Journal of Cell Science 113, 1471-1480 (2000) 1471 Printed in Great Britain © The Company of Biologists Limited 2000 JCS1243 Nup2p is located on the nuclear side of the nuclear pore complex and coordinates Srp1p/importin-α export Jennifer K. Hood, Jason M. Casolari and Pamela A. Silver* Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School and The Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA *Author for correspondence (e-mail: [email protected]) Accepted 10 February; published on WWW 21 March 2000 SUMMARY Proteins bearing canonical nuclear localization sequences displaced from the nuclear rim to the nuclear interior are imported into the nucleus by the importin/karyopherin- in ∆nup2 cells. We do not observe any biochemical α/β heterodimer. Recycling of the importin-α subunit to the interaction between Cse1p and Nup2p. Instead, we find cytoplasm requires the action of Cas, a member of the that Nup2p binds directly to Srp1p. We have localized importin-β superfamily. In the yeast Saccharomyces Nup2p to the interior face of the nuclear pore complex, and ceresivisiae, the essential gene CSE1 encodes a Cas have shown that its N terminus is sufficient for targeting homologue that exports the yeast importin-α protein Nup2p to the pore, as well as for binding to Srp1p. Taken Srp1p/Kap60p from the nucleus. In this report, we describe together, these data suggest that Nup2p is an important a role for the FXFG nucleoporin Nup2p, and possibly the NPC docking site in the Srp1p export pathway. related Nup1p, in the Cse1p-mediated nuclear export pathway. Yeast cells lacking Nup2p or containing a particular temperature-sensitive mutation in NUP1 Key words: Nup2p, Srp1p, Cse1p, Nucleoporin, Yeast, Nuclear accumulate Srp1p in the nucleus. Similarly, Cse1p is export INTRODUCTION rounds of protein import, the import receptor must be recycled to the cytoplasm. Importin-β can exit the nucleus on its own Proteins move into and out of the nucleus through nuclear pore (Izaurralde et al., 1997; Kose et al., 1999), but Srp1p/importin- complexes (NPCs) embedded in the nuclear membrane. Their α must be exported by the exportin Cse1p/CAS (Hood and transport requires soluble receptor proteins of the Silver, 1998; Künzler and Hurt, 1998; Kutay et al., 1997; importin/exportin superfamily. These receptors shuttle Solsbacher et al., 1998). Cse1p/CAS binds to Srp1p/importin- between the nucleus and the cytoplasm, making contacts with α with Ran-GTP, forming a cooperative export complex that is NPC components, or nucleoporins, as they traverse the pore dissociated by Ran-GTP hydrolysis once it reaches the (reviewed by Pemberton et al., 1998; Adam, 1999). The cytoplasm (Kutay et al., 1997; Solsbacher et al., 1998). association of transport receptors with their cargoes and with The actual mechanism of nucleocytoplasmic translocation is the NPC is regulated by the nucleotide-bound state of the Ran not well understood, although a fairly detailed picture of the GTPase in the nuclear and cytoplasmic compartments of the structure of the NPC and the position of many of its cell (Marelli et al., 1998; Nakielny et al., 1999; Seedorf et al., components within that structure is available, especially for the 1999; reviewed by Moore, 1998; Adam, 1999). As a result, the yeast Saccharomyces cerevisiae (reviewed by Stoffler et al., Ran cycle dictates the direction of transport. 1999). Many nucleoporins contain repeats of the short amino Proteins that contain classical nuclear localization signals acid sequences FG, FXFG or GLFG. Numerous interactions (NLSs) of the SV40 or bipartite type are imported into the between nucleoporin repeats and importins/exportins have nucleus by importin-β, also called Kap95p in yeast (Görlich et been reported (Albertini et al., 1998; Fornerod et al., 1997; al., 1995a; Imamoto et al., 1995; Moroianu et al., 1995). This Hellmuth et al., 1998; Iovine et al., 1995; Radu et al., 1995; transport pathway is unique in that the association of importin- Shah et al., 1998; Titov and Blobel, 1999), but it is unclear β with NLS substrates requires an adapter protein, importin-α, which of these are significant for nuclear transport in vivo. also called Srp1p in yeast (Enenkel et al., 1995; Görlich et al., The yeast proteins Nup1p, Nup2p and Nsp1p belong to the 1995b). The trimeric import complex assembles in the class of nucleoporins that contain amino acid repeats of the cytoplasm, crosses the NPC, and then dissociates in the nucleus FXFG variety. NUP1 and NSP1 are essential in most strain due to the binding of Ran-GTP to Kap95p/importin-β (Görlich backgrounds, while NUP2 is nonessential in an otherwise wild- et al., 1996b; Rexach and Blobel, 1995). To allow further type background (Loeb et al., 1993). Genetic interactions 1472 J. K. Hood, J. M. Casolari and P. A. Silver between these three genes suggest that they have some University, Waltham, MA, USA). These were raised against a peptide