Karyopherin Enrichment at the Nuclear Pore Complex Attenuates Ran Permeability Suncica Barbato*, Larisa E

SHORT REPORT Karyopherin enrichment at the nuclear pore complex attenuates Ran permeability Suncica Barbato*, Larisa E. Kapinos*, Chantal Rencurel and Roderick Y. H. Lim‡

ABSTRACT of it. Selective Kap transport is underpinned by multivalent Ran is a small GTPase whose nucleotide-bound forms cycle through interactions with numerous phenylalanine-glycine (FG)-repeat-rich, nuclear pore complexes (NPCs) to direct nucleocytoplasmic transport intrinsically disordered nucleoporins (FG Nups) that line the NPC (NCT). Generally, Ran guanosine triphosphate (RanGTP) binds cargo- channel (Sakiyama et al., 2016). For instance, the classical 97 kDa β carrying karyopherin receptors (Kaps) in the nucleus and releases them import receptor karyopherin subunit 1 (KPNB1, hereafter referred to β into the cytoplasm following hydrolysis to Ran guanosine diphosphate as Kap 1) (Cingolani et al., 1999) engages up to ten FG repeats (RanGDP). This generates a remarkably steep Ran gradient across the (Bayliss et al., 2000, 2002; Bednenko et al., 2003; Isgro and Schulten, nuclear envelope that sustains compartment-specific cargo delivery 2005). Otherwise, the FG Nups are considered to adopt barrier-like and accumulation. However, because NPCs are permeable to small characteristics, such as polymer brushes (Lim et al., 2007; Rout et al., molecules of comparable size, it is unclear how an uncontrolled mixing 2000), gel-like meshworks (Frey and Görlich, 2007; Labokha et al., of RanGTP and RanGDP is prevented. Here, we find that an NPC- 2013) or variations of these (Yamada et al., 2010). Still, Kap-cargo enriched pool of karyopherin subunit beta 1 (KPNB1, hereafter referred complexes are considerably larger than the non-specific size cut-off. β to as Kapβ1) selectively mediates Ran diffusion across the pore but not Furthermore, Kap 1 recruits adaptor proteins from the karyopherin α α passive molecules of similar size (e.g. GFP). This is due to RanGTP subunit family (KPNA, hereafter referred to as Kap ) that bind having a stronger binding interaction with Kapβ1 than RanGDP. For this directly to a diverse repertoire of NLS-cargos such as transcription reason, the RanGDP importer, nuclear transport factor 2, facilitates the factors (Pumroy and Cingolani, 2015; Xu and Massagué, 2004). return of RanGDP into the nucleus following GTP hydrolysis. Hence, our understanding of how NPCs reconcile physical size Accordingly, the enrichment of Kapβ1 at NPCs may function as a exclusion with biochemical selectivity to mediate NCT remains retention mechanism that preserves the sharp transition of RanGTP incomplete. and RanGDP in the nucleus and cytoplasm, respectively. One peculiarity concerns Ran (Melchior et al., 1993; Moore and Blobel, 1993), which controls the site of cargo release, accumulation KEY WORDS: RanGTP, Karyopherin, Nucleocytoplasmic transport, and recycling of Kaps to underpin NCT directionality across Nuclear pore complex the nuclear envelope (Weis, 2003). This is sustained by the interconversion of its two nucleotide-bound forms, RanGTP and INTRODUCTION RanGDP, which are localized to the nucleus and cytoplasm, Nucleocytoplasmic transport (NCT) describes the selective exchange respectively (Görlich et al., 1996). With a molecular mass of of macromolecules between the nucleus and cytoplasm in eukaryotes 25 kDa, Ran is below the NPC size limit for non-specific (Görlich and Kutay, 1999). This is mediated by conduits of 50–60 nm molecules. Also, neither RanGDP nor RanGTP interact with the FG diameter within the nuclear envelope, known as nuclear pore repeats (Rexach and Blobel, 1995). Yet, the concentration of RanGTP complexes (NPCs) (Eibauer et al., 2015; Kim et al., 2018; von is estimated to be at least 200 times higher in the nucleus than in the Appen et al., 2015). Given their considerable size, NPCs are cytoplasm (Görlich et al., 2003; Kalab et al., 2002; Smith et al., 2002). permeable to passive molecules below ∼40 kDa, whereas larger non- Thus, how an uncontrolled mixing of RanGTP and RanGDP is specific macromolecules are generally withheld (Popken et al., 2015; prevented at NPCs remains unknown. Importantly, a disruption in the Timney et al., 2016). Meanwhile, three main groups of protein are Ran gradient results in the loss of NCT directionality (Nachury and selectively trafficked across the NPC central channel to sustain NCT. Weis, 1999) and has been linked to apoptosis (Wong et al., 2009), These are transport receptors known as karyopherins (Kaps), signal- hyperosmotic stress (Kelley and Paschal, 2007) and disease specific cargos and the Ran GTPase that harmonizes the process. (Eftekharzadeh et al., 2018). Aprioriexclusive NPC access is reserved for Kaps (Kimura and In the nucleus, RanGTP binds Kapβ1 to disassemble NLS-cargo– Imamoto, 2014; Tran et al., 2007). These include importins that Kapα–Kapβ1 complexes (Chi et al., 1996; Görlich et al., 1996; deliver cargos bearing nuclear localization signals (NLS) (Boulikas, Rexach and Blobel, 1995). This serves to facilitate the nuclear 1994; Cokol et al., 2000) into the nucleus, and exportins that usher retention of NLS-cargos whose return to the cytoplasm is hindered in cargos containing nuclear export signals (NES) (Xu et al., 2012) out the absence of FG Nup binding. On the other hand, RanGTP–Kapβ1 retains its interactions with the FG Nups to return through NPCs (Kapinos et al., 2017). At the cytoplasmic periphery, RanGTP is Biozentrum & The Swiss Nanoscience Institute, University of Basel, 4056 Basel, hydrolyzed to RanGDP by RanGTPase-activating protein 1 Switzerland. *These authors contributed equally to this work (RanGAP1) together with the Ran-binding proteins RanBP1 and RanBP2 (Lounsbury and Macara, 1997; Vetter et al., 1999). This ‡ Author for correspondence ([email protected]) frees Kapβ1, which is then able to seek out the next NLS-cargo. Still, R.Y.H.L., 0000-0001-5015-6087 Ran seems to accumulate at NPCs (Abu-Arish et al., 2009; Smith et al., 2002; Wong et al., 2009; Yang and Musser, 2006), suggesting

