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• W2131878394 endingPage "1995" @default.
• W2131878394 startingPage "1982" @default.
• W2131878394 abstract "Article1 April 1999free access Nup153 is an M9-containing mobile nucleoporin with a novel Ran-binding domain Sara Nakielny Sara Nakielny Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104-6148 USA Search for more papers by this author Sarah Shaikh Sarah Shaikh Faculty of Medicine, University of Calgary, Calgary, Alberta, T2N 4N1 Canada Search for more papers by this author Brian Burke Brian Burke Faculty of Medicine, University of Calgary, Calgary, Alberta, T2N 4N1 Canada Search for more papers by this author Gideon Dreyfuss Corresponding Author Gideon Dreyfuss Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104-6148 USA Search for more papers by this author Sara Nakielny Sara Nakielny Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104-6148 USA Search for more papers by this author Sarah Shaikh Sarah Shaikh Faculty of Medicine, University of Calgary, Calgary, Alberta, T2N 4N1 Canada Search for more papers by this author Brian Burke Brian Burke Faculty of Medicine, University of Calgary, Calgary, Alberta, T2N 4N1 Canada Search for more papers by this author Gideon Dreyfuss Corresponding Author Gideon Dreyfuss Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104-6148 USA Search for more papers by this author Author Information Sara Nakielny1, Sarah Shaikh2, Brian Burke2 and Gideon Dreyfuss 1 1Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104-6148 USA 2Faculty of Medicine, University of Calgary, Calgary, Alberta, T2N 4N1 Canada *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:1982-1995https://doi.org/10.1093/emboj/18.7.1982 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We employed a phage display system to search for proteins that interact with transportin 1 (TRN1), the import receptor for shuttling hnRNP proteins with an M9 nuclear localization sequence (NLS), and identified a short region within the N-terminus of the nucleoporin Nup153 which binds TRN1. Nup153 is located at the nucleoplasmic face of the nuclear pore complex (NPC), in the distal basket structure, and functions in mRNA export. We show that this Nup153 TRN1-interacting region is an M9 NLS. We found that both import and export receptors interact with several regions of Nup153, in a RanGTP-regulated fashion. RanGTP dissociates Nup153–import receptor complexes, but is required for Nup153–export receptor interactions. We also show that Nup153 is a RanGDP-binding protein, and that the interaction is mediated by the zinc finger region of Nup153. This represents a novel Ran-binding domain, which we term the zinc finger Ran-binding motif. We provide evidence that Nup153 shuttles between the nuclear and cytoplasmic faces of the NPC. The presence of an M9 shuttling domain in Nup153, together with its ability to move within the NPC and to interact with export receptors, suggests that this nucleoporin is a mobile component of the pore which carries export cargos towards the cytoplasm. Introduction Transport of proteins and RNA–protein (RNP) complexes between the nucleus and the cytoplasm is essential for both constitutive and regulated cellular activities. While some proteins and RNP complexes undergo unidirectional transport across the nuclear envelope, for example import of a subset of proteins and export of mRNA, tRNA and rRNA, others undergo bidirectional trafficking in the course of their maturation and/or function (reviewed in Nakielny et al., 1997; Görlich, 1998; Izaurralde and Adam, 1998; Mattaj and Englmeier, 1998). For example, spliceosomal U snRNAs are exported, processed in the cytoplasm, and re-imported into the nucleus as mature snRNPs (Nakielny et al., 1997; Mattaj and Englmeier, 1998), and many hnRNP proteins shuttle continuously in and out of the nucleus in order to perform their probable function in the export of mRNA (reviewed in Piñol-Roma, 1997). Regulation of both unidirectional and bidirectional transport adds a further level of complexity to the nucleocytoplasmic traffic (reviewed in Cole and Saavedra, 1997; Nigg, 1997). All cargos get into and out of the nucleus through