144 Research Article The nucleoporin-like NLP1 (hCG1) promotes CRM1-dependent nuclear protein export

Inga Waldmann, Christiane Spillner and Ralph H. Kehlenbach* Department of Biochemistry I, Faculty of Medicine, Georg-August-University of Go¨ttingen, Humboldtallee 23, 37073, Go¨ttingen, Germany *Author for correspondence ([email protected])

Accepted 25 July 2011 Journal of Cell Science 125, 144–154 ß 2012. Published by The Company of Biologists Ltd doi: 10.1242/jcs.090316

Summary Translocation of transport complexes across the nuclear envelope is mediated by nucleoporins, of the nuclear pore complex that contain phenylalanine-glycine (FG) repeats as a characteristic binding motif for transport receptors. CRM1 (exportin 1), the major export receptor, forms trimeric complexes with RanGTP and proteins containing nuclear export sequences (NESs). We analyzed the role of the nucleoporin-like protein 1, NLP1 (also known as hCG1 and NUPL2) in CRM1-dependent nuclear transport. NLP1, which contains many FG repeats, localizes to the nuclear envelope and could also be mobile within the nucleus. It promotes the formation of complexes containing CRM1 and RanGTP, with or without NES-containing cargo proteins, that can be dissociated by RanBP1 and/or the cytoplasmic nucleoporin Nup214. The FG repeats of NLP1 do not play a major role in CRM1 binding. Overexpression of NLP1 promotes CRM1-dependent export of certain cargos, whereas its depletion by small interfering RNAs leads to reduced export rates. Thus, NLP1 functions as an accessory factor in CRM1-dependent nuclear protein export.

Key words: NLP1, hCG1, Nuclear export, CRM1, Nucleus, Nup214

Introduction mediates transport of hundreds of different proteins and certain Exchange of molecules between the cytoplasm and the nucleus RNAs out of the nucleus (for a review, see Hutten and occurs through nuclear pores that are embedded in the nuclear Kehlenbach, 2007). The first CRM1-dependent NESs were envelope. The nuclear pore complex (NPC) is a giant protein identified in the HIV-1 Rev protein, which serves as an adaptor assembly with many copies of approximately 30 individual for nuclear export of unspliced viral RNAs (Fischer et al., 1995)

Journal of Cell Science nucleoporins (Nups) [for a review see Wente and Rout and and in the inhibitor of the catalytic subunit of cAMP-dependent references therein (Wente and Rout, 2010)]. Four transmembrane protein kinase, PKI (Wen et al., 1995). Recently, the crystal Nups that anchor the NPC in the double lipid bilayer of the structure of CRM1 in a complex with export cargos was nuclear envelope have been described in vertebrate cells (Chadrin determined (Dong et al., 2009; Monecke et al., 2009). An et al., 2010; Gerace et al., 1982; Hallberg et al., 1993; Mansfeld important component of export complexes is the small GTP- et al., 2006; Stavru et al., 2006). Approximately 15 different binding protein Ran in its GTP-bound form, which binds structural Nups form subcomplexes that participate in the cooperatively to CRM1, together with export cargos (Fornerod formation of characteristic NPC building blocks such as the et al., 1997a). Similar to nuclear import complexes, CRM1- nuclear basket and nuclear or cytoplasmic rings. Finally, containing export complexes can interact with FG Nups. Among approximately 10 Nups are rich in phenylalanine–glycine (FG) these is Nup214, a nucleoporin that localizes to the cytoplasmic repeats, motifs that mediate the interaction of the NPC with side of the NPC and that is required for efficient export of some, soluble nuclear transport receptors. This interaction is the basis of but not all, CRM1 cargos (Bernad et al., 2006; Bernad et al., all models of the mechanisms for the selective translocation of 2004; Hutten and Kehlenbach, 2006). More recently, Nup98 macromolecules across the NPC (for reviews, see Goldfarb, was described as a CRM1-interacting nucleoporin that promotes 2009; Terry and Wente, 2009; Wa¨lde and Kehlenbach, 2010; nuclear export (Oka et al., 2010). Wente and Rout, 2010). The majority of the soluble factors In this study, we investigated the role of the ill-defined belong to the superfamily of importin-b-like transport receptors. nucleoporin-like protein NLP1 (also known as NUPL2) in They are called importins or exportins, depending on the major , which has a presumptive ortholog, Rev-interacting direction of cargo transport, and are collectively also referred to protein (Rip1p; also known as Nup42), in yeast. Rip1p was as karyopherins (for a review, see Fried and Kutay, 2003). The originally identified in a yeast two-hybrid screen, searching for prototype importin is importin-b itself, which, through the yeast proteins that bind to the viral protein HIV-1 Rev (Stutz adaptor protein importin-a, interacts with proteins containing a et al., 1995). Later, the protein human candidate 1 classical nuclear localization signal (cNLS). The major exportin (hCG1) was described (Van Laer et al., 1997). hCG1 has 55% is exportin 1, better known as CRM1 (Fornerod et al., 1997a; amino acid to Rip1p over the entire length of Fukuda et al., 1997; Kehlenbach et al., 1998; Neville et al., 1997; the proteins (423 aa in humans) and can substitute for Rip1p Ossareh-Nazari et al., 1997; Stade et al., 1997). CRM1 interacts functions in yeast (Strahm et al., 1999). hCG1 was also identified with proteins containing a nuclear export sequence (NES) and as NLP1 (nucleoporin-like protein 1), again as a protein NLP1 in CRM1-dependent export 145