Received 19 August 2019; Accepted 13 December 2019 that it does not freely diffuse through the NPC like other non-specific Journal of Cell Science

1 SHORT REPORT Journal of Cell Science (2020) 133, jcs238121. doi:10.1242/jcs.238121 molecules of similar size (Timney et al., 2016). RanGDP then recruits against samples re-populated with exoKapα–Kapβ1 (20 µM:10 µM) a dedicated import factor, i.e. nuclear transport factor 2 (NUTF2, (Fig. 2A). Unexpectedly, exoRanGDP retention was the lowest in hereafter referred to as NTF2) (Ribbeck et al., 1998; Smith et al., nuclei harboring exoKapα–Kapβ1, i.e. 25% less than in control 1998), which returns RanGDP to the nucleus. Upon re-entry, the samples that lacked exoKaps (Fig. 2B,C). In comparison, nuclei that chromatin-bound enzyme regulator of chromosome condensation 1 contained exoKapβ1 alone showed 20% more exoRanGDP retention (RCC1, also referred to as Ran guanine nucleotide exchange factor or than control cells. This signified that exoKapα–Kapβ1 does not RanGEF) (Klebe et al., 1995b; Renault et al., 2001) recharges impede exoRanGDP outflow at the NPC as effectively as exoKapβ1 RanGDP to RanGTP to complete the cycle. In this manner, NCT alone. Nevertheless, we did obtain exoRanGDP fluorescence at the cargo delivery and the recycling of Kaps are regulated by RanGAP1 nuclear envelope of control cells, which suggested residual binding and RanGEF as well as the controlled exchange of RanGTP and with either endogenous transport receptors or other NPC components RanGDP across NPCs (Abu-Arish et al., 2009; Izaurralde et al., 1997; (Görlich et al., 1996; Partridge and Schwartz, 2009; Schrader et al., Kalab et al., 2006, 2002). 2008). Following these observations, we rationalized that NPCs More recently, a steady-state enrichment of Kapβ1 was uncovered might effectively impede exoRanGDP movement based on its in FG Nup layers (Kapinos et al., 2014; Schoch et al., 2012; Vovk biochemical interactions with exoKapβ1. et al., 2016; Wagner et al., 2015; Zahn et al., 2016) and in NPCs Subsequently, binding affinity measurements between exoKapβ1 (Görlich et al., 1995; Kapinos et al., 2017; Lowe et al., 2015). We also and either exoRanGDP or a GDP-bound, non-hydrolyzable Ran found that depleting this Kapβ1 pool abolishes NPC barrier function mutant comprising a Gln69 to Leu point mutation (RanQ69L-GDP) against large non-specific cargos (see Kapinos et al., 2017), which resulted in Kd values of 0.5±0.07 µM or 1.3±0.15 µM, respectively suggests that Kaps serve as bona fide constituents of the NPC barrier (Fig. S1D), consistent with literature values (Forwood et al., 2008; mechanism. This motivated our present study, in which we show that Lonhienne et al., 2009). In comparison, binding between Kapα and NPC-bound Kapβ1 selectively retains RanGTP and RanGDP at the Kapβ1isstronger(Kd=0.2 µM) (Bednenko et al., 2003; Catimel et al., NPC but not passive molecules of similar size (e.g. GFP). This is due 2001; Kapinos et al., 2017). For this reason, exoRanGDP is unable to to binding interactions with Kapβ1 at the pore, which are stronger for outcompete exoKapα for exoKapβ1. Indeed, the nuclear and nuclear RanGTP and weaker for RanGDP. In comparison, NPCs that lack envelope exoRanGDP signals are at least ∼30% higher for exoKapβ1 Kaps show unrestricted Ran movement, i.e. a Ran ‘leak’. Therefore, than exoKapα·Kapβ1 (Fig. 2B,C). Therefore, we reasoned that RanGTP outflow depends on the hydrolysis of RanGTP to RanGDP, exoKapβ1 in the NPC might serve to restrict exoRanGDP outflow. By whereas RanGDP import requires NTF2. These results explain how contrast, a lack of binding of exoRanGDP to exoKapα–Kapβ1results Kaps might serve to maintain the Ran gradient by regulating the in a higher outflow of exoRanGDP from the nucleus. movement of RanGTP/GDP through NPCs. To validate the latter, we used GFP (∼26 kDa), which is similar in size compared with Ran but does not bind to FG Nups or Kaps and, RESULTS AND DISCUSSION therefore should not be retained at the nuclear envelope. Indeed, Reduction of Kapβ1 weakens the NPC barrier against neither exoKapβ1 nor exoKapα–Kapβ1 could stem the outflow of RanGDP GFP from the nucleus (Fig. S1E,F), which is consistent with in vivo A pool