very large proteinaceous structures termed nuclear pore complexes (NPCs) which span the nuclear envelope (reviewed in Rout and Wente, 1994; Davis, 1995; Doye and Hurt, 1997). In higher eukaryotes, the NPC is ∼125 MDa, and in yeast it is ∼66 MDa (Doye and Hurt, 1997; Yang et al., 1998). Each NPC is comprised of 50–100 different proteins, termed nucleoporins, that assemble in multiple copies to form the complex. Biochemical, genetic and genome sequencing approaches have led to the identification of ∼30 yeast nucleoporins or NPC-associated proteins, while about half as many higher eukaryotic nucleoporins have been characterized. Many of the nucleoporins identified so far have one sequence motif in common, the FG (phenylalanine–glycine) repeat. This motif generally is repeated multiple times, and is found within at least two distinct sequence contexts, FXFG and GLFG, where X is an amino acid with a small or polar side chain (Rout and Wente, 1994; Davis, 1995; Doye and Hurt, 1997). As yet, the function(s) of FG repeats is unknown. Progress in characterizing the components and principles of nucleocytoplasmic transport has been rapid over the last few years. Basic tenets have been established, namely that import and export of proteins and RNP complexes are generally dictated by specific protein and/or RNA signals in the cargo which are recognized by soluble receptors (Nakielny et al., 1997; Görlich, 1998; Izaurralde and Adam, 1998; Mattaj and Englmeier, 1998). The receptors are generally large proteins (∼100 kDa), and are related to each other in primary sequence (Fornerod et al., 1997b; Görlich et al., 1997). They mediate association with, and translocation through, the NPC, in a process that is temperature dependent and requires the small GTPase Ran (reviewed in Rush et al., 1996; Dahlberg and Lund, 1998; Melchior and Gerace, 1998). Central to one function of Ran in nucleocytoplasmic transport is the maintenance of Ran in a predominantly GTP-bound form in the nucleus, and in a GDP-bound form in the cytoplasm. This is achieved by distinct and restricted subcellular localizations of the only known guanine nucleotide exchange factor for Ran, RCC1, and the only known Ran GTPase-activating protein, RanGAP. RCC1 is a nuclear protein, while RanGAP is restricted to the cytoplasm and to the cytoplasmic side of the NPC (Matunis et al., 1996; Rush et al., 1996; Mahajan et al., 1997; Dahlberg and Lund, 1998; Melchior and Gerace, 1998). Nuclear RanGTP dissociates import receptor–cargo complexes and therefore functions in the release of imported cargo into the nucleoplasm (Rexach and Blobel, 1995; Görlich et al., 1996b; Izaurralde et al., 1997b; Siomi et al., 1997). It has exactly the opposite effect on export receptor–cargo complexes, i.e. RanGTP facilitates their formation (Fornerod et al., 1997a; Kutay et al., 1997a, 1998; Arts et al., 1998). When the export complex reaches the cytoplasm, Ran-binding protein 1 (RanBP1) cooperates with RanGAP to effect the release of cargo from the complex with the concomitant conversion of RanGTP to RanGDP (Bischoff and Görlich, 1997; Kutay et al., 1997a, 1998). Return of RanGDP to the nucleus is mediated by the NTF2 protein (Ribbeck et al., 1998). While these general principles of nucleocytoplasmic transport have been established, the detailed molecular mechanism of translocation within the NPC remains mysterious. Large proteins can be translocated in the absence of RanGTP hydrolysis and any other NTP hydrolysis (Izaurralde et al., 1997b; Kose et al., 1997; Nakielny and Dreyfuss, 1997; Richards et al., 1997; reviewed in Dahlberg and Lund, 1998), so the driving force is not yet understood. Several nuclear import signal—receptor systems have been described. Classical, basic nuclear localization signal (NLS)-containing proteins are imported by a dimeric complex comprising an adaptor, importin α, which binds the NLS, and the receptor, importin β, that mediates translocation through the NPC (reviewed in Nigg, 1997; Görlich, 1998; Izaurralde and Adam, 1998; Mattaj and Englmeier, 1998). The majority of the other six or more import signal–receptor systems described so far reveal that the adaptor can be dispensed with. For