interacting with HIV-1 Rev in a two-hybrid screen (Farjot et al., CRM1 concentration. In the presence of leptomycin B (LMB), 1999). A characteristic of the yeast and the human proteins is a selective CRM1 inhibitor (Wolff et al., 1997), NES–GFP2– the abundance of FG motifs, which are also found in FG cNLS, is localized entirely in the nucleus in control cells and in nucleoporins. Although the yeast protein Rip1p is not essential, it cells overexpressing CRM1–HA, indicating that nuclear export seems to be required for export of certain heat shock mRNAs of the reporter protein indeed involves CRM1 as an export factor (Saavedra et al., 1997; Stutz et al., 1997). Another study and also that its nuclear import was not inhibited under our suggested a role of Rip1p in nuclear export of heat shock and experimental conditions. Very similar observations were non-heat shock mRNAs at elevated temperatures (Vainberg et al., made with another shuttling protein, the negative cofactor 2 b 2000). Rip1p was found to also interact with CRM1 in two- (NC2b; see supplementary material Fig. S1A,B). NC2b employs hybrid assays (Neville et al., 1997), as well as in vitro using importin-a and/or -b and importin 13 as import- and CRM1 purified proteins (Floer and Blobel, 1999). A Rip1p-deletion as export receptors, respectively (Kahle et al., 2009). Notably, strain did not exhibit a profound defect in CRM1-dependent under conditions of overexpression of CRM1 substrates, nuclear protein export (Stade et al., 1997). Hence, the functional the cytoplasmic localization of the endogenous CRM1 cargo relevance of Rip1p–CRM1–RanGTP complexes (Floer and RanBP1 was not compromised, suggesting that the CRM1 system Blobel, 1999) remains unclear. The human protein NLP1 was not completely overloaded (supplementary material Fig. interacts with the mRNA export factor TAP (Katahira et al., S1C). Together, these results demonstrate that in HeLa 1999) and functions in nuclear export of Hsp70 mRNA (Kendirgi cells, CRM1 concentrations are not saturating for export of et al., 2005). Similar to the yeast protein, an interaction between overexpressed nucleocytoplasmic shuttling proteins. We next NLP1 and CRM1 was detected in two-hybrid assays (Farjot et al., analyzed the function of NLP1, a putative CRM1-binding 1999). Nothing is known, however, about the function of NLP1 protein, as a potential accessory factor that might stimulate in nuclear export of protein cargos in mammalian cells. Here, we nuclear protein export under certain conditions. describe a supportive role of NLP1 in CRM1-dependent export. NLP1 interacts with CRM1 in a RanGTP- and export Results cargo-dependent manner CRM1 concentrations are rate-limiting for nuclear export of The yeast ortholog of NLP1, Rip1p/Nup42p, forms trimeric artificial reporter proteins complexes with CRM1 and RanGTP. Export cargos such as HIV- 1-Rev, however, appeared to be excluded from such complexes The cellular concentration of nuclear import factors of the (Floer and Blobel, 1999). In yeast two-hybrid assays, human importin-b superfamily is rate-limiting for efficient transport of NLP1 has previously been described to interact with CRM1 proteins into the nucleus (Yang and Musser, 2006). We (Farjot et al., 1999). A direct binding or a functional role in investigated whether the concentration of CRM1, the major nuclear export, however, has not been reported. We therefore set nuclear export factor, is also rate-limiting, and analyzed the out to analyze the binding of NLP1 to CRM1 using recombinant subcellular localization of nuclear shuttling proteins in control proteins, and to investigate the role of this protein in CRM1- HeLa cells and in cells overexpressing CRM1. As a reporter dependent export. First, we expressed NLP1 fused to the maltose protein, we used an artificial shuttling protein, a double GFP Journal of Cell Science binding protein (MBP) and immobilized it on beads. CRM1 and containing an N-terminal NES and a C-terminal cNLS (NES– RanQ69L, a Ran mutant that is insensitive to the GTPase- GFP2–cNLS). This protein localized predominantly in the activating protein RanGAP (Klebe et al., 1995), loaded with GTP nucleus in ,70% of transfected control cells (Fig. 1A,B). were then added in combination with an NES peptide or with Strikingly, co-transfection of hemagglutinin-tagged CRMI GST–snurportin 1 (GST–SPN1) as a CRM1-dependent export (CRM1–HA) resulted in a clear shift of the reporter protein cargo (Dong et al., 2009; Monecke et al., 2009; Paraskeva et al., towards the cytoplasm, suggesting that nuclear export of 1999). Very little binding of CRM1 to NLP1 was observed in the overexpressed NES–GFP2–cNLS is limited by the cellular absence of RanQ69L–GTP, whether or not an export cargo was present in the reaction (Fig. 2A, lanes 1, 2, 5, 6). Addition of RanQ69L–GTP alone resulted in detectable levels of CRM1 binding to NLP1 (lanes 3, 7). Including either GST–SPN1 (lane 4) or the NES–peptide (lane 8) in the reaction further increased CRM1 binding to MBP–NLP1, suggesting the formation of tetrameric complexes. Binding of GST–SPN1 was only detected when RanGTP was present in the reaction (compare lanes 2 and 4). Importantly, CRM1 and RanQ69L–GTP bound to immobilized MBP–NLP1 but not to MBP alone when added to the reaction together with an export cargo (lanes 9 and 10), demonstrating the specificity of the interaction. In a complementary experiment, we immobilized GST–Ran–GTP and analyzed binding of CRM1, MBP–NLP1 and SPN1. Again, Fig. 1. CRM1 concentrations are rate-limiting for nuclear protein export. HeLa cells were co-transfected with plasmids coding for CRM1–HA the strongest signals were observed when CRM1 was added together with MBP–NLP1 and His–SPN1 (Fig. 2B, lane 6). Less or an empty control vector and NES–GFP2–cNLS and treated with LMB (5 nM) for 2.5 hours as indicated. (A) Microscopic analysis. Scale bar: CRM1 was detected when either His–SPN1 (lane 4) or MBP– 10 mm. (B) Quantification of cells with a predominant nuclear signal (N.C) NLP1 (lane 5) was omitted from the reaction. Only background of the reporter protein. Error bars show the standard deviation of the mean of binding was detected when the immobilized GST–Ran had been three (+ LMB) or six (–LMB) independent experiments. loaded with GDP (data not shown) or when unfused GST had 146 Journal of Cell Science 125 (1)

increasing concentrations of CRM1. Bound NLP1 was then detected with a specific anti-NLP1 antibody. As shown in Fig. 2C, only background levels of NLP1 binding to immobilized RanGDP were observed, even at the highest CRM1 concentration. With immobilized RanGTP, by contrast, increasing CRM1 concentrations resulted in increased binding of NLP1. Especially at lower CRM1 concentrations, the addition of His–SPN1 to the reaction further promoted binding of NLP1 (together with CRM1) to RanGTP. With or without SPN1, half- maximal binding was observed at CRM1 concentrations in the low nanomolar range. These results show that NLP1 can form trimeric complexes with RanGTP and the export receptor CRM1. CRM1 cargos such as SPN1 or an NES peptide can join these complexes, suggesting that NLP1 functions in CRM1-mediated nuclear export rather than in re-import of CRM1 back into the nucleus. We previously showed that wild-type RanGTP in a complex with CRM1 and the nucleoporin Nup214 is resistant to RanGAP- promoted GTP hydrolysis (Hutten and Kehlenbach, 2006). Likewise, a ternary complex of yeast CRM1, RanGTP and Rip1p/Nup42p was protected against RanGAP (Floer and Blobel, 1999). In RanGAP assays, a C-terminal fragment of Nup214 strongly reduced the ability of RanGAP to promote GTP hydrolysis on Ran in the presence of CRM1 (Fig. 3A), confirming our previous results (Hutten and Kehlenbach, 2006). Similarly, the addition of NLP1 to the reaction inhibited GTP hydrolysis. By contrast, Nup88, a nucleoporin that does not interact with CRM1, was not active in this assay, as shown before (Hutten and Kehlenbach, 2006). This result supports the notion that trimeric complexes containing RanGTP, CRM1 and NLP1 can form. In a late step of transport, such trimeric complexes (and of course tetrameric ones containing an export cargo) have to dissociate. The RanGTP-binding protein RanBP1 has previously been implicated in terminal steps of nuclear export (Kehlenbach