of endogenous Kapβ1andKapα (endoKapβ1 and endoKapα, observations of small passive cargos of equivalent size (Abu-Arish respectively; hereafter collectively referred to as endoKaps) is generally et al., 2009; Timney et al., 2016). This is a key finding because it retained at the nuclear envelope after cell permeabilization with shows that the NPC size exclusion limit for non-specific cargos does digitonin (Kapinos et al., 2017). Hence, we asked whether endoKaps not preclude Kap occupancy at the pore. can impede the movement of exogenous RanGDP (exoRanGDP) through NPCs (Fig. 1A). For comparison, we incubated permeabilized RanGTP efflux depends on GTP hydrolysis −1 cells in Ran mix to deplete the pool of endoKaps from the NPC RanGDP is converted by RanGEF into RanGTP (kcat=3.5 s , see (Kapinos et al., 2017), which resulted in a reduction of endoKapβ1 Fig. S2A) (Klebe et al., 1995a), which binds Kapβ1 in the nucleus (∼50%) and endoKapα (∼80%) within the nucleus and nuclear to displace Kapα during NCT. Given that the binding of RanGTP to envelope, respectively (Fig. 1B,C). Then, we incubated both samples in Kapβ1 is significantly stronger (Kd=0.035 µM) (Bednenko et al., 5 µM exoRanGDP for 1 h until equilibration was reached within the 2003; Hahn and Schlenstedt, 2011; Kapinos et al., 2017) than nucleus and its exterior. This was followed by a short wash with PBS RanGDP to Kapβ1, we wondered how their efflux would differ. (3×5 min) to investigate the extent of exoRanGDP nuclear retention Upon verifying that RanGEF, RanGAP1 and RanBP2 were present in the presence and absence of endoKaps. In comparison to the following permeabilization (Fig. S2B,C), we again entrapped permeabilized cell control (and under the same conditions), exoRanGDP with 10 µM exoKapβ1 or exoKapα–Kapβ1 and exoRanGDP was reduced by ∼50% in both the nucleus and nuclear added an energy-regenerating mixture comprising 2 mM GTP, envelope when endoKaps were depleted (Fig. 1B,C). Meanwhile, to 0.1 mM ATP, 4 mM creatine phosphate and 20 U/ml creatine kinase ensure the efficacy of our assay, we confirmed that nuclear retention of (Ribbeck et al., 1998) to enable RanGEF activity (Fig. 3A). exoRanGDP following its incubation is considerably larger than its Although exoRanGDP was withheld by exoKapβ1 because they remainder after the washing step (Fig. S1A–C). Taken together, our bound to each other (Fig. 2), we observed a dramatic 50% reduction results suggest that the presence of endoKaps in NPCs impedes the of its fluorescence inside the nucleus and at the nuclear envelope outflow of exoRanGDP from the nucleus. when energy mix was added, indicating that exoRanGDP was converted to exoRanGTP (Fig. 3B–D). Indeed, the same was true in Kapβ1 but not Kapα–Kapβ1 restricts RanGDP movement terms of the nuclear signal when exoKapα–Kapβ1 was used, except through NPCs that the signal at the nuclear envelope was slightly higher. Still, On the basis of physiological estimates, we next filled endoKap- because both exoRanGDP–Kapβ1 and exoRanGTP–Kapβ1 bind reduced nuclei with 5 µM exoRanGDP (Görlich et al., 2003) FG Nups, we questioned whether exoRanGTP efflux was promoted −1 followed by 10 µM exogenous Kapβ1(exoKapβ1) (Eisele et al., by RanGAP1, which hydrolyses GTP at a rate of kcat=2.1 s (Klebe

2010) that re-populated the NPCs. These nuclei were compared et al., 1995a). Journal of Cell Science

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Fig. 1. Depletion of Kapβ1 from the NPC facilitates the release of RanGDP from the nucleus. (A) Experimental sequence. (B) EndoKapα and endoKapβ1are retained at the nuclear envelope (NE) after digitonin permeabilization. Ran mix reduces endoKapα and endoKapβ1 at the NE to release exoRanGDP from the nucleus. Scale bars: 10 µm. (C) Quantification of the endoKapβ1, endoKapα and exoRanGDP fluorescence signals in digitonin-permeabilized cells with and without Ran mix. n≥3 experiments each, with a total of 120, 63 and 39 cells for non-permeabilized cells, digitonin-permeabilized (control) cells and Ran mix, respectively. ****P<0.0001; Student’s t-test. Box plots denote the median, first, and third quartiles. Error bars denote standard deviation, including outliers. See Table S1 for details.