example, transportin (Kap104p in yeast) is distantly related to importin β, and mediates the import of a subset of hnRNP proteins bearing an M9-type NLS that is rich in glycine and aromatic residues (Siomi and Dreyfuss, 1995; Weighardt et al., 1995; Aitchison et al., 1996; Pollard et al., 1996; Bonifaci et al., 1997; Fridell et al., 1997; Siomi et al., 1997, 1998; Truant et al., 1998). Transportin binds directly to the M9-containing cargo (Nakielny et al., 1996). Characterization of nuclear export pathways has so far revealed four export signal–receptor systems. Exportin 1 (also known as CRM1) mediates the export of leucine-rich nuclear export signal (NES)-bearing proteins (Fornerod et al., 1997a; Fukuda et al., 1997; Neville et al., 1997; Ossareh-Nazari et al., 1997; Stade et al., 1997), and exportin 2 (also known as CAS1) exports importin α, which contains a novel NES (Boche and Fanning, 1997; Kutay et al., 1997a; Herold et al., 1998). Nuclear export of tRNA is mediated by exportin-t which binds directly to an as yet undefined NES in tRNA (Arts et al., 1998; Kutay et al., 1998). The molecular mechanism of a regulated export process was recently uncovered. The export receptor Msn5 recognizes only the phosphorylated form of the transcription factor Pho4 and thereby mediates its export from the nucleus of yeast under phosphate-rich growth conditions (Kaffman et al., 1998). Towards understanding the detailed molecular mechanism of translocation through the NPC, a map revealing the localization of individual nucleoporins within the NPC is being established, and interactions of transport receptor–cargo complexes that may be relevant to the translocation process are being uncovered. Within the vertebrate NPC, RanBP2/Nup358 (Wilken et al., 1995; Wu et al., 1995; Yokoyama et al., 1995), CAN/Nup214/p250 and Nup84 (Kraemer et al., 1994; Panté et al., 1994; Bastos et al., 1997; Fornerod et al., 1997b) have been localized to filaments that extend from the cytoplasmic face. A complex of nucleoporins, Nup62/58/54/45, lies within the central region of the pore complex (Guan et al., 1995), and Nup98 (Radu et al., 1995), Nup93 (Grandi et al., 1997) and Nup153 (Sukegawa and Blobel, 1993; Panté et al., 1994) are components of the so-called nuclear basket that consists of filaments extending into the nucleoplasm, connected at their ends by a small ring. Finally, Tpr is localized to fibers that extend from the nuclear basket into the nucleoplasm (Cordes et al., 1997). Import receptor interactions with nucleoporins are generally mediated by the nucleoporin FG repeat regions (for examples, see Iovine et al., 1995; Kraemer et al., 1995; Radu et al., 1995; Rexach and Blobel, 1995; Hu et al., 1996; Shah et al., 1998). In the course of characterizing the TRN1 import pathway, we identified a novel interaction with the nucleoporin Nup153 within a region devoid of FG repeats. Further characterization of this region of Nup153 established that it is an M9-type NLS. Consistent with the presence of an M9 shuttling domain in Nup153, we find that this nucleoporin is mobile within the NPC, as anti-Nup153 antibodies injected into the cytoplasm rapidly concentrate in the nucleus in a rim pattern. The identification of a receptor–nucleoporin interaction that is independent of FG repeats led us to examine the interactions of other import and export receptors with Nup153. We report that transport receptors can interact with several regions of Nup153. These interactions are regulated by RanGTP, which generally prevents import receptor interactions and, in contrast, promotes export receptor interactions. In addition, we have identified a novel Ran-binding domain that contains the zinc finger motif of Nup153. Results Transportin 1 interacts with the N-terminus of Nup153 in a Ran-regulated manner To search for proteins that interact with TRN1, we employed a phage display screen. In this system for detecting protein–protein interactions, GST–TRN1 was immobilized on glutathione–Sepharose beads and incubated with bacteriophage T7 that display proteins expressed from a HeLa cDNA library on their surface (see Materials and methods). After four rounds of selection, 55 clones were sequenced. Sixteen