functional overlap: the 175 amino acids at the N terminus of consisting of repeats of the amino acid sequence FSFG; they Nup2p are essential for viability in strains that express mutant recognize the three FXFG-containing yeast nucleoporins Nup1p, versions of Nup1p or Nsp1p that lack the amino termini of Nup2p and Nsp1p. Mouse monoclonal anti-GST antibodies were from these proteins (Loeb et al., 1993). In addition to this N-terminal Santa Cruz Biotechnology. domain, the Nup2p protein possesses a central domain that Immunofluorescence contains the FXFG repeats and a C-terminal Ran-binding Cells were grown to mid-log phase in 10 ml liquid yeast extract- domain similar to that found in the yeast Ran-binding proteins peptone-dextrose (YEPD) cultures at 25°C, then fixed in 3.7% Yrb1p and Yrb2p (Dingwall et al., 1995). Nup2p is of special formaldehyde for 30 minutes and processed for immunofluorescence interest because it is the only yeast nucleoporin that contains as previously described (Hood and Silver, 1998). Rabbit polyclonal such a Ran-binding domain. anti-Srp1p antibodies were used at 1:1000 dilution overnight. Both Nup1p and Nup2p have previously been shown to FITC-conjugated anti-rabbit secondary antibodies (Jackson interact with Srp1p in coimmunoprecipitation experiments ImmunoResearch) were used at 1:1000 dilution with a 1 hour (Belanger et al., 1994). In this work we have investigated the incubation. role of Nup2p in the Srp1p export pathway and have found Visualization of GFP/YFP-tagged proteins in live cells evidence to support the model that Nup2p serves as an initial Cells were grown to mid-log phase in liquid synthetic complete docking site for the formation of the Cse1p/Ran-GTP/Srp1p medium or in ‘dropout’ medium to select for the retention of plasmids. export complex. Fluorescent signal was observed using a Nikon fluorescence microsope and filters specific for GFP (Chroma Technology Corp.) or EYFP (Omega). Images were captured by a Princeton Instruments MATERIALS AND METHODS Micromax digital camera using Metamorph imaging software (Universal Imaging Corp.). Yeast strains and plasmids The ∆nup2-4::URA3 (MAT α, ura3-5, leu2-3, 112, trp1-289), ∆nup2- Expression and purification of GST-tagged proteins from 5::HIS3 (MAT α, ura3-52, leu2-3, 112, his3∆200, ade2, trp1∆63), E. coli nup1-8 (pCEN LEU2 nup1-8 in MAT α, his3∆200, trp1-1, ura3-5, pGEX expression plasmids were transformed into BL-21 E. coli. 5- leu2-3, 112, nup1-2::LEU2) and nup1-21 (pCEN LEU2 nup1-21 in ml LB cultures containing 100 µg/ml ampicillin (LB/AMP) were MAT α, his3∆200, trp1-1, ura3-5, leu2-3, 112, nup1-2::LEU2) strains grown overnight at 37°C, then diluted into 1 L LB/AMP and allowed + were previously described (Loeb et al., 1993). An ADE version of to grow at 37°C to an OD600 of 0.4. Isopropyl-1-thio-β- ∆nup2-5::HIS3 (PSY1946; MAT a, ura3, leu2, his3, trp1) was D-galactopyranoside (IPTG) was added to 0.1 mM to induce generated by crossing ∆nup2-5 to PSY1052 (MAT a, ura3, leu2, trp1, expression for 3 hours at 37°C. Cultures were centrifuged and his3). The CSE1-GFP:TRP1 and XPO1-GFP:TRP1 strains have been pellets frozen overnight at −20°C or in liquid nitrogen. Cell previously described (Hood and Silver, 1998). ∆nup2-4::URA3 CSE1- pellets were resuspended in ice-cold PBS containing 0.5 mM GFP:TRP1 and ∆nup2-4::URA3 XPO1-GFP:TRP1 strains were phenylmethylsulfonyl fluoride (PMSF) and 3 µg/ml each pepstatin A, created by transforming BstEII-linearized pPS1610 (pRS304 CSE1- leupeptin, aprotinin and chymostatin. Cells were lysed by sonication GFP) or XcmI-linearized pRS304 XPO1-GFP, respectively, into (3× 30 seconds with 1 minute on ice between sonications), Triton X- ∆nup2-4::URA3. The NUP2-EYFP:URA3 (PSY1835) strain was 100 was added to 1%, and lysates were clarified by centrifugation. constructed by M. Damelin (Damelin and Silver, 2000). Lysates were incubated for 30 minutes at room temperature with 1 ml The GST-Nup2p E. coli expression plasmid (pPS1944) was created 50% glutathione-Sepharose slurry (Amersham Pharmacia Biotech) by cloning the NUP2 ORF into the BamHI/XhoI sites of pGEX 4T-1 prewashed with PBS. Beads were washed 3× with 10 ml PBS/1% (Amersham Pharmacia Biotech). Sequence encoding amino acids 1- Triton X-100, then bound GST fusion proteins were eluted with 5× 556 of Nup2p was cloned into the BamHI/XhoI sites of pGEX 4T-1 500 µl of 10 mM reduced glutathione/50 mM Tris, pH 8. to generate the GST-Nup2∆RBD E. coli expression plasmid (pPS1945). The GAL-Nup2p-GFP plasmid (pPS1946) and the Thrombin cleavage of GST-Srp1p truncated versions (Nup2∆C-GFP, pPS1947; Nup2∆N-GFP, Purified GST-Srp1p was dialyzed overnight in thrombin cleavage pPS1948) were constructed by PCR-amplifying the corresponding buffer (150 mM NaCl, 25 mM Tris, pH 8, 2.5 mM CaCl2, 0.1% β- regions of the NUP2 ORF with a BamHI site at the N terminus and a mercaptoethanol, 10% glycerol), then again for 4 hours in fresh buffer.