Journal of Cell Science et al., 1999). We therefore tested the ability of RanBP1 to disassemble NLP1-containing complexes. As before, tetrameric complexes containing immobilized MBP–NLP1, CRM1, RanQ69L–GTP and an NES-peptide were assembled. After the reaction, RanBP1 was added, and binding of CRM1 and Ran to NLP1 was analyzed. Clearly, RanBP1 dissociated the pre- Fig. 2. NLP1 interacts with CRM1 and export cargos in a RanGTP- assembled complexes because the levels of bound CRM1 and dependent manner. (A) MBP–NLP1 or MBP were immobilized on beads Ran were strongly reduced (Fig. 3B). Very similar observations and incubated in the presence or absence of CRM1, NES peptide, GST–SPN1 were made with SPN1 as a CRM1 cargo (data not shown). and RanQ69L, loaded with GTP, as indicated. Note that we usually use the Together, our results demonstrate the ability of NLP1 to interact RanGAP-insensitive mutant RanQ69L (Klebe et al., 1995) for this type of with CRM1 and RanGTP, with or without export cargos, experiment, although this is not required, as no RanGAP is present in the suggesting a role in nuclear protein export. Such complexes are reactions. (B) Wild-type GST–Ran loaded with GTP or GST was immobilized expected to be stable until they are dissociated by the concerted on beads and incubated in the presence or absence of MBP–NLP1, CRM1 and action of cytoplasmic RanBP1 and RanGAP. His–SPN1, as indicated. Bound proteins were subjected to SDS-PAGE, followed by Coomassie Blue staining (A,B). The input in B corresponds to Nup214 is a well-characterized binding partner of CRM1 and 10% of the material used for the binding reaction. (C) ELISA assay. is involved in nuclear export of a subset of proteins (Bernad et al., GST–Ran was loaded with either GDP or GTP, immobilized on wells of a 2006; Fornerod et al., 1997a; Hutten and Kehlenbach, 2006). microtiter plate and incubated with increasing concentrations of CRM1 with According to our previous results, it functions at a late stage of MBP–NLP1 in the presence or absence of His–SPN1, as indicated. Bound nuclear export, i.e. before disassembly of the RanBP1-mediated NLP1 was detected with an anti-NLP1-antibody. export complex (Hutten and Kehlenbach, 2006; Kehlenbach et al., 1999). We therefore asked whether NLP1 and Nup214 interact with CRM1 in a similar manner. CRM1–NLP1 been immobilized (Fig. 2B, lane 8). To analyze the interaction of complexes were assembled on immobilized wild-type GST– CRM1–NLP1 complexes with RanGTP and RanGDP in a more Ran, and increasing concentrations of an FG-rich C-terminal quantitative manner, we performed an ELISA-type binding assay. fragment of Nup214 that is known to interact with CRM1 GST–Ran loaded with either GDP or GTP was bound to (Fornerod et al., 1996) were added. Clearly, the Nup214 fragment microtiter plate wells and incubated with MBP–NLP1 and prevented NLP1 from binding to the GST–Ran–CRM1 complex NLP1 in CRM1-dependent export 147

Fig. 3. RanGAP-resistant NLP1–CRM1–RanGTP complexes are dissociated by RanBP1 and Nup214. (A) RanGAP assay. Increasing concentrations of MBP–NLP1 (full-length) or fragments of Nup214 or Nup88 were incubated with CRM1 and [c,32P]RanGTP. GTP hydrolysis was initiated by the addition of RanGAP. (B) MBP–NLP1 was immobilized on beads and incubated with CRM1, RanQ69L–GTP and an NES peptide. After complex formation, RanBP1 was added for 60 minutes. (C) GST–Ran, loaded with GTP, was immobilized on beads and incubated with CRM1, MBP–NLP1 (5 mg each), an NES peptide and increasing amounts (2.5–10 mg) of MBP–Nup214 (aa 1859–2090). Bound proteins were analyzed by SDS-PAGE and Coomassie Blue staining (B,C).

(Fig. 3C), suggesting that binding of the two proteins to CRM1 role in CRM1 binding. C-terminal fragments [amino acids (aa) is mutually exclusive. Furthermore, the Nup214 fragment 165–423 and 205–423], which contain the majority of the FG could dissociate a pre-assembled NLP1–CRM1–RanGTP complex repeats, showed weak binding or no binding at all. An N-terminal (data not shown). Thus, nuclear export complexes might fragment containing only two FG repeats (aa 1–204), by contrast, sequentially bind to NLP1 and Nup214 during transport, before clearly interacted with CRM1 in an SPN1- and RanGTP- complex disassembly at the cytoplasmic site of the NPC by dependent manner. The putative coiled-coil region of NL RanBP1. (aa 165–204) seems to contribute to CRM1-binding (compare the full-length protein, aa 1–423 and fragment 1–423D165–204, FG repeats are not crucial for the NLP1–CRM1 interaction fragments 1–204 and 1–165 and fragments 165–423 and 205– CRM1 has been shown to interact with a number of FG Nups 423). In the deletion mutant lacking the coiled-coil region, a (Neville et al., 1997). For Nup214, the FG-repeat-containing C- mutation of two FG repeats in the N-terminal part of NLP1 did terminal part of the protein is required for high-affinity not lead to reduced CRM1 binding. Together, our biochemical interaction with CRM1 (Fornerod et al., 1997b). We therefore analysis shows that NLP1 can engage in interactions that are analyzed the region(s) in NLP1 that are required for CRM1 relevant for CRM1-dependent nuclear export. Interestingly, the binding. Various fragments and deletion mutants of NLP1 were FG repeats, the classic binding motif for nucleoporin–transport expressed as either MBP or GST fusions, depending on the receptor interactions, do not seem to be absolutely required for

Journal of Cell Science solubility of the proteins. The proteins were immobilized on CRM1 binding. We therefore generated an NLP1 mutant in beads and analyzed for CRM1 binding as described above. which all phenylalanine residues within FG motifs were replaced Fig. 4A,B summarizes our results. The details of the binding with serine residues (NLP1FG-less). Clearly, this mutant interacted experiments are shown in the supplementary material Fig. S2. with CRM1 in a Ran–GTP- and cargo-dependent manner, albeit Surprisingly, the FG repeats of NLP1 do not seem to play a major to a lesser extent than the wild-type protein (Fig. 4C).

Fig. 4. FG repeats are not crucial for NLP1–CRM1 complexes. (A) MBP- or GST-tagged fragments of NLP1. Orange box, zinc finger region; yellow box (C–C), putative coiled-coil region. FG motifs are depicted as vertical bars. Binding to CRM1 (as analyzed in B) was qualitatively assessed (+++, strong; ++, intermediate; +, weak; –, no binding). (B) NLP1 fragments were immobilized on beads and incubated with CRM1 in the presence or absence of His–SPN1 and RanQ69L–GTP, as indicated. See supplementary material Fig. S2 for entire

gels. (C) Full-length NLP1FG-less was fused to MBP, immobilized as in B and incubated with CRM1 in the presence or absence of GST–SPN1 and RanQ69L– GTP, as indicated. Binding of CRM1 was analyzed by SDS-PAGE and Coomassie Blue staining (B,C). 148 Journal of Cell Science 125 (1)