of the selected clones coded for members of the hnRNP protein family, or for proteins with RNP domains, demonstrating that the screen conditions had selected effectively for cargos of TRN1 (Table I). In addition, 13 of the clones coded for ribosomal proteins. These may be non-specific interactions, or they may be bona fide TRN1 interactors, since TRN1 recently has been reported to also import ribosomal proteins in vitro (Jäkel and Görlich, 1998). Table 1. Clones selected by phage display screening with GST–TRN1 as bait Clone No. of times selected hnRNP A1 4 hnRNP A2 3 FBRNP 4 TAFII68/RBP56 4 hnRNP M 1 Nup153 1 Ribosomal proteins 13 Inserts were sequenced from 55 phage that were selected after four cycles of screening. The table lists clones coding for bona fide TRN1 interactors (hnRNPA1 and A2) together will clones coding for proteins that are good candidates for real TRN1 interactors. FBRNP (fetal brain RNA-binding protein; Takiguchi et al., 1993) is related to hnRNP A1, and TAFII68/RBP56 (TBP-associated factor 68/RNA-binding protein 56; Bertolotti et al., 1996; Morohoshi et al., 1996) encodes an RNP motif protein. One clone coded for a short region (amino acids 247–290) within the N-terminus of Nup153, a component of the NPC (Table I and Figure 1A). Since this clone was selected a second time in a different phage display screen with GST–TRN1 as the bait (data not shown), we decided to characterize this interaction further. Figure 1.Schematic representation of human Nup153, and sequence of the M9-like region. (A) Nup153 can be visualized in three domains (McMorrow et al., 1994). The N-terminal (N) domain has NLS/NPC targeting activity and six FG motifs, the middle (M) domain contains four zinc finger motifs of the C2C2 type and two FG motifs, and the C-terminal (C) domain contains 25 FG motifs. The TRN1-binding region (amino acids 247–290), identified in the phage display screen with GST–TRN1 as bait, and the M9-like NLS (amino acids 235–300) are indicated. (B) Alignment of hnRNP A1 M9 and the Nup153 M9-like region. The alignment was performed using the ClustalW program. Identical residues are boxed in black, similar residues are boxed in gray, and the amino acid range of each sequence is indicated. Download figure Download PowerPoint Almost all transport receptor–nucleoporin interactions reported to date are restricted to the FG repeat domains of the nucleoporins (Iovine et al., 1995; Kraemer et al., 1995; Radu et al., 1995; Rexach and Blobel, 1995; Hu et al., 1996; Shah et al., 1998), although p62 interacts with importin β via a coiled-coil motif (Percipalle et al., 1997). The N-terminal region of Nup153 which we identified in the phage display screen is devoid of FG repeats, or indeed of any recognizable motif (Sukegawa and Blobel, 1993; McMorrow et al., 1994). This, together with the fact that the conditions employed for the screen selected for TRN1 cargos, led us to test the possibility that Nup153 may be a novel cargo for the TRN1 import pathway. We confirmed the TRN1–Nup153 interaction by an alternative protein binding assay. A fusion of pyruvate kinase (PK) and Nup153[235–300] was produced by translation in vitro in the presence of [35S]methionine, and tested for binding to GST–TRN1 (Figure 2A). TRN1 interacted with Nup153, and the interaction was competed effectively by recombinant M3, a C-terminal portion of A1 that spans the M9 NLS (Siomi and Dreyfuss, 1995). The interaction of Nup153 with TRN1 and its competition by M3 is similar to the interaction of M9 with TRN1 (Figure 2A). Figure 2.(A) Nup153[235–300] binds to TRN1 in vitro, and the interaction is competed by the M3 NLS of A1. Pyruvate kinase (PK) fusions were translated in vitro in the presence of [35S]methionine, and incubated with GST (2 μg) or GST–TRN1 (2 μg) loaded glutathione–Sepharose beads, in the absence or presence of recombinant zzM3 (15 μg). Bound PK fusions were eluted by boiling the beads in SDS–PAGE sample buffer, and analyzed by SDS–PAGE and autoradiography. (B) Nup153[230–305] interacts specifically with TRN1 and the interaction is disrupted by RanGTP. The import receptors TRN1 and importin β were translated in vitro in the presence of [35S]methionine, and