To validate our hypothesis, we used RanQ69L-GDP, which can be exoRanGDP (Fig. 2), exoKapβ1 impedes the outflow of RanQ69L- converted into its GTP-bound form by RanGEF (Fig. S2A) but GDP leading to signal increase in the nuclear envelope and nucleus cannot be hydrolyzed by RanGAP1 (Klebe et al., 1995a). Similar to (Fig. S3) due to their binding (Fig. S1D). Also, RanQ69L-GDP Journal of Cell Science

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Fig. 2. Binding to Kapβ1 restricts RanGDP diffusion through NPCs. (A) Experimental sequence. (B) Re-populating NPCs with exoKapβ1 retains exoRanGDP in Ran mix-treated nuclei. Re-population of NPCs with exoKapα–Kapβ1 does not impede the outflow of exoRanGDP. Scale bars: 10 µm. (C) Quantification of the exoRanGDP fluorescence signal in the nucleus and nuclear envelope (NE) under each of the above conditions. n=3 with a total of 56, 65 and 70 cells for PBS control, exoKapβ1 and exoKapα–Kapβ1, respectively. **P<0.01; ****P<0.0001; Student’s t-test. Box plots denote the median, first and third quartiles. Error bars denote standard deviation, including outliers. See Table S1 for details. Journal of Cell Science

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Fig. 3. RanGTP efflux requires Kapβ1-binding and GTP hydrolysis. (A) Experimental sequence. (B) ExoRanGDP is retained in the nucleus in the absence of energy mix. When energy mix is added, RanGEF converts exoRanGDP to exoRanGTP, which is released from the nucleus in a GTP hydrolysis-dependent manner (see Fig. S4 for the retention of non-hydrolyzable mutant RanQ69L-GTP in the nucleus). Scale bars: 10 µm. (C) ExoKapα–Kapβ1 resulted in similar observations with respect to exoRanGDP/GTP as shown in B. Scale bars: 10 µm. (D) Quantification of the exoRanGDP and exoRanGTP fluorescence signals in the nucleus and nuclear envelope (NE) under each of the above conditions. For exoKapβ1, n=3 experiments were carried out, with a total of 65 and 55 cells for PBS control and ‘energy mix’ experiments, respectively. For exoKapα–Kapβ1, n=3 experiments were carried out, with a total of 85 and 77 cells for PBS control and ‘energy mix’ experiments, respectively. **P<0.05; ***P<0.001; ****P<0.0001; Student’s t-test. Box plots denote the median, first and third quartiles. Error bars denote standard deviation, including outliers. See Table S1 for details.

leaked out non-specifically in the presence of exoKapα–Kapβ1 NTF2 facilitates RanGDP re-import into the nucleus compared to control. Yet, when energy mix was present, the nuclear NTF2 binds to RanGDP with a Kd=75–240 nM (Chaillan- signal for RanQ69L-GTP was only slightly reduced by 10% (Fig. S4) Huntington et al., 2000), and regulates the re-import of RanGDP to compared to that of exoRanGTP (∼50%; Fig. 3D). Hence, in the the nucleus (Ribbeck et al., 1998) based on interactions between absence of hydrolysis, RanQ69L-GTP remains bound to Kapβ1 NTF2 and the FG Nups (Wagner et al., 2015). We therefore asked if residing at NPCs and does not depart from the nucleus. NTF2 can return exoRanGDP to the nucleus after exoRanGTP Journal of Cell Science

5 SHORT REPORT Journal of Cell Science (2020) 133, jcs238121. doi:10.1242/jcs.238121 hydrolysis in the presence of exoKapβ1 at the NPCs (Fig. 4A). As followed by hydrolysis back to exoRanGDP at the NPC (by before (Fig. 3), exoRanGDP was significantly reduced when energy RanGAP1). In marked contrast, the exoRanGDP signal in the mix was supplied, due to its conversion to exoRanGTP (by RanGEF) nucleus increased almost 200% over control when 4 µM exogenous

Fig. 4. NTF2 returns RanGDP to the nucleus. (A) Experimental sequence. (B) NTF2 facilitates exoRanGDP import back into the nucleus. Scale bars: 10 µm. (C) Quantification of the exoRanGDP and exoRanGTP fluorescence signals within the nucleus and the nuclear envelope (NE) under each of the above conditions. n=3 experiments each, with a total of 39, 53 and 44 cells for PBS control, energy mix and energy mix plus NTF2 experiments, respectively. See Table S1 for details. (D) Left: Uncontrolled mixing in NPCs that lack Kaps. Right: Kapβ1 selectively mediates Ran movement in the NPC. The dotted line separates conditions under which RanGDP (left) and RanGTP (right) bind to Kapβ1. See text for details. Journal of Cell Science