NLP1 is found at the nuclear envelope and could be mobile within the nucleoplasm In a proteomic analysis, mammalian NLP1 was suggested to be a component of the NPC, occurring at a copy number of 16 (Cronshaw et al., 2002). In NLP1-overexpressing HeLa cells, the protein was originally detected in the nucleus, being excluded from nucleoli (Farjot et al., 1999). A myc-tagged version of NLP1 also localized to the nuclear envelope in transfected cells (Le Rouzic et al., 2002). However, a thorough analysis of the subcellular and subnuclear localization of the endogenous protein has not been performed. We therefore raised antibodies against NLP1 in rabbits and also tested two anti-NLP1 antibodies raised in guinea pigs (data not shown). None of these antibodies yielded specific and reliable signals in indirect immunofluorescence assays, although they specifically recognized NLP1 in western blotting. We therefore resorted to a biochemical fractionation approach and also re-investigated the localization of NLP1 using various tagged proteins in overexpressing cells. As shown in Fig. 5A, endogenous NLP1 largely co-fractionated with nucleoporins, as detected with the anti-FG-Nup antibody mAb414. Only small amounts of NLP1 could be detected in the cytosolic fraction. When we expressed GFP–NLP1, RFP–NLP1, NLP1–RFP, HA–NLP1 or NLP1–HA, low expressing cells showed a clear signal at the nuclear envelope (Fig. 5B, arrows). Many cells also showed a clear nuclear staining, excluding the nucleoli (Fig. 5B and data not shown). In highly overexpressing cells, NLP1 was also detectable in the cytoplasm. In control cells, potential binding sites for NLP1 in the NPC are probably occupied by the endogenous protein. We therefore expressed GFP–NLP1 in cells where the endogenous protein had been depleted by specific siRNAs. The localization of GFP– NLP1 in such cells was very similar to that observed in control cells, with some cells exhibiting a weak signal for GFP–NLP1 at Fig. 5. NLP1 is a mobile nuclear protein. (A) HeLa cells were subjected to subcellular fractionation and proteins in the total (t), cytoplasmic (c) and the nuclear rim and others a pan-nuclear signal (Fig. 5B, right nuclear (n) fraction were analyzed by SDS-PAGE followed by western Journal of Cell Science panel). We next investigated whether nucleoplasmic GFP–NLP1 blotting to detect NLP1 (rabbit anti-NLP1), a-tubulin and a set of is a mobile protein by performing fluorescence recovery after nucleoporins (monoclonal antibody 414). (B) HeLa cells that had been treated photobleaching (FRAP) assays. As an example of a mobile with the specific siRNA 9 to deplete endogenous NLP1, or left untreated, protein, we expressed GFP2–GST–cNLS, which is efficiently were transfected with a plasmid coding for GFP–NLP1 and analyzed by transported into the nucleus. A small nuclear region of GFP– fluorescence microscopy. Note that cells with a nuclear rim (white arrows) can be seen under both conditions. Scale bars: 10 m. Cell lysates were NLP1- or GFP2–GST–cNLS-expressing cells was bleached and m the recovery of fluorescence was recorded. As shown in Fig. 5C, analyzed by western blotting for levels of endogenous NLP1 and, as a loading the fluorescence in the bleached area was completely restored control, Uba2. (C) FRAP analysis of nuclear GFP–NLP1 or GFP2–GST– after ,40 seconds for GFP–NLP1 and after ,15 seconds for cNLS. The error bars show the standard deviation from the mean recovery of fluorescence in the bleached area of 10 cells. GFP2–GST–cNLS, suggesting that GFP–NLP1 can move within the nucleus, albeit with a lower mobility compared with GFP2– GST–cNLS. This reduced mobility could be explained, for were co-transfected with a construct coding for NLP1–HA, example, by transient interactions of NLP1 with nucleic acids. however, the percentage of cells showing a clear nuclear Together, our results suggest that NLP1 interacts with the nuclear localization of NES–GFP2–cNLS dropped to ,50%. Such envelope, in accordance with previous observations (Farjot et al., changes in the subcellular localization of NES–GFP2–cNLS 1999; Le Rouzic et al., 2002). Depending on the expression level, could result from stimulated export or inhibited import. To it might also be found in the nuclear interior, similar to many distinguish between these two possibilities, we incubated other nucleoporins. transfected cells in the presence of LMB. This treatment resulted in a strong nuclear accumulation of NES–GFP2–cNLS, NLP1 promotes CRM1-dependent nuclear export and also occurred in cells expressing NLP1–HA (Fig. 6B). In light of our initial results showing that the concentration Hence, overexpression of NLP1–HA did not inhibit nuclear of CRM1 is rate-limiting for nuclear export (Fig. 1), we next import of NES–GFP2–cNLS but rather stimulated its nuclear expressed tagged versions of NLP1 in HeLa cells and analyzed export. Similar observations were made with NC2b–GFP2 as a their effects on nuclear transport. In cells that had been co- reporter protein and N- or C-terminally RFP-tagged NLP1. transfected with an empty HA vector, our artificial reporter Again, co-expression of NLP1 fusion proteins resulted in a clear protein NES–GFP2–cNLS localized mainly to the nucleus shift of NC2b–GFP2 towards the cytoplasm, compared with cells (Fig. 6A,B), similar to our results shown in Fig. 1. When cells expressing unfused RFP (Fig. 6C,D). As above, treatment of cells NLP1 in CRM1-dependent export 149

Fig. 6. NLP1 promotes CRM1-dependent nuclear protein export. (A,B) HeLa cells were co-transfected with plasmids coding for NLP1–HA or an empty control vector (contr.) and

NES–GFP2–cNLS and treated with or without LMB, as indicated. The subcellular localization of the reporter protein was analyzed by microscopy (A) and quantified (B), showing the mean percentage of cells with a predominant nuclear signal (N.C). Error bars show the standard deviation from the mean of three (+ LMB) or six (–LMB) independent experiments. (C,D) HeLa cells were co-transfected with plasmids coding

for NC2b–GFP2 and RFP, RFP–NLP1 or NLP1–RFP and treated with or without LMB, as indicated. The subcellular

localization of NC2b–GFP2 was analyzed by microscopy (C) and quantified (D). Error bars show the standard deviation from the mean of the percentage of cells with a predominant nuclear signal (N.C) from three independent experiments. (E) Cells were co-transfected with plasmids coding for NC2b–

GFP2 and either RFP or RFP–NLP1, as indicated. After constant bleaching of a cytoplasmic region, the loss of nuclear GFP fluorescence was analyzed by FLIP in 10 cells. Error bars show the variation from the mean of two independent experiments. Scale bars: 10 mm.

with LMB led to a clear nuclear accumulation of NC2b–GFP2 Our results, described so far, suggest that NLP1 stimulates under all conditions, indicating stimulated nuclear export in CRM1-dependent nuclear export under conditions where the cells expressing RFP–NLP1 or NLP1–RFP. Next, we analyzed export receptor itself is rate-limiting. This limitation might result Journal of Cell Science the kinetics of nuclear export in control cells and in cells from low concentrations of CRM1 and/or from comparatively overexpressing NLP1. To this end, we performed fluorescence low affinities of export cargos to the receptor. loss in photobleaching (FLIP) assays in cells co-expressing NC2b–GFP2 and either RFP or RFP–NLP1. A cytoplasmic Depletion of NLP1 inhibits CRM1-dependent nuclear export region in RFP-positive cells was constantly bleached and the loss In in vitro reactions using digitonin-permeabilized cells, CRM1 of GFP fluorescence in the nuclear compartment was measured and Ran were the only exogenous factors that were required as an indicator of the efficiency of nuclear export of NC2b– for efficient nuclear export (Kehlenbach et al., 1998). Under GFP2. As shown in Fig. 6E, cells co-expressing RFP–NLP1 these conditions, however, NLP1 is still present in nuclei of exhibited faster export kinetics than cells co-expressing RFP. permeabilized cells (see Fig. 5A). To specifically address the NLP1 is a nuclear-envelope-associated protein that could question of whether NLP1 is required for CRM1-dependent potentially also shuttle between the nuclear envelope and the export, we performed siRNA experiments, reducing the cellular nuclear interior. Given the stimulatory effect of NLP1 on nuclear concentration of the protein by RNA interference. In a similar export in vivo, we argued that recombinant NLP1 might also setup, we previously showed that depletion of nucleoporin promote CRM1-dependent export in permeabilized cells. HeLa Nup214, but not Nup358 reduced CRM1-dependent nuclear cells that stably expressed GFP–NFAT [nuclear factor of protein export (Hutten and Kehlenbach, 2006). As judged by activated T cells (Kehlenbach et al., 1998)] were permeabilized western blotting, two different siRNAs against NLP1 resulted in with digitonin and subjected to nuclear export reactions in the a clear reduction of the NLP1 concentration, although traces of presence of Ran and CRM1, with or without additional NLP1. the protein were still detectable (Fig. 7A and data not shown). Clearly, NLP1 promoted nuclear export of GFP–NFAT Other proteins that are involved in various steps of (supplementary material Fig. S3). This stimulation of export nucleocytoplasmic transport were not affected by the siRNA was specific, as the loss of nuclear fluorescence in the presence of treatment (Fig. 7A). We first investigated nuclear export of our NLP1 required ATP. Furthermore, nuclear export under all reporter protein GFP–NFAT (Hutten and Kehlenbach, 2006). conditions was inhibited by wheat germ agglutinin, a general Cells were treated with ionomycin to induce nuclear import of inhibitor of nuclear transport (data not shown), demonstrating the protein. Control cells and siRNA-treated cells showed the that the permeability barrier of the NPC was not compromised same partial nuclear import of GFP–NFAT after 1 minute and during the reaction. complete nuclear localization of the reporter protein after 2 150 Journal of Cell Science 125 (1)