incubated with glutathione–Sepharose beads loaded with 5 μg of GST, GST–A1 or GST–Nup153[230–305], in the absence or presence of RanQ69L-GTP (10 μg). Bound receptors were eluted by boiling the beads in SDS–PAGE sample buffer, and analyzed by SDS–PAGE and autoradiography. Download figure Download PowerPoint One feature of a receptor–import cargo interaction is its response to RanGTP, which binds to the receptor and triggers dissociation of the receptor–cargo complex (Rexach and Blobel, 1995; Görlich et al., 1996b; Izaurralde et al., 1997b; Siomi et al., 1997). RanGTP thereby serves to release cargo into the nucleoplasm. We therefore tested the effect of RanQ69L-GTP [a point mutant of Ran that cannot hydrolyze GTP at a significant rate, (Klebe et al., 1995)] on the Nup153–TRN1 interaction. GST–Nup153[230–305] interacted with 35S-labeled TRN1, and binding was almost completely abolished in the presence of RanQ69L-GTP (Figure 2B). Thus, the Nup153–TRN1 and the A1–TRN1 interaction respond identically to RanGTP. We also tested the receptor specificity of the Nup153–TRN1 interaction. Importin β does not bind to this region of Nup153 under conditions that support efficient TRN1 binding (Figure 2B). The N-terminus of Nup153 contains a functional M9-type NLS We tested for NLS activity of Nup153[235–300] by first fusing this portion of Nup153 to a reporter protein, PK. In cells transfected with DNA encoding PK alone, PK localizes to the cytoplasm (Siomi and Dreyfuss, 1995; Nakielny and Dreyfuss, 1996; data not shown). However, a fusion of Nup153[235–300] and PK localizes completely to the nucleus of the transfected cells (Figure 3). This region of Nup153 therefore has NLS activity in vivo. Figure 3.Nup153[235–300] is a transcription-sensitive NLS in vivo. HeLa cells were transfected with DNA encoding myc-A1 or myc-PK–Nup153[235–300]. Approximately 24 h post-transfection, cells were incubated in the absence or presence of 5 μg/ml actinomycin D for 4 h, and the subcellular localizations of the transfected proteins were analyzed by immunofluorescence microscopy using a monoclonal anti-myc antibody, 9E10. Download figure Download PowerPoint An interesting property of the M9 NLS is its sensitivity to the activity of RNA polymerase II (pol II). In cells incubated with pol II inhibitors such as actinomycin D, A1 accumulates in the cytoplasm and M9 has been found to be the sensor of pol II inhibition (Piñol-Roma and Dreyfuss, 1992; Siomi et al., 1997). PK–Nup153[235–300] also relocates to the cytoplasm in response to inhibition of pol II transcription (Figure 3). The relocalization of the Nup153 fusion is more dramatic than that of A1 (Piñol-Roma and Dreyfuss, 1992), and is more similar to the response of PK-M9 to transcription inhibition (Siomi et al., 1997). This is probably due to the RNA-binding activity of A1, which allows it to bind to RNA in the nucleus, or to other interactions of A1 with nuclear proteins. Intranuclear binding of full-length A1 presumably renders it less sensitive to relocalization to the cytoplasm (Siomi et al., 1997). To confirm further the conclusion that Nup153 contains an M9-type NLS, we analyzed the behavior of recombinant GST–Nup153[230–305] in the in vitro protein import assay. In this assay, HeLa cells are incubated with digitonin such that the cytoplasmic membrane is perforated, but the nuclear membrane remains intact (Adam et al., 1990). The digitonin-permeabilized cells are washed to remove soluble cell components, and then incubated with import factors (either recombinant proteins or provided by cell extracts such as reticulocyte lysate), an energy-regenerating system and recombinant import cargo that can be detected by immunofluorescence. We analyzed the import of three cargos, GST–M9 (imported by the TRN1 pathway), GST–IBB (IBB is the importin β-binding domain of importin α, and is sufficient to direct a heterologous protein to the nucleus via the importin β pathway; Görlich et al., 1996a; Weis et al., 1996), and GST–Nup153[230–305] (test cargo). In the presence of reticulocyte lysate and an energy-regenerating system, all three cargos accumulated in the nuclei of the permeabilized cells, although the import of