MI:0096) MINT-7951276: pom121 (uniprotkb:Q5EWX9) physically interacts (MI:0914) with nup155 (uniprotkb:Q7ZWL0), nup205 (uniprotkb:Q642R6), nup93 (uniprotkb:Q7ZX96), Importin beta (uniprotkb:P52297) and nup62 (uniprotkb:Q91349) by pull down (MI:0096) MINT-7951306, MINT-7951362: pom121 (uniprotkb:Q5EWX9) physically interacts (MI:0914) with nup155 (uniprotkb:Q7ZWL0), nup160 (uniprotkb:P83722), nup93 (uniprotkb:Q7ZX96), Importin beta (uniprotkb:P52297) and nup62 (uniprotkb:Q91349) by pull down (MI:0096)

1. Introduction lates these processes. Ran mediates nucleocytoplasmic transport in interphase and mitotic spindle assembly in metaphase [2–5]. The Nuclear pore complexes (NPCs) mediate selective transport import receptor importin b is involved in this regulation. It binds across the nuclear envelope (NE) and are formed by nucleoporins. to its cargos via nuclear localization signals (NLS), usually via the In metazoa, NPCs disassemble at the onset of mitosis and reassem- adaptor protein importin a, and releases them upon RanGTP bind- ble around chromatin upon mitotic exit [1]. The GTPase Ran regu- ing [6]. Ran is also necessary for post-mitotic nuclear reassembly [7–

* Corresponding author. Fax: +49 62213878211. 11]. A Ran mutant unable to hydrolyse GTP (RanQ69L) inhibits nu- ** Correspondence to: Wolfram Antonin, Friedrich Miescher Laboratory of the Max clear assembly and induces formation of cytoplasmic pore complex Planck Society, Spemannstrasse 39, 72076, Tübingen, Germany. Fax: +49 structures, annulate lamellae [10]. Either excess or lack of importin 7071601801. a or b blocks nuclear assembly [10,12,13]. E-mail addresses: [email protected] (I.W. Mattaj), wolfram.antonin@tuebingen. Detailed information regarding the mechanisms by which Ran mpg.de (W. Antonin). 1 These authors contributed to the work equally. affects nuclear assembly is lacking. At the onset of post-mitotic

0014-5793/$36.00 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2010.07.008 S. Yavuz et al. / FEBS Letters 584 (2010) 3292–3298 3293

A a.a. ER nucleoplasm/cytoplasm 1-1050 pom121 213-334 1 334-452 2 334-619 3 4 213-452 RanQ69L 15 μM cyt cyt +RanQ69L B C C 1 2 3 4 1 2 3 4 kDa kDa pom NPL NLS pom NPL ctrl pom pom NPL NLS pom NPL input 205 pom pom NLS mut. Mel-28 116 Nup205 170 97 Nup160 β

80 Nup155 130

66 Nup133 130 Nup93 95 72 Nup62

α Impβ 95

Impα 55 55 ∗ 34 baits ∗ ∗ ∗ ∗ ∗ 26

Fig. 1. Interaction of importin a/b and nucleoporins with Pom121. (A) Schematic representation of Xenopus Pom121 and GST-tagged Pom121 baits. Putative NLS sites are red. (B) GST-Pom121 baits or GST-gp210 fragment bait as a control (C) were incubated with Xenopus egg cytosol either alone or with 10 lM RanQ69L. Thrombin eluates were analyzed on a 10% SDS–PAGE gel and silver-stained. Arrows; importin a and b. (C) Control bait (GST-GFP, ctrl), GST-Pom121 213–452 (pom), GST-Pom121 213–452 NLS mutant and GST-Pom121 213–452 containing nucleoplasmin NLSs (NPL NLS) were incubated Xenopus egg cytosol either alone or with 15 lM RanQ69L. Thrombin eluates were either analyzed on a 8% SDS–PAGE gel and immunoblotted using the indicated antibodies or on a 4–12% SDS–PAGE gel and Coomassie stained (Ã indicates bait).