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NTF2 (exoNTF2) was included with the energy mix (Fig. 4B,C). previously (Kapinos et al., 2017, 2014; Schoch et al., 2012; Wagner et al., Thus, NTF2 replenishes exoRanGDP in the nucleus to close the Ran 2015). Kapβ1 was eluted in 10 mM TrisHCl pH 7.5, 100 mM NaCl, 1 mM cycle following exoRanGTP hydrolysis. DTT-containing buffer and concentrated to 15–20 μM. A full-length human Kapα construct (pCMVTNT-T7-KPNA2; Addgene plasmid #26678) was cloned using EcoRI/BamII restriction enzymes into the pQE70 vector with a Implications of Ran transport regulation in NPCs His6 tag at its C-terminus and a short linker (-GSRSHHHHHH) that does not We have tested specific conditions related to NCT to study how the affect the complex formation of this protein with Kapβ1. Human Kapα was mixing of RanGDP and RanGTP is minimized at NPCs. A key purified and eluted in 20 mM TrisHCl pH 7.5, 100 mM NaCl, 2 mM DTT, finding is that an enrichment of Kapβ1 within NPCs attenuates the 10% glycerol. A plasmid (pQE32) with a full-length human RanQ69L permeability of Ran but does not impede passive molecules of construct was a gift from Ulrike Kutay (ETH Zurich, Zurich, Switzerland) comparable size, e.g. GFP. By contrast, Ran freely diffuses through (Kutay et al., 1997). Using site-directed mutagenesis, RanWT was derived the NPC when Kapβ1 is absent. This is consistent with simulations from this clone using the following primers: 5′-GTATGGGACACAGCCG- that have shown that the Ran gradient is sensitive to changes in NPC GCCAGGAGAAA TTCGGTGGACTG-3′ and 5′-CAGTCCACCGAATT- permeability and that a retention mechanism may be required to TCTCCTGGCC GGCTGTGTCCCATAC-3′. establish the steep Ran gradient (Becskei and Mattaj, 2003; Görlich RanWT or RanQ69L were purified using a Ni-NTA column (Roche) over – et al., 2003; Kopito and Elbaum, 2009). This may also explain an imidazole gradient (10 500 mM) and then dialyzed into a 10 mM HEPES buffer pH 7.2 with 100 mM NaCl as described previously (Kapinos observations of Ran accumulation at NPCs (Abu-Arish et al., 2009; et al., 2017). Purified proteins were incubated for 30 min at 4°C with 10 mM Smith et al., 2002; Wong et al., 2009; Yang and Musser, 2006). EDTA. Then 25 mM MgCl2 was added together with 1 mM GTP or GDP These findings further underscore the role of Kapβ1 as an essential that ensured its binding to nucleotide-free Ran. Finally, GTP or GDP-loaded constituent of the NPC barrier mechanism i.e. Kap-centric control RanWT or RanQ69L proteins were dialyzed into PBS buffer, pH 7.2 (Kapinos et al., 2017, 2014; Lim et al., 2015) (Fig. 4D). Clearly, the (Invitrogen, Lifesciences), in the presence of 1 mM MgCl2 and purified size of Ran is below the non-specific size limit (Paine et al., 1975; using size-exclusion column (Superdex 200 HiLoad 16/60; GE). Finally, Popken et al., 2015; Timney et al., 2016) and does not bind to FG these proteins were concentrated to 35-45 µM in PBS containing 1 mM repeats. Hence, the FG Nups alone are insufficient to prevent a MgCl2. mixing of RanGTP and RanGDP. Rather, the binding of RanGTP and The full-length rat NTF2 was cloned, expressed and purified as before β (Wagner et al., 2015). Typical stock concentration of NTF2 was ∼250– RanGDP to Kap 1 at the NPC would minimize their mixing. μ β 300 M in PBS (Invitrogen, Lifesciences). RanGEF construct was obtained Nevertheless, this depends on the distribution of Kap 1 complexes from GenScript in pUC57 vector and re-cloned into pPEP-TEV plasmid that co-exist in the NPC at steady-state, which remains poorly using XbaI/BamHI restriction enzymes for the further expression. It was defined. RanGDP might interact with Kapβ1 (and not Kapα–Kapβ1) purified as described before for Kapβ1 (Kapinos et al., 2014; Schoch et al., long enough to be converted into RanGTP by RanGEF in the 2012). The protein quality was verified using 12% SDS PAGE. All purified nucleoplasm. Subsequently, RanGTP binds with stand-alone