Fig. 7. Depletion of NLP1 inhibits CRM1- dependent protein export. (A) HeLa cells were treated with siRNA 8 against NLP1 and cell lysates were analyzed by SDS-PAGE and western blotting. (B,C) HeLa NFAT cells were treated with siRNA 8 or 9 against NLP1 and nuclear import of GFP– NFAT was induced with ionomycin. After washing the cells to remove the ionomycin, nuclear export was analyzed immediately (0 h) or after 1 hour, by microscopy Scale bar: 10 mm. (C) Quantification of cells showing a predominant nuclear localization of GFP–NFAT after 1 hour. Error bars show the standard deviation from the mean of three independent experiments. (D) HeLa cells were treated with siRNA 9 against NLP1 and

transfected with a plasmid coding for NC2b–GFP2. Nuclear export of NC2b–GFP2 was analyzed by FLIP, bleaching a cytoplasmic region and recording the loss of nuclear GFP fluorescence over time. Error bars show the standard deviation from the mean of four independent experiments.

minutes, suggesting that nuclear import is not affected by the hydrophobic binding pocket for interaction with different cargos, depletion of NLP1 (supplementary material Fig. S4A). After a forcing NES peptide sequences to adapt their conformation to the change of medium to remove the ionomycin, nuclear export was rather rigid binding-site on CRM1 (Gu¨ttler et al., 2010). allowed to proceed. A clear nuclear GFP–NFAT signal was Depending on the spacing of key hydrophobic residues, the present in ,40% of the control cells after 1 hour. In cells treated affinities of NES-containing cargos for their cognate receptor with two different siRNAs this figure was 60–65%, indicating a CRM1 can vary dramatically, with low-affinity cargos prevailing modest, yet reproducible inhibition of nuclear export under (Cook et al., 2007; Kutay and Gu¨ttinger, 2005). Functionally, conditions of reduced NLP1 concentrations (Fig. 7B,C). After 2 weak affinities seem to be important for efficient disassembly of hours of export, differences between control cells and siRNA- export complexes on the cytoplasmic side of the NPC (Engelsma treated cells were less pronounced (data not shown). As an et al., 2004). Furthermore, they prevent cargos from binding to Journal of Cell Science alternative reporter for nuclear export, we used NC2b–GFP2. CRM1 in the cytoplasm in the absence of RanGTP (Kutay and This protein localizes predominantly to the nucleus (compare Gu¨ttinger, 2005). The consequences of different affinities on the Fig. 6) and obvious changes in this localization upon treatment of subcellular localization of individual nucleocytoplasmic shuttling cells with siRNAs against NLP1 were not expected. We therefore proteins has not been investigated so far. Our results now show resorted to a kinetic analysis of NC2b–GFP2 export by FLIP. As that the cellular CRM1 concentrations are rate limiting for shown in Fig. 7D, depletion of NLP1 by specific siRNAs led to a nuclear export in HeLa cells, at least under conditions of small, yet reproducible reduction of the nuclear export rate overexpression of cargo proteins. Elevated CRM1 levels have of NC2b–GFP2. Because NLP1 and its yeast homolog have been observed in many tumor cells including cervical cancers previously been implicated in RNA export (Saavedra et al., 1996; (Noske et al., 2008; van der Watt et al., 2009) and could also be Stutz et al., 1997; Vainberg et al., 2000), we also compared effective in our HeLa cells. Hence, export of a subset of control cells and NLP1-depleted cells by in situ hybridization, endogenous proteins in primary cells could well be limited under detecting Poly(A)+ mRNA. No obvious differences in mRNA certain conditions by the available pool of the export receptor, localization were detected under our experimental conditions allowing the cells to regulate export simply by adjusting the (supplementary material Fig. S4B), suggesting that the observed CRM1 concentration. Under conditions where many NES- effects on CRM1-dependent export did not result from indirect containing proteins compete for binding sites on CRM1, the effects on mRNA export. In summary, reduced NLP1 formation of export complexes containing low-affinity CRM1 concentrations resulted in decreased export of certain nuclear substrates could also be promoted by accessory factors. One such proteins. NLP1, however, does not seem to be absolutely required factor is the Ran-binding protein RanBP3, which enhances the for transport, as export still occurred at a substantial rate upon affinity of CRM1 for cargo proteins and for RanGTP. As a depletion of the protein. consequence, RanBP3 stimulates CRM1-dependent export in permeabilized cells (Englmeier et al., 2001; Lindsay et al., 2001). Discussion Another cofactor in CRM1-dependent export is the nucleoporin Limiting CRM1 concentrations: a means to Nup98, which also interacts with the export receptor in a regulate transport? RanGTP-dependent manner (Oka et al., 2010). Here, the authors CRM1 is an export receptor that interacts with hundreds or even used antibody-injection experiments and suggested a role of thousands of cargo proteins. Strikingly, CRM1 uses the same Nup98 in export of overexpressed transport cargos in living cells. NLP1 in CRM1-dependent export 151