NPC assembly, the nucleoporin Mel-28 and the Nup107–160 com- 2.3. GST-pulldown assay plex form pre-pore structures on chromatin that are thought to be precursors of NPCs [14–18]. The transmembrane nucleoporin GST-tagged Pom121 (1 mg) fragments bound to 25 ll GSH-Se- Pom121 is then recruited as NPC assembly progresses linking it to pharose were incubated with either puriﬁed recombinant proteins the formation of a closed NE [19]. In this work, we investigated or Xenopus egg extract [19] which was diluted 1:1 in PBS and how Pom121’s function in NPC assembly is regulated. cleared at 100 krpm for 30 min. Unbound proteins were removed and beads were washed several times with PBS and eluted with thrombin protease which cleaves between the GST and the baits. 2. Materials and methods 2.4. RNAi 2.1. Molecular cloning Knockdown of human Pom121 was performed using HiPerfect Full-length Xenopus laevis Pom121 and fragments thereof were transfection reagent (Qiagen) and chemically synthesized siRNA cloned in a vector with a gp210 signal sequence and EGFP (a gift of duplexes (CAGUGGCAGUGGACAUUCA [21], Ambion Inc., USA) that Dr. Jan Ellenberg). NLS mutant and nucleoplasmin NLS mutant targets both Pom121 isoforms and corresponds to 540–559 bp in Pom121 fragments were generated by site-directed mutagenesis the BC008794.2 mRNA isoform and 1217–1236 bp in the (Stratagene, USA). Transport reporters were generated by the NM_172020.1 mRNA isoform. Luciferase siRNAs were used as a insertion of nucleoplasmin NLS and Rev NES into pDendra2 vector negative control (UCGAAGUAUUCCGCGUACG, MWG, Germany). by site-directed mutagenesis. See Supplementary data for remaining materials and methods.

2.2. Antibodies 3. Results and discussion

Rabbit antibodies against human Pom121, Xenopus importin a 3.1. Pom121 interacts with importin /b and several nucleoporins and GFP were raised using a GST human Pom121 fragment (aa 448–647), 6Â His-Xenopus importin a and 6Â His-GFP, respec- Pom121 has a single N-terminal transmembrane domain fol- tively. Antibodies against Xenopus Nup205, Nup160, Nup93 and lowed by a extramembranous region containing two putative Nup155 have been described [17,20]. mAb414 (Covance, USA), bipartite NLSs (Fig. 1A) which interact with importin a and impor- anti-His (Qiagen, Germany) and importin b antibody (Abcam, UK) tin b [18]. To investigate their impact on importin interactions we were purchased. performed GST pulldowns with fragments of Xenopus Pom121 3294 S. Yavuz et al. / FEBS Letters 584 (2010) 3292–3298

Fig. 2. Localization of wild-type (wt) and mutant Xenopus Pom121. A signal sequence and EGFP were fused to all Pom121 constructs. Localization of proteins was examined in transiently transfected U2OS cells by confocal microscopy. Scale bar: 10 lm.

containing either one or both NLS sites and Xenopus egg extracts Pom121 NLS sites were substituted by nucleoplasmin bipartite NLS (Fig. 1B). Importin a/b interacts with the regions of Pom121 con- [22] (see Fig. S1B for sequences). Binding of Nup155 and Nup93 taining either NLS site. Addition of the RanGTP mutant RanQ69L was efficiently restored, while Nup205, Nup160, Nup133 and abolished importin b binding and reduced importin a binding Nup62 binding remained significantly reduced (Fig. 1C). Recombi- (Fig. 1B and C). Binding of importin b required importin a nant Nup155, Nup93 and Nup107 also bound directly to Pom121 (Fig. S1A). Mutating both Pom121 NLS sites abolished importin while the recombinant Mel-28 fragment did not (Fig. S1C). ab binding (Fig. 1C). Taken together, these results show that Pom121 interacts with We tested the effect of the NLS sites on Pom121’s interactions importin a/b and nucleoporins including Nup160, Nup133, with other nucleoporins. In GST pulldown experiments we ob- Nup155, Nup93 and Nup205 in an NLS-dependent manner. Rasala served that nucleoporin binding to Pom121 was salt sensitive et al. [18] observed the binding of importin a/b, Nup160, Nup133, (Fig. 1C and Fig. S1E–F). While Nup155 bound to Pom121 during Nup205 and Nup93 but not Nup155 and Nup62 probably because 150 mM salt washes (Fig. S1E), binding of Nup93 and Nup62 was they used a slightly different Xenopus Pom121 fragment. Support- detected only at 110 mM (Fig. S1F) and binding of Nup205, ing our findings, human Pom121 recruits Nup205 and Nup62 [23]. Nup160 and Nup133 at 75 mM (Fig. 1C). These nucleoporin interactions under the less stringent conditions were nevertheless spe- 3.2. The NLS sites of Pom121 are required for NE localization cific since another nucleoporin, Mel-28, did not bind (Fig. 1C). Addition of RanQ69L decreased binding of Nup205 and Nup62 Next, we examined the localization of EGFP-tagged full length while Nup160, Nup155, Nup133 and Nup93 were largely unaf- and truncated Xenopus Pom121 in transiently transfected human fected. Mutation of the either one of the two or both NLS sites re- U2OS cells (Fig. 2). Full-length Xenopus Pom121, expressed at a duced nucleoporin binding (Fig. 1C, lane 3, Fig. S1G, lanes 4 and 5). low level, localized to the NE. An N-terminal Pom121 fragment To test if an NLS sequence per se is sufficient for these interactions, (aa 1–689) encompassing both NLS sites, also localized to the NE. S. Yavuz et al. / FEBS Letters 584 (2010) 3292–3298 3295