Kapβ1 recombinant proteins were shock-frozen and stored at −80°C. or Kapα–Kapβ1toformRanGTP–Kapβ1. Following GTP hydrolysis by RanGAP1, RanGDP then departs from the NPC into Protein labeling the cytoplasm, being the more weakly bound component of the Recombinant proteins were labeled with fluorescent dyes (1:5 ratio) for 2 h complex. Incoming Kapα might further promote RanGDP release by at room temperature in light-protected vials. AlexaFluor 647 maleimide binding Kapβ1. For these reasons, it is compelling that RanGDP (Invitrogen, Lifesciences) was used to label RanWT or RanQ69L. AlexaFluor 488 maleimide (Invitrogen, Lifesciences) was used to label requires NTF2 to be expeditiously shuttled back through NPCs β α β Kap 1. Atto 550 maleimide (Sigma-Aldrich) was used to label Kap .PD enriched with Kap 1 (Wagner et al., 2015). MiniTrap G-25 sample preparation spin columns (GE Healthcare, But why would it be crucial to prevent a mixing of the two Lifesciences) were used to remove the excessive dye. The degree of nucleotide-bound forms of Ran? On a mechanistic level, the mixing labelling (DOL) was calculated following Nanodrop UV-Vis spectrometry of RanGTP and RanGDP reduces the Ran gradient. This can to measure the respective dye and protein absorptions. dramatically alter the cellular distribution of Kaps, as shown for exportin-t (Kuersten et al., 2002) leading to cargo mislocalization Permeabilized cell assay (Wong et al., 2009) and, potentially, in extreme cases, an inversion of HeLa cells (ATCC® CCL-2™; authenticated and confirmed to be NCT (Nachury and Weis, 1999). Nevertheless, the sensitivity of cells contamination-free on 17.4.2019) were washed with PBS and then to a mixing of RanGTP and RanGDP is unclear, and several permeabilized with 40 µg/ml digitonin (5 min incubation time). After questions remain. How much of this mixing is tolerable in cells? Is permeabilization, cells were washed with PBS three times for 5 min. Cells were then incubated with Ran mix (2 mM GTP, 0.1 mM ATP, 4 mM creatine the enzymatic activity of RanGEF and RanGAP1 sufficient to in vivo phosphate, 20 U/ml creatine kinase, 5 µM RanGDP, 4 µM NTF2 and 1 mM maintain the Ran gradient ? How do cells recover from DTT) for 1 h, followed by three washes with PBS for 5 min each. Upon this, changes to the Ran gradient, such as in response to hyperosmotic 5 µM RanGDP-AlexaFluor 647 (DOL=1) or 5 µM RanQ69L-GDP– stress (Kelley and Paschal, 2007)? Might the mixing of RanGTP and AlexaFluor 647 was added to cells for 1 h at room temperature. Depending RanGDP be cause or consequence of defects in Kap transport on the assay, some samples underwent a triple washing step in PBS for 5 min efficiency, cargo directionality and accumulation? Indeed, other each. Following this, permeabilized cells were immediately fixed with 4% small essential cargos (<40 kDa), such as histones (Mühlhäusser formalin, stained with DAPI, mounted on the sample glass using Vectashield et al., 2001) and ribosomal proteins (Jakel and Görlich, 1998), utilize (Vector Labs) and imaged. For re-population assays of exogenous Kap, cells dedicated Kaps for nuclear import. Hence, we postulate that Kap were not washed after exoRanGDP incubation but were directly incubated β α– β enrichment at the NPC selectively restricts the uncontrolled mixing with exoKap 1, exoKap Kap 1 or PBS for 1 h at room temperature. Subsequently, the cells were washed and fixed except when energy mix of small essential proteins across the nuclear envelope. (2 mM GTP, 0.1 mM ATP, 4 mM creatine phosphate, 20 U/ml creatine kinase) (Lowe et al., 2015) was added for 1 h, or PBS as a control. After a MATERIALS AND METHODS triple washing step in PBS for 5 min each the cells were also immediately Protein expression and purification fixed with 4% formalin and mounted on the microscope slide using All exogenous proteins, such as human Kapβ1, Kapα, wild-type Ran Vectashield medium. In the NTF2 assay, a similar procedure was followed but