In our study, we analyzed the role of a nucleoporin-like protein, CRM1, RanGTP and NLP1 might initially form in the nucleus or NLP1, in CRM1-mediated export. NLP1 had previously been on the nuclear side of the NPC and accommodate a fourth implicated in nuclear export of certain mRNAs (Katahira et al., component, a high- or low-affinity NES cargo. In such trimeric 1999; Kendirgi et al., 2005). Although it had been shown to and tetrameric complexes, Ran is resistant to RanGAP-induced interact with CRM1 in two-hybrid assays, its function in protein GTP hydrolysis. RanGAP, which promotes GTP hydrolysis by export in mammalian cells has remained unclear. We now show more than 1000-fold (Bischoff et al., 1994) is restricted to the that NLP1 promotes CRM1-dependent export of certain cargos in cytoplasm but could occasionally enter the nucleus, leading to vitro and in vivo. Together with other accessory factors, NLP1 deleterious effects on the Ran gradient. In that sense, NLP1 might contribute to the control of nuclear export of distinct cargo would help to protect nuclear Ran from GTP hydrolysis. proteins. Furthermore, it would compensate for low affinities of NESs, which on their own are not sufficient for tight CRM1 binding, NLP1 promotes CRM1-dependent export at least not under conditions where high-affinity cargos are NLP1 was originally so named because of the characteristic FG competing for the same binding site. How could such a repeats, as they also occur in certain nucleoporins. Indeed, the mechanism facilitate export of low-affinity cargos, when also protein was identified as a component of the NPC in a proteomic high-affinity CRM1 substrates, such as SPN1, would profit from analysis (Cronshaw et al., 2002). In one study, the yeast homologue enhanced export complex formation? In the simplest scenario, Rip1p/Nup42p has been found to reside in the nucleus and at the certain NES cargos could directly interact with NLP1, prior to nuclear and the cytoplasmic face of the NPC (Strahm et al., 1999) export complex formation, resulting in a stabilization of the and in another study exclusively on the cytoplasmic side tetrameric (cargo–NLP1–CRM1–RanGTP) complex. Indeed, our of the NPC (Rout et al., 2000). Our results now suggest that initial results suggest that the HIV-1 Rev protein, a CRM1 cargo mammalian NLP1 is associated with the nuclear envelope and could of comparatively low affinity (Paraskeva et al., 1999), can also be mobile within the nucleus, similar to Nup98. The levels of directly interact with NLP1, independent of CRM1 or RanGTP intranuclear NLP1 could well depend on the expression level of the (data not shown). In such a complex, the NES of the cargo NLP1 gene. To unequivocally demonstrate the existence of an protein might be exposed upon NLP1 binding, promoting intranuclear pool of NLP1 and further analyze its significance, subsequent CRM1 interaction. Alternatively, NLP1 could level specific antibodies that are suitable for immunofluorescence or the differences in affinities as observed in direct cargo–CRM1 immunoelectron microscopy will be required. interactions. In that case, increasing the cellular concentration Overexpression as well as siRNA depletion experiments point of NLP1 (or regulating its activity by post-translational to a role of NLP1 in CRM1-mediated nuclear protein export. The modifications) would have a more profound effect on low- results of the overexpression experiments can be easily explained affinity CRM1 cargos than on high-affinity substrates. A similar if we assume that a nuclear pool of NLP1 does exist under observation was made for RanBP3, which increased the binding physiological conditions and could thus support complex of a GST–NES substrate to CRM1 while decreasing binding of formation as discussed below. If a nuclear pool only resulted SPN1 (Englmeier et al., 2001). from overexpression of the protein, we would have to assume that After complex formation in the nucleus, the FG-rich C-

Journal of Cell Science the levels of endogenous NLP1 at the nuclear envelope are not terminal region of NLP1, which is not required for CRM1 saturated in all cells. Upon overexpression, additional binding binding, could facilitate translocation of the export complex sites at the nuclear envelope and/or nuclear pore are then through the transport channel of the NPC. Subsequently, occupied by NLP1, resulting in stimulation of CRM1-dependent disassembly of export complexes is the terminal step of export. However, the siRNA depletion experiments in which transport on the cytoplasmic side of the NPC. In the simplest we observed a small reduction of CRM1-dependent transport, model, this is accomplished by the concerted action of RanBP1 or suggest that NLP1 is not absolutely required for nuclear export. the RanGTP binding domains of the cytoplasmic nucleoporin Instead, it functions as an accessory factor, e.g. for proteins that Nup358 (RanBP2) and the GTPase-activating protein RanGAP on their own have a low affinity for the export receptor. In cells (Kehlenbach et al., 1999). For export complexes containing that do not overexpress transport receptors as many cancer cells NLP1 as an integral component, an additional factor could come do (Noske et al., 2008; van der Watt et al., 2009), limiting CRM1 into play. Nup214, a bona fide FG nucleoporin that also localizes concentrations might prevail. It will be interesting to investigate to the cytoplasmic side of the NPC, binds to CRM1–RanGTP– the functional relevance of NLP1 in such cells in the future. NES complexes with high affinity (Hutten and Kehlenbach, In binding assays, we could demonstrate that: (1) NES cargos 2006). Binding of NLP1 and Nup214 to such complexes is as well as RanGTP enhanced binding of CRM1 to NLP1; (2) mutually exclusive (Fig. 3C), and Nup214 could replace NLP1 in NLP1 enhanced binding of cargo to CRM1–RanGTP complexes; the complex. It will be interesting to compare the interaction of and (3) CRM1 (indirectly) enhanced binding of NLP1 to CRM1 with the two proteins at the structural level, because FG RanGTP. These results suggest that distinct trimeric and repeats appear to be important for Nup214 binding but not for tetrameric export complexes can assemble in the nucleus. NLP1 binding. The export complex, now tethered to Nup214, Strikingly, the FG repeats of NLP1 do not play a major role in would be well positioned for disassembly by RanBP1 and CRM1 binding, although an FG-rich region of Nup214 seems to RanGAP, a large portion of which stably associates with Nup358 compete for the same binding site on CRM1. It will be interesting (Mahajan et al., 1997; Matunis et al., 1996). Nup358 also to compare the interaction mode of CRM1 with the two proteins contains four Ran-binding domains that are functionally in detail. equivalent to that of RanBP1. Hence, soluble cytoplasmic High-affinity NES cargos might form trimeric export factors would be dispensable for export complex dissociation, complexes with RanGTP and CRM1 and be exported as in line with previous observations (Kehlenbach et al., 1999). such. Alternatively, a trimeric ‘pre-export complex’, containing Interestingly, depletion of NLP1 reduced CRM1-dependent 152 Journal of Cell Science 125 (1)