A DAPI α-Pom121hs mAb414 GFP-Pom121xl NLS mut. C

control siRNA kDa Pom121 xl wt Pom121 Pom121 xl NLS mut. Pom121 170 GFP 130 55 Pom121hs α-tub siRNA 43

B 1.2

0.8

0.6 24 hrs 48 hrs 0.4 72 hrs

0.2

Normalized cell number 0 control Pom121 control Pom121 control Pom121 siRNA siRNA siRNA siRNA siRNA siRNA

U2OS U2OS-Pom121 wt U2OS-Pom121 NLS mutant

Fig. 3. An NLS mutant of Xenopus Pom121 decreases cell viability (A) U2OS cells stably expressing EGFP-Pom121 NLS mutant were transfected with human Pom121 or control siRNAs. After 72 h, cells were ﬁxed and stained with anti-human Pom121 and mAb414. Scale bar: 10 lm. (B) U2OS cells and U2OS cells expressing wt or NLS mutant Xenopus EGFP-Pom121 were transfected with human Pom121 or control siRNAs. After 24, 48 and 72 h, surviving cells were trypsinized and counted using a hemocytometer and normalized to control siRNA cell numbers. Error bars; SD from four experiments. (C) U2OS cells stably expressing wild-type and NLS mutant Xenopus Pom121 were harvested and analyzed by SDS–PAGE (4–12% gel) followed by immunoblotting using antibodies against GFP and a-tubulin (loading control).

However, a shorter N-terminal fragment (aa 1–282) that lacks the but expression of the NLS mutant exacerbated this defect NLS sites localized predominantly to cytoplasmic membranes. A (Fig. 3B) despite similar expression levels (Fig. 3C). To our surprise, full-length protein in which both NLS sites were mutated localized in spite of its effect on cell viability, the NLS mutant Xenopus to the cytoplasmic membranes (construct 5) in a manner similar to Pom121 restored mAb414 levels at the NE indistinguishably from the truncated fragment. Pom121 variants in which either NLS site wild-type (Fig. 3A and Fig. S2C). However, it was unclear whether was mutated also localized to cytoplasmic membranes (constructs these NPCs were functional. 3 and 4). The NLS sites are therefore important for Pom121 localization to the NE. 3.4. Transport kinetics in NLS mutant-expressing cells