(RanWT) and RanQ69L were cloned, expressed and purified as described with the addition of NTF2 into the energy mix. Endogenous proteins Journal of Cell Science

7 SHORT REPORT Journal of Cell Science (2020) 133, jcs238121. doi:10.1242/jcs.238121 were detected using the following primary antibodies (all Abcam): Kapβ1 References (ab2811, 1:200), Kapα (ab6036, 1:200), RanGAP1 (ab2081, 1:500), Abu-Arish, A., Kalab, P., Ng-Kamstra, J., Weis, K. and Fradin, C. (2009). Spatial Biophys. J. RanGEF (ab54600, 1:200) and RanBP2 (ab64276, 1:2000). distribution and mobility of the Ran GTPase in live interphase cells. 97, 2164-2178. doi:10.1016/j.bpj.2009.07.055 Bayliss, R., Littlewood, T. and Stewart, M. (2000). Structural basis for the Confocal imaging and analysis interaction between FxFG nucleoporin repeats and importin-beta in nuclear Fluorescence images were obtained at room temperature using an LSM700 trafficking. Cell 102, 99-108. doi:10.1016/S0092-8674(00)00014-3 upright confocal microscope with an oil-immersed 63×/1.4 NA PLAN APO Bayliss, R., Littlewood, T., Strawn, L. A., Wente, S. R. and Stewart, M. (2002). GLFG and FxFG nucleoporins bind to overlapping sites on importin-beta. J. Biol. objective and multialkali photomultiplier (PMT) detector type (Zeiss). Chem. 277, 50597-50606. doi:10.1074/jbc.M209037200 Quantification of fluorescence intensity was performed using CellProfiler Becskei, A. and Mattaj, I. W. (2003). The strategy for coupling the RanGTP gradient software (Kamentsky et al., 2011). DAPI staining was used to define a to nuclear protein export. Proc. Natl. Acad. Sci. USA 100, 1717-1722. doi:10. region of interest (ROI). To define the nuclear envelope, the DAPI ROI was 1073/pnas.252766999 reduced by five pixels and simultaneously expanded by five pixels, i.e. pixel Bednenko, J., Cingolani, G. and Gerace, L. (2003). Importin beta contains a μ ‘ ’ ‘ COOH-terminal nucleoporin binding region important for nuclear transport. J. Cell size=0.04 m×0.04 µm), yielding the nuclear envelope ROI ; the nucleus Biol. ’ 162, 391-401. doi:10.1083/jcb.200303085 ROI , therefore, is the area within the reduced ROI. Both regions were then Boulikas, T. (1994). Putative nuclear localization signals (NLS) in protein used to quantify the mean fluorescence intensity of exoRan at the nuclear transcription factors. J. Cell. Biochem. 55, 32-58. doi:10.1002/jcb.240550106 envelope and within the nucleus. In each case, when calculating the fraction Catimel, B., Teh, T., Fontes, M. R. M., Jennings, I. G., Jans, D. A., Howlett, G. J., of exoRan, the Ran fluorescence intensity was normalized to the signal Nice, E. C. and Kobe, B. (2001). Biophysical characterization of interactions obtained from the PBS-treated sample (control); the number of analyzed involving importin-alpha during nuclear import. J. Biol. Chem. 276, 34189-34198. cells is specified in the figure legends. doi:10.1074/jbc.M103531200 Chi, N. C., Adam, E. J. H., Visser, G. D. and Adam, S. A. (1996). RanBP1 stabilizes the interaction of Ran with p97 nuclear protein import. J. Cell Biol. 135, 559-569. Microscale thermophoresis doi:10.1083/jcb.135.3.559 RanGDP or RanQ69L-GDP binding to Kapβ1 was verified by microscale Chaillan-Huntington, C., Braslavsky, C. V., Kuhlmann, J. and Stewart, M. (2000). Dissecting the interactions between NTF2, RanGDP, and the nucleoporin thermophoresis (MST). Increasing concentrations of RanGDP or RanQ69L- J. Biol. Chem. β XFXFG repeats. 275, 5874-5879. doi:10.1074/jbc.275.8.5874 GDPwereaddedtoKap 1 (100 nM) labeled with maleimide conjugated to Cingolani, G., Petosa, C., Weis, K. and Müller, C. W. (1999). Structure of importin- Alexa Fluor 488 (Kapβ1-488) (DOL=2). The final test solutions contained β bound to the IBB domain of importin-α. Nature 399, 221-229. doi:10.1038/20367 Kapβ1-488 (50 nM) and Ran at varying concentrations (0.00031 µM, Cokol, M., Nair, R. and Rost, B. (2000). Finding nuclear localization signals. EMBO 0.000625 µM, 0.00125 µM, 0.0025 µM, 0.005 µM, 0.01 µM, 0.019 µM, Rep. 1, 411-415. doi:10.1093/embo-reports/kvd092 0.039 µM, 0.078 µM, 0.156 µM, 0.3125 µM, 0.625 µM, 1.25 µM, 2.5 µM, Eftekharzadeh, B., Daigle, J. G., Kapinos, L. E., Coyne, A., Schiantarelli, J., Carlomagno, Y., Cook, C., Miller, S. J., Dujardin, S., Amaral, A. S. et al. (2018). 5 µM or 10 µM). These solutions were loaded into glass capillaries (Monolith Tau protein disrupts nucleocytoplasmic transport in Alzheimer’s disease. Neuron NT.115 capillary, standard treatment MO-K002, NanoTemper) and the change 99, 925-940.e7. doi:10.1016/j.neuron.2018.07.039 of normalized fluorescence (‰ Fnorm) was measured (60% laser power, 100% Eibauer, M., Pellanda, M., Turgay, Y., Dubrovsky, A., Wild, A. and Medalia, O. LED power, 30 s laser on/10 s laser off) using Nanotemper Monolith NT.115 (2015). Structure and gating of the nuclear pore complex. Nat. Commun. 