GST–Nup153[230–305] appears less robust than that of GST–M9 and GST–IBB (Figure 4, column 1). This N-terminal region of Nup153 is, therefore, a bona fide NLS that functions in vitro (Figure 4) as well as in vivo (Figure 3). To identify the specific import pathway taken by GST–Nup153[230–305], we tested the effects of competitors of the importin β pathway [maltose-binding protein (MBP)–IBB] and of the TRN1 pathway (His-A1). MBP–IBB blocks GST–IBB import, and has no effect on the import of either GST–M9 or GST–Nup153[230–305] (Figure 4, columns 2 and 3), while His-A1 abolishes the import of both GST–M9 and GST–Nup153[230–305], but has no effect on GST–IBB import (Figure 4, column 4). Altogether, the data presented in Figures 1,2,3,4 indicate that Nup153 contains a functional M9-type NLS that allows for its nuclear import by the TRN1 import pathway. Figure 4.Nup153[230–305] is imported by the TRN1 pathway in vitro. Digitonin-permeabilized HeLa cells were incubated with reticulocyte lysate, an energy-regenerating system and the indicated GST fusion protein import cargo. Import was allowed to proceed for 20 min in the absence of competitor (−), or in the presence of an ∼10× molar excess (over cargo) of maltose-binding protein (MBP), MBP–IBB or His-A1. Assays were terminated by fixing the cells in formaldehyde, and the localization of the import cargo was analyzed by immunofluorescence microscopy using a monoclonal anti-GST antibody. Download figure Download PowerPoint Nup153 is a mobile component of the NPC The identification of an M9 NLS within Nup153 suggests that this domain, like A1 M9, may confer nuclear export as well as import on Nup153 (Michael et al., 1995). To address this possibility, we injected an anti-Nup153 antibody, together with control IgG, into the cytoplasm of mammalian cells and monitored the localization of the antibody molecules 1 h post-injection. While the control antibody remained at the site of injection, the anti-Nup153 antibody decorated the rim of the nucleus (Figure 5A). In contrast to the anti-Nup153 antibody, an antibody to another nuclear protein, lamin A, does not redistribute when injected into the cytoplasm (Figure 5A). Figure 5.Nup153 is mobile within the NPC. (A) Anti-Nup153 monoclonal antibody together with non-immune rabbit IgG were microinjected into the cytoplasm of NRK cells. In a separate experiment, anti-lamin A antibody was injected into the cytoplasm. The cells were fixed and processed for immunofluorescence microscopy either immediately after microinjection (0 h) or following a 1 h incubation at 37°C (1 h), and then labeled only with fluorescein- and rhodamine-conjugated secondary antibodies in order to detect the microinjected rabbit and mouse IgGs directly. (B) Anti-Nup153 monoclonal antibody was microinjected into the cytoplasm of NRK cells, and 1 h post-injection the cells were fixed and permeabilized with either digitonin (0.004%), which leaves the nuclear membranes intact, or Triton X-100 (0.2%), and labeled with rabbit anti-lamin A followed by fluorescein- and rhodamine-conjugated secondary antibodies. Download figure Download PowerPoint When we attempted to detect the cytoplasmically injected Nup153 antibody in cells permeabilized with digitonin (which perforates only the plasma membrane, leaving the nuclear membrane intact), the nuclear envelope-associated Nup153 antibody was not accessible to the secondary detection antibody, just as a lamin A antibody cannot detect lamin A that is localized on the nucleoplasmic side of the nuclear envelope (Figure 5B). Only when the nuclear membrane is permeabilized using Triton X-100, is the Nup153 antibody accessible to the secondary antibody (Figure 5B). The cytoplasmic Nup153 antibody is able, therefore, to traverse the NPC and accumulate at the nucleoplasmic side of the membrane. The antibody moves into the nucleus rapidly, since significant accumulation at the rim is detected as early as 20 min post-injection (data not shown). Since all microinjections were performed in the presence of cycloheximide to inhibit new protein synthesis, these observations indicate that Nup153 is exposed transiently to the cytoplasm, and are consistent with the concept that