3.3. NLS mutant Xenopus Pom121 causes decreased viability in cells To examine the cause of NLS mutant-induced cell death, we analysed nuclear transport in these cells. We generated a nuclear To gain functional insight into the importin a/b-Pom121 inter- transport reporter containing an NLS and a nuclear export signal action we tested the behavior of the NLS mutant in the absence of (NES) which shuttled between the nucleus and cytoplasm endogenous Pom121. Removal of human Pom121 by RNAi affects (Fig. S3A, construct 1). NPCs as judged by the NPC marker mAb414 ([19,21,24], see also We examined nuclear import kinetics after human Pom121 Fig. S2A). Sequence divergence between human and Xenopus RNAi in cells which expressed wild-type or NLS mutant Xenopus Pom121 is sufﬁcient that the siRNAs targetting human Pom121 Pom121 along with the transport reporter. Nuclear accumulation had no effect on Xenopus Pom121 (data not shown). In contrast of the reporter was analysed by time-lapse microscopy after nucle- to non-transgenic U2OS cells, removal of endogenous Pom121 ar export was blocked. The rate of nuclear import was slower in the from wild-type Xenopus Pom121-expressing cells did not decrease cells expressing NLS mutant Pom121 than in those expressing mAb414 levels (Fig. S2C, lower panels). This indicates that Xenopus wild-type Pom121 or U2OS cells alone (Fig. 4A and Fig. S3B and Pom121 can restore the effect of loss of human Pom121 on NPCs. C). While nuclear import of the reporter proceeded largely to com- Consistently, Xenopus Pom121 interacted with human nucleopo- pletion in wild-type Pom121 expressing cells, import in the NLS rins in GST pulldowns (Fig. S1D). mutant Pom121-expressing cells became saturated when the nu- We observed a dramatic decrease in cell viability upon deple- clear signal reached a level similar to the cytoplasmic signal. To tion of human Pom121 after 72 h (Fig. 3B). Interestingly, expres- quantify this import defect we measured the ratio of cytoplasmic sion of wild-type Xenopus Pom121 did not rescue this phenotype to nuclear ﬂuorescence in randomly chosen cells (Fig. 4B). The ratio 3296 S. Yavuz et al. / FEBS Letters 584 (2010) 3292–3298

A Pom121 wt + reporter

0 min 20 min 63.4 min

Pom121 NLS mut. + reporter

0 min 42.8 min 92.7 min

1.2 1 0.8 0.6

signal 0.4 0.2 0 Normalized fluorescence 0102030405060708090100110120130

Time (min)

B 0 min 90 min 0.9

Pom121 0.8 wt 0.7

a + a

reporter i 0.6

0.5

ence l

a 0.4

o t 0.3

Pom121 fluoresc

C mic/Nuc r NLS mut. 0.2 + reporter 0.1

0 Wild-type NLS mutant

Fig. 4. Nuclear import is reduced in Pom121 NLS mutant-expressing cells. (A) U2OS cells expressing wt or NLS mutant EGFP-Xenopus Pom121 were transfected with human Pom121 siRNAs then with the transport reporter construct at 24 h. Twenty-four hours later, Leptomycin B was added and reporter nuclear accumulation measured. Fluorescence signal was normalized to the average maximal mean intensity reached in the nucleus as described in [28] (wt Pom121-expressing blue, NLS mutant-expressing cells red. Error bars show the SD). (B) Cell were treated as in (A) and ﬁxed at indicated time points after Leptomycin B addition. Localization of the reporter was analysed and cytoplasmic versus nuclear ﬂuorescent signal plotted. The boxes delineate interquartile range and the horizontal line indicates the median. Scale bars: 10 lm.

between the nuclear and cytoplasmic signals was roughly 4:1 in was 66 nm while in Pom121 depleted NLS mutant-expressing cells, wild-type Xenopus Pom121-expressing cells and between 3:1 it was roughly 100 nm (Fig. 5E). In addition to this NE spacing de- and 1.25:1 in the NLS mutant-expressing cells (Fig. 4B, right panel), fect, in NLS mutant-expressing cells, both in the presence and ab- confirming the defect. These data suggest that NPCs are defective sence of endogenous human Pom121, we observed membrane in cells expressing NLS mutant Pom121. stacks in the cytoplasm that were electron dense and exhibited irregular membrane spacing with frequent narrowing of the lumen 3.5. Ultra-structural analysis of Pom121 NLS mutant-expressing cells (Fig. 5B). Immunogold labeling of cryosections confirmed that wild-type Pom121 localized specifically to NPCs (Fig. 5C and D). We examined both the NE and NPCs and the abundant cytoplas- In contrast, the NLS mutant was predominantly localized in cyto- mic aggregates of mAb414 staining seen in Pom121 NLS mutant- plasmic NPC-free annulate lamellae-like membrane stacks. Inter- expressing cells (Fig. 3A) by electron microscopy. In NLS mutant- estingly, although no NPC-like structures were observed, mAb414 expressing cells, following depletion of human Pom121, the spac- antigens co-localized with those structures (Fig. 5C, right). ing between the inner and outer nuclear membranes was dis- Taken together these results indicate that the NLS sites of rupted and irregular (Fig. 5A right panel, lower) in contrast to Pom121 are important for its function in the NPC. Interactions be- control cells that had no morphological defects (Fig. 5A). To quan- tween Pom121 and several nucleoporins are reduced upon muta- tify this, we measured the distance between the inner and outer tion of these sites. In addition, mutation or deletion of the NLS nuclear membrane every 10 lm along a cross-section of the NE. sites causes mislocalization of the protein to cytoplasmic mem- In control NLS mutant-expressing cells the average NE spacing branes. Intriguingly, expression of this NLS mutant Pom121 in S. Yavuz et al. / FEBS Letters 584 (2010) 3292–3298 3297