6, 7532. (NanoTemper). doi:10.1038/ncomms8532 Eisele, N. B., Frey, S., Piehler, J., Görlich, D. and Richter, R. P. (2010). Ultrathin nucleoporin phenylalanine-glycine repeat films and their interaction with nuclear RanGEF activity assay transport receptors. EMBO Rep. 11, 366-372. doi:10.1038/embor.2010.34 A Synergy H1 Hybrid Multi-Mode Monochromator Fluorescence Microplate Forwood, J. K., Lonhienne, T. G., Marfori, M., Robin, G., Meng, W., Guncar, G., Reader (BioTek) was used to measure the enzymatic activity of RanGEF to Liu, S. M., Stewart, M., Carroll, B. J. and Kobe, B. (2008). Kap95p binding exchange RanGDP to RanGTP (Klebe et al., 1995a). 1 µl of 3 µM RanGEF induces the switch loops of RanGDP to adopt the GTP-bound conformation: implications for nuclear import complex assembly dynamics. J. Mol. Biol. 383, (final concentration: 30 nM) was added to 100 µl of 5 µM RanGDP (WT or 772-782. doi:10.1016/j.jmb.2008.07.090 Q69 L mutant) and 200 µM N-methylanthraniloyl-tagged GTP (Mant-GTP; Frey, S. and Görlich, D. (2007). A saturated FG-repeat hydrogel can reproduce the Sigma-Aldrich) in PBS at 25°C. As control, we repeated the experiments permeability properties of nuclear pore complexes. Cell 130, 512-523. doi:10. without RanGEF in order to monitor the non-catalyzed rate of the GDP-GTP 1016/j.cell.2007.06.024 exchange. The fluorescent signal was monitored at two wavelengths: 335 nm Görlich, D. and Kutay, U. (1999). Transport between the cell nucleus and the Annu. Rev. Cell Dev. Biol. (excitation at 292 nm; internal tryptophan fluorescence) and 450 nm cytoplasm. 15, 607-660. doi:10.1146/annurev.cellbio. 15.1.607 (excitation at 370 nm; Mant nucleotide fluorescence). Fluorescence change Görlich, D., Vogel, F., Mills, A. D., Hartmann, E. and Laskey, R. A. (1995). Distinct was normalized to control experiments carried out without RanGEF. functions for the two importin subunits in nuclear protein import. Nature 377, 246-248. doi:10.1038/377246a0 Acknowledgements Görlich, D., Panté, N., Kutay, U., Aebi, U. and Bischoff, F. R. (1996). Identification We thank Mikel Ghelfi for technical assistance. of different roles for RanGDP and RanGTP in nuclear protein import. EMBO J. 15, 5584-5594. doi:10.1002/j.1460-2075.1996.tb00943.x ̈ Competing interests Gorlich, D., Seewald, M. J. and Ribbeck, K. (2003). Characterization of Ran-driven cargo transport and the RanGTPase system by kinetic measurements and The authors declare no competing or financial interests. computer simulation. EMBO J. 22, 1088-1100. doi:10.1093/emboj/cdg113 Hahn, S. and Schlenstedt, G. (2011). Importin beta-type nuclear transport Author contributions receptors have distinct binding affinities for Ran-GTP. Biochem. Biophys. Res. Conceptualization: L.E.K., R.Y.H.L.; Methodology: R.Y.H.L.; Validation: L.E.K.; Commun. 406, 383-388. doi:10.1016/j.bbrc.2011.02.051 Formal analysis: S.B., L.E.K.; Investigation: S.B.; Resources: C.R.; Writing - original Isgro, T. A. and Schulten, K. (2005). Binding dynamics of isolated nucleoporin repeat draft: S.B., L.E.K.; Writing - review & editing: R.Y.H.L.; Visualization: S.B., L.E.K.; regions to importin-beta. Structure 13, 1869-1879. doi:10.1016/j.str.2005.09.007 Supervision: R.Y.H.L.; Project administration: R.Y.H.L.; Funding acquisition: R.Y.H.L. Izaurralde, E., Kutay, U., von Kobbe, C., Mattaj, I. W. and Görlich, D. (1997). The asymmetric distribution of the constituents of the Ran system is essential for EMBO J. Funding transport into and out of the nucleus. 16, 6535-6547. doi:10.1093/emboj/ S.B. acknowledges financial support from the National Centre of Competence in 16.21.6535 Jakel, S. and Görlich, D. (1998). Importin beta, transportin, RanBP5 and RanBP7 Research (NCCR) in Molecular Systems Engineering. R.Y.H.L.’s research group mediate nuclear import of ribosomal proteins in mammalian cells. EMBO J. 17, received support through a grant from the Schweizerischer Nationalfonds zur 4491-4502. doi:10.1093/emboj/17.15.4491 Förderung der Wissenschaftlichen Forschung (Swiss National Science Kalab, P., Weis, K. and Heald, R. (2002). Visualization of a Ran-GTP gradient in Foundation), grant no. 31003A_170041. interphase and mitotic Xenopus egg extracts. Science 295, 2452-2456. doi:10. 1126/science.1068798 Supplementary information Kalab, P., Pralle, A., Isacoff, E. Y., Heald, R. and Weis, K. (2006). Analysis of a Supplementary information available online at RanGTP-regulated gradient in mitotic somatic cells. Nature 440, 697-701. doi:10. http://jcs.biologists.org/lookup/doi/10.1242/jcs.238121.supplemental 1038/nature04589 Journal of Cell Science

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