export of NC2b and NFAT, two proteins that are also affected by at a final concentration of 100 nM using Oligofectamine (Invitrogen), according to the instructions of the manufacturer. Control cells were treated with transfection reagent the depletion of Nup214 (Stephanie Roloff and R.H.K., only. After 24 hours of siRNA treatment, cells were transfected with reporter protein unpublished observations) (Hutten and Kehlenbach, 2006). constructs of interest and analyzed after 20–24 hours. Together, our data suggest a new function for NLP1 in promoting CRM1-dependent nuclear export of a subset of proteins. Antibodies Antibodies against NLP1 were raised in rabbits by injecting His–NLP1 1–204 and affinity purified using MBP–NLP1 coupled to CNBr beads. For the detection Materials and Methods of HA-epitope-tagged proteins by immunofluorescence, a monoclonal mouse Cell culture and transfections anti-HA antibody (16B12, Covance) was used. The rabbit anti-Nup214 antibody HeLa P4 cells (Charneau et al., 1994) and HeLa NFAT cells (Hutten and (Hutten and Kehlenbach, 2006) and the goat anti-RanGAP antibody (Hutten et al., Kehlenbach, 2006) were grown in DMEM (Gibco) containing 4.5 g/l glucose, 10% 2008; Pichler et al., 2002) were described previously. The mouse anti-lamin A/C, FCS, 2 mM glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin. Cells anti-Ran, anti-RanBP1 and anti-RanBP3 antibodies were obtained from BD were transfected using the calcium phosphate method (Ausubel et al., 1994) or Bioscience, the mouse anti-a-tubulin antibody was obtained from Sigma. The Polyfect transfection reagent (Qiagen) according to the instructions of the anti-CRM1 antibody was raised in goat against a C-terminal decapeptide as manufacturer and cultured for 48 hours. described before (Kehlenbach et al., 1998). The goat-anti Uba2 antibody was a kind gift from Frauke Melchior (University of Heidelberg, Germany). For Plasmids immunofluorescence, donkey anti-mouse Alexa Fluor 594 and donkey anti-rabbit Constructs coding for CRM1–HA (Hilliard et al., 2010), NC2b–GFP2 (Kahle et al., Alexa Fluor 594 (1:1000; Molecular Probes) were used as secondary antibodies. 2009), NES–GFP2–cNLS [Rev(47–116)–GFP2–cNLS] and Rev68–90–GFP2– For immunoblotting, HRP-coupled donkey anti-goat, donkey anti-mouse or M9core (Hutten et al., 2008; Hutten et al., 2009) have been described donkey anti-rabbit IgG (1:5000; Dianova) were used as secondary antibodies. previously. Human NLP1 was PCR amplified from cDNA and cloned into pMal-C2 using BamHI and HindIII, generating MBP–NLP1. NLP1 fragments were Indirect immunofluorescence and microscopy PCR amplified from MBP–NLP1 and cloned into pMal-C2 or pGEX-KG using Immunofluorescence staining was performed as described before (Hutten and BamHI and HindIII. Deletion mutants (MBP–NLP1D165–204) and point mutants Kehlenbach, 2006). Cells were analyzed using a Zeiss Axioskop 2 microscope with (MBP–NLP1 FGFG14,15,95,96AAAA, D165–204) were generated by site-directed a 1006 Plan-Neofluar 1.3 NA water-corrected objective and appropriate filter mutagenesis. A mutant NLP1 (NLP1FG-less) with all phenylalanine residues within settings and processed using AxioVision Rel. 4.8 LE and Adobe Photoshop 6.0. the FG motifs replaced by serine residues was synthesized by GeneArt For quantification of subcellular localization of reporter proteins, cells were (Regensburg, Germany). For construction of GFP–NLP1, RFP–NLP1, NLP1– grouped into two categories: N.C (majority of the reporter protein in the nucleus) RFP and NLP1–HA, NLP1 sequences were amplified from MBP–NLP1 and and others (either N5C; equal distribution between nucleus and cytoplasm or cloned into pEGFP-C1, pmRFP-C1, pmRFP-N1 (Clontech) or pcDNA3.1(+), C.N; predominant cytoplasmic localization). Quantification was performed from containing an HA tag. The plasmid coding for the FRAP reporter GFP2–GST– at least three independent experiments, counting more than 100 cells with similar cNLS was kindly provided by Sonja Neimanis (University of Go¨ttingen, expression levels. Statistical significance of the data was analyzed by a two-tailed, Germany). For generation of His–SPN1 and GFP–SPN1, SPN1 sequences were heteroscedastic Student’s t-test. P-values ,0.05 were considered as statistically PCR-amplified from GST–SPN1 (Strasser et al., 2004) and cloned into pET30b or significant. pEGFP-C1, respectively. Constructs were verified by DNA sequencing. Live cell imaging Protein expression and purification FRAP and FLIP analyses were performed at 37˚C with a LSM 510 Meta confocal MBP–NLP1 and GST–NLP1 fusions were expressed in Escherichia coli microscope (Zeiss) using a LCI Plan-Neofluar 636 1.3 NA water-corrected BL21(DE3) cells upon induction with 500 mM isopropyl-b-D- objective in a temperature-controlled chamber. Cells were grown on LabTec- thiogalactopyranosid (IPTG) for 3.5 hours at 25 C. Cells were lysed in PBS, ˚ chambers (Nunc) and transferred to CO2-free medium (Invitrogen) before the 2 mM EDTA, 1% Triton X-100, 5 mM dithiothreitol (DTT), 1 mg/ml each of analysis. The mobility of GFP–NLP1 was analyzed by FRAP. After 20 scans at aprotinin, leupeptin and pepstatin, and centrifuged for 30 minutes at 100,000 g. low laser intensity (1.2% 488-nm laser transmission), an area of 10630 pixels After diluting the supernatant 1:5 with PBS, 10 mM MgCl2, 5 mM DTT, 1 mg/ml (1.3564.15 mm) in the nucleus was bleached (100% transmission of the 488-nm Journal of Cell Science each of aprotinin, leupeptin and pepstatin, proteins were purified by affinity laser, 40 iterations). Subsequently, scanning of the bleached area was continued at chromatography using amylose–resin beads (New England Biolabs) for MBP- low laser intensity for 380 pictures (154 mseconds/picture). Data analysis was tagged fragments or glutathione–Sepharose (GE Healthcare) for GST fusion preformed with LSM software (Zeiss) and Excel (Microsoft), normalizing the proteins. After several washing steps, proteins were eluted with either 20 mM original fluorescence in the bleached area to one. FLIP analysis of nuclear export glutathione or 10 mM maltose in 10 mM Hepes pH 8.0, 5% glycerol, 150 mM was performed essentially as described before (Hilliard et al., 2010). NaCl, 1 mM DTT and 1 mg/ml of protease inhibitors. His–NLP1 (1–204) was expressed as described for MBP–NLP1. For purification, a bacterial pellet was lysed in 50 mM Tris pH 8.0, 2% Triton X-100, 2 mM DTT and 1 mg/ml each Nuclear transport in HeLa-cells expressing GFP–NFAT of aprotinin, leupeptin and pepstatin. After centrifugation for 20 minutes at Nuclear import of GFP–NFAT (Hutten and Kehlenbach, 2006) was induced by the 100,000 g, the pellet was resuspended in 50 mM Tris pH 8.0, 4 M urea, 1 mM addition of 300 nM ionomycin (Sigma) and cells were incubated at 37˚C for DTT and protease inhibitors and centrifuged as above. The resulting pellet was various periods of time, fixes with 4% formaldehyde and analyzed by fluorescence solubilized in binding buffer (50 mM Tris pH 8.0, 8 M urea, 1 mM DTT and microscopy. Export of GFP–NFAT was analyzed as described previously (Hutten protease inhibitors). After centrifugation as above, the supernatant was added to and Kehlenbach, 2006). Ni-NTA agarose beads (Qiagen) and incubated for 1 hour. After several washing steps, the protein was eluted with binding buffer containing 300 mM imidazole Binding studies and dialyzed against PBS. Precipitated protein was used for antibody production. MBP or GST fusion proteins (5 mg) were immobilized on 20 ml amylose–agarose GST–Ran and His–SPN1 were expressed in BL21(DE3) upon induction with or glutathione–agarose beads that had been preincubated with 10 mg/ml BSA in 500 mM isopropyl b-D-1-thiogalactopyranoside and purified according to standard TPB. The beads were incubated with 5 mg of each protein of interest or 10 mM protocols. GST–SPN1 (Strasser et al., 2004), RanBP1 (Kehlenbach et al., 2001), NES peptide [NS2 protein of minute virus of mice, CVDEMTKKFGTLTIHDTEK RanGAP (Mahajan et al., 1997), CRM1–His (Guan et al., 2000), Ran and (Askjaer et al., 1999)] in 300 ml TPB containing 2 mg/ml BSA. After 75 minutes RanQ69L (Melchior et al., 1995), MBP–Nup88 (aa 500-741) and MBP–Nup214 at 4˚C, beads were washed three times with TPB. Bound proteins were eluted with (aa1859–2090) (Hutten and Kehlenbach, 2006) were purified as described before. SDS sample buffer and subjected to SDS-PAGE, followed by Coomassie Blue His–Nup214 was expressed in BL21(DE3) and purified as described above for staining or western blotting. MBP–NLP1 using Ni-NTA agarose beads and 300 mM imidazole for protein elution. All proteins were dialyzed against transport buffer (TPB; 20 mM Hepes– ELISA KOH, pH 7.3, 110 mM potassium acetate, 2 mM magnesium acetate, 1 mM A 96-well plate (Immuno 96 MicroWellTM Solid Plates; Nunc) was coated EGTA, 2 mM DTT and 1 mg/ml each of aprotinin, leupeptin, pepstatin), frozen in overnight at 4˚C with 300 ng GST–Ran per well loaded with GDP or GTP in 50 ml liquid nitrogen and stored at –80˚C. Ran was loaded with GTP, GDP or 32 TPB. The wells were blocked with 3% BSA in TPB for 30 minutes at room [c- P]GTP as described previously (Kehlenbach et al., 1999). temperature and 300 ng MBP–NLP1 and increasing concentrations of CRM1–His, with or without 300 ng His–SPN1, were added. After incubation for 75 minutes at RNA interference room temperature and several washing steps, bound NLP1 was detected using the Cells were transfected with small interfering RNAs (siRNAs) from Ambion (NLP1 rabbit anti-NLP1 antibody (1:5000 in TPB + 3% BSA) and HRP-conjugated siRNA8, 59-agguaauaauagacguggatt-39, corresponding to nucleotides 120–138 and donkey anti-rabbit IgG (1:2000 in TPB + 3% BSA) as secondary antibody. For the NLP1 siRNA9, 59-gagcuucaacuaacaggaatt-39, corresponding to nucleotides 257–275) colorimetric reaction, the TMB substrate reagent set from Millipore was used. The NLP1 in CRM1-dependent export 153