A Control siRNA Pom121 siRNA

U2OS

U2OS Pom121 wt

U2OS Pom121 NLS mutant

B Pom121 NLS mut. C Nucleus

Pom121 wt

Cytoplasm

D Cytoplasm

90 Pom121 80 NLS mutant 70 60 NE 50 Cyt. 40 30 20 10

number of gold particles number 0 wild-type NLS mutant

E 140 120 100 80 ctrl Pom121 60 siRNA 40 NE spacing (nm) 20 0 NLS mutant

Fig. 5. NLS mutant Pom121 affects NE and cytoplasmic membrane morphology. (A) TEM of the NE of U2OS cells or U2OS cells expressing wt or NLS mutant EGFP-Xenopus Pom121 that were transfected with human Pom121 or control siRNAs and analysed 48 hours later. Arrowheads indicate NPCs embedded in the NE. (B) TEM of cytoplasmic membranes in U2OS cells expressing EGFP-Xenopus Pom121 NLS mutant. (C) Double immunogold labelling of U2OS cells expressing wt or NLS mutant EGFP-Xenopus Pom121 with antibodies against GFP (10 nm gold) or mAb414 antigens (20 nm gold). Scale bars: 200 nm. (D) Quantification of anti-GFP particles. (E) Quantification of NE spacing in NLS mutant cells prepared as in (A) lower panel. Error bars, SD, NLS mutant control cells, n = 14, NLS mutant Pom121 siRNA cells n = 16. the absence of endogeneous Pom121 affects nuclear import effi- cytoplasmic membrane stacks that are reminiscent of annulate ciency and NE spacing. Although we cannot detect NE and NPC lamellae but lack pore-like structures might indicate that this mu- localization of this mutant at steady-state, we speculate that the tant is defective in NPC function and/or assembly. It is unclear how NLS mutant might act through a dynamic localization to NPCs or these membrane stacks are formed but we suggest that the NLS by sequestering other nucleoporins to cytoplasmic membranes. mutant Pom121 initiates NPC insertion by narrowing the spacing Weaker interactions between NLS mutant Pom121 and NPC com- between the stacked ER membranes and attracting other NPC com- ponents could also lead to the formation of defective NPCs in ponents. It is known that overexpression of wild-type rat Pom121 which the nucleoporin composition is altered. The presence of leads to a large increase in annulate lamellae [25,26]. We speculate 3298 S. Yavuz et al. / FEBS Letters 584 (2010) 3292–3298 that the NLS mutant initiates the process of NPC formation but that [10] Walther, T.C., Askjaer, P., Gentzel, M., Habermann, A., Griffiths, G., Wilm, M., the loss of NLS-mediated nucleoporin interactions blocks NPC Mattaj, I.W. and Hetzer, M. (2003) RanGTP mediates nuclear pore complex assembly. Nature 424, 689–694. insertion into the ER at a point after lumenal narrowing but prior [11] Hughes, M., Zhang, C., Avis, J.M., Hutchison, C.J. and Clarke, P.R. (1998) The role to fusion between the apposed ER membranes. The NLS mutant of the ran GTPase in nuclear assembly and DNA replication: characterisation thus suggests a role for Pom121 in NPC membrane domain forma- of the effects of Ran mutants. J. Cell Sci. 111, 3017–3026. [12] Hachet, V., Köcher, T., Wilm, M. and Mattaj, I.W. (2004) Importin alpha tion or stabilisation. 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