Nup153 is a mobile component that shuttles between the nuclear and cytoplasmic faces of the NPC. However, we note that a careful analysis of the transport properties of Nup153 in the heterokaryon shuttling assay reveals very slow shuttling (data not shown). It is possible that Nup153 rarely moves sufficiently far out of the body of the pore for a significant number of molecules to accumulate in a heterologous nucleus, or that it rarely dissociates from the cytoplasmic face of the NPC. Import and export receptors interact with N- and C-terminal regions of Nup153 The identification of a novel type of receptor–nucleoporin interaction that is independent of FG repeats led us to examine the interactions of other transport receptors with Nup153. First, we analyzed the interactions between full-length in vitro translated Nup153 and GST–TRN1 or GST–importin β. Both import receptors interact with full-length Nup153, and these interactions are significantly reduced or abolished when the binding assays are performed in the presence of RanQ69L-GTP (Figure 6). The importin β–Nup153 interaction detected here is in agreement with a report that importin β and Nup153 are found in a complex in Xenopus oocyte extracts, and this interaction is abolished by the presence of the non-hydrolyzable GTP analog, GMP-PNP (Shah et al., 1998). Figure 6.Full-length Nup153 interacts with TRN1 and importin β in vitro, in a RanGTP-regulated manner. Nup153 was translated in vitro in the presence of [35S]methionine, and incubated with glutathione–Sepharose beads loaded with 40 pmol of GST–TRN1 or GST–importin β, in the absence or presence of a 2×, 5× or 10× molar excess (over GST–receptor) of RanQ69L-GTP. Bound Nup153 was eluted by boiling the beads in SDS–PAGE sample buffer, and analyzed by SDS–PAGE and autoradiography. Download figure Download PowerPoint Next, we produced purified recombinant GST–Nup153 in three domains (Figure 1), the N-terminal 609 amino acids containing the M9-type NLS and six FG motifs (GST–Nup153-N), the middle portion of the protein comprising amino acids 610–895, which contains four zinc fingers and two FG motifs (GST–Nup153-M), and the C-terminal domain, amino acids 896–1475, which contains 25 of the FG motifs (GST–Nup153-C). These recombinant GST–Nup153 fusions were incubated with in vitro translated TRN1, importin β or CRM1. Both import receptors interact with the N- and C-terminal domains of Nup153, and TRN1 also interacts, albeit much more weakly, with the middle domain (Figure 7). All TRN1–Nup153 interactions are reduced or abolished in the presence of RanQ69L-GTP, while only the importin β–Nup153 N-terminus interaction is sensitive to RanQ69L-GTP. By contrast, the export receptor CRM1 interacts weakly or not at all with Nup153. However, in the presence of RanQ69L-GTP, CRM1 binds to both the N- and C-terminal portions of Nup153 (Figure 7). Figure 7.Import and export receptors interact with several regions of Nup153 in a RanGTP-regulated manner. Import receptors TRN1 and importin β (A), and the export receptor CRM1 (B), were translated in vitro in the presence of [35S]methionine, and incubated with glutathione–Sepharose beads loaded with 5 μg of GST or GST fused to the N-terminal (N), middle (M) or C-terminal (C) domains of Nup153 (see Figure 1), in the absence or presence of RanQ69L-GTP (6 μg). Bound receptors were eluted by boiling the beads in SDS–PAGE sample buffer, and analyzed by SDS–PAGE and autoradiography. The input lanes show 1/15 of the protein used in each binding assay. Download figure Download PowerPoint We also performed receptor–Nup153 binding assays in which only recombinant, purified proteins were used. Similarly to in vitro translated receptors, both recombinant TRN1 and recombinant importin β interact with the N- and C-terminal domains of Nup153 (Figure 8). However, in the a" @default.
• W2131878394 created "2016-06-24" @default.
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• W2131878394 date "1999-04-01" @default.
• W2131878394 modified "2023-09-27" @default.
• W2131878394 title "Nup153 is an M9-containing mobile nucleoporin with a novel Ran-binding domain" @default.
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