absorbance was measured at 420 nm using a plate reader (Appliskan, Thermo Farjot, G., Sergeant, A. and Mikaelian, I. (1999). A new nucleoporin-like protein Electron Corporation). interacts with both HIV-1 Rev nuclear export signal and CRM-1. J. Biol. Chem. 274, 17309-17317. Fischer, U., Huber, J., Boelens, W. C., Mattaj, I. W. and Lu¨hrmann, R. (1995). The Cell fractionation HIV-1 Rev activation domain is a nuclear export signal that accesses an export HeLa cells were trypsinized, washed with PBS and permeabilized with 0.01% pathway used by specific cellular RNAs. Cell 82, 475-483. digitonin in PBS with 1 mg/ml each of aprotinin, leupeptin and pepstatin. Nuclei Floer, M. and Blobel, G. (1999). Putative reaction intermediates in Crm1-mediated were separated from cytosol by centrifugation at 1000 g for 5 minutes and washed nuclear protein export. J. Biol. Chem. 274, 16279-16286. twice with PBS. Fractions corresponding to 100,000 cells were analyzed by SDS- Fornerod, M., Boer, J., van Baal, S., Morreau, H. and Grosveld, G. (1996). PAGE, followed by western blotting. Interaction of cellular proteins with the leukemia specific fusion proteins DEK-CAN and SET-CAN and their normal counterpart, the nucleoporin CAN. Oncogene 13, SDS-PAGE and western blotting 1801-1808. Proteins were analyzed by SDS-PAGE and western blotting using standard Fornerod, M., Ohno, M., Yoshida, M. and Mattaj, I. W. (1997a). CRM1 is an export methods. The ECL system (Millipore) was used for visualization of proteins. receptor for leucine-rich nuclear export signals. Cell 90, 1051-1060. Fornerod, M., van Deursen, J., van Baal, S., Reynolds, A., Davis, D., Murti, K. G., Fransen, J. and Grosveld, G. (1997b). The human homologue of yeast CRM1 is in a RanGAP-assays dynamic subcomplex with CAN/Nup214 and a novel nuclear pore component Nup88. RanGAP assays were performed as described previously (Askjaer et al., 1999; EMBO J. 16, 807-816. Kehlenbach et al., 2001), with 16 nM RanGAP and increasing concentrations of Fried, H. and Kutay, U. (2003). Nucleocytoplasmic transport: taking an inventory. MBP–NLP1, MBP–Nup214 (aa 1859–2090) or MBP–Nup88 (aa 500–741). Cell. Mol. Life Sci. 60, 1659-1688. Fukuda, M., Asano, S., Nakamura, T., Adachi, M., Yoshida, M., Yanagida, M. and Nishida, E. (1997). CRM1 is responsible for intracellular transport mediated by the In situ hybridization nuclear export signal. Nature 390, 308-311. In situ hybridization was performed as described previously (Hutten and Gerace, L., Ottaviano, Y. and Kondor-Koch, C. (1982). Identification of a major Kehlenbach, 2006). polypeptide of the nuclear pore complex. J. Cell Biol. 95, 826-837. Goldfarb, D. S. (2009). At loose ends: a guide to the translocation channel of the nuclear Acknowledgements pore complex for dummies. In Nuclear Transport (ed. R. H. Kehlenbach), pp 54-68. We thank Frauke Melchior, Detlef Doenecke, Ralf Ficner and Volker Austin, Texas: Landes Bioscience. Guan, T., Kehlenbach, R. H., Schirmer, E. C., Kehlenbach, A., Fan, F., Clurman, Cordes for valuable reagents, Saskia Hutten for preparation of Nup88 B. E., Arnheim, N. and Gerace, L. (2000). Nup50, a nucleoplasmically oriented fragments and Blanche Schwappach and Ruth Geiss-Friedlander for nucleoporin with a role in nuclear protein export. Mol. Cell Biol. 20, 5619-5630. helpful comments on the manuscript. Gu¨ttler, T., Madl, T., Neumann, P., Deichsel, D., Corsini, L., Monecke, T., Ficner, R., Sattler, M. and Go¨rlich, D. (2010). NES consensus redefined by structures of PKI-type and Rev-type nuclear export signals bound to CRM1. Nat. Struct. Mol. Biol. Funding 17, 1367-1376. The project was partially funded by Deutsche Hallberg, E., Wozniak, R. W. and Blobel, G. (1993). An integral membrane protein of Forschungsgemeinschaft [grant number KE 660/9-1 to R.H.K.]. the pore membrane domain of the nuclear envelope contains a nucleoporin-like region. J. Cell Biol. 122, 513-521. Hilliard, M., Frohnert, C., Spillner, C., Marcone, S., Nath, A., Lampe, T., Supplementary material available online at Fitzgerald, D. J. and Kehlenbach, R. H. (2010). The anti-inflammatory http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.090316/-/DC1 prostaglandin 15-deoxy-delta(12,14)-PGJ2 inhibits CRM1-dependent nuclear protein export. J. Biol. Chem. 285, 22202-22210. Hutten, S. and Kehlenbach, R. H. (2006). Nup214 is required for CRM1-dependent References nuclear protein export in vivo. Mol. Cell. Biol. 26, 6772-6785. Askjaer, P., Bachi, A., Wilm, M., Bischoff, F. R., Weeks, D. L., Ogniewski, V., Hutten, S. and Kehlenbach, R. H. (2007). CRM1-mediated nuclear export: to the pore Ohno, M., Niehrs, C., Kjems, J., Mattaj, I. W. et al. (1999). RanGTP-regulated and beyond. Trends Cell Biol. 17, 193-201.

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