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RESEARCH ARTICLE 2641 Evidence for a nuclear passage of nascent polypeptide-associated complex subunits in

Jacqueline Franke1, Barbara Reimann2, Enno Hartmann3, Matthias Köhler1,4 and Brigitte Wiedmann2,* 1Max Delbrück Center for Molecular Medicine, Robert-Rössle-Strasse 10, D-13122 Berlin, Germany 2Department of , Humboldt University, Charité, Hessische Straße 3-4, D-10115 Berlin, Germany 3Center for Biochemistry and Molecular Chemistry, Georg-August University, Heinrich-Düker-Weg 12, D-37073 Göttingen, Germany 4Franz-Volhard-Klinik, Humboldt University, Charité, Wiltbergstraße 50, D-13122 Berlin, Germany *Author for correspondence (e-mail: [email protected])

Accepted 6 April 2001 Journal of Cell Science 114, 2641-2648 (2001) © The Company of Biologists Ltd

SUMMARY

The nascent polypeptide-associated complex (NAC) has nuclear import factors. Translocation into the nucleus was been found quantitatively associated with in the only observed when binding to ribosomes was inhibited. by means of cell fractionation or fluorescence We identified a of the -binding NAC microscopy. There have been reports, however, that subunit essential for nuclear import via the single NAC subunits may be involved in transcriptional Kap123p/Pse1p-dependent import route. We hypothesize regulation. We reasoned that the cytosolic location might that newly translated NAC travel into the nucleus only reflect a steady state equilibrium and therefore to bind stoichiometrically to ribosomal subunits and investigated the yeast NAC proteins for their ability to enter then leave the nucleus together with these subunits to the nucleus. We found that single subunits of yeast NAC concentrate in the cytosol. can indeed be transported into the nucleus and that this transport is an active process depending on different Key words: NAC, , Saccharomyces cerevisiae

INTRODUCTION NAC may function in cotranslational import into mitochondria (George et al., 1998; Fünfschilling and Rospert, The nascent polypeptide-associated complex (NAC) is an 1999). evolutionarily conserved . Three NAC subunits In addition to the functions of ribosome-bound NAC in the are present in yeast: Egd2p, Btt1p and Egd1p. Egd2p is cytosol, single NAC subunits may interact with DNA thereby homologous to αNAC proteins in (Shi et al., 1995) regulating (Parthun et al., 1992; Moreau et al., and (Wiedmann et al., 1994) and a protein family in 1998). Such a function would imply that at least some NAC Archea (J.F. et al., unpublished). Btt1p and Egd1p are protein is located transiently within the nucleus and that the homologues of the mammalian βNAC (BTF 3) (Hu et al., 1994; NAC subunits contain signals for their active translocation Parthun et al., 1992; Wiedmann et al., 1994). Whereas Egd2p into the nucleus. Indeed, the NAC α-subunit has been found and Egd1p are expressed at about stoichiometric levels in the nuclei of serum-deprived osteoblastic cells in mice compared with ribosomes, the concentration of Btt1p is about (Yotov, et al., 1998). However, verification of this finding 100-times lower (George et al., 1998; Reimann et al., 1999). has been difficult (Beatrix et al., 2000). NAC subunit The proteins form tight heteromeric complexes even at 500 translocation into nuclei should require import receptors. mM salt. After cell fractionation, more than 95% of the Egd2p According to our present knowledge, nuclear import is found bound to ribosomes via Egd1p or Btt1p (J.F. et al., substrates bind to soluble receptors of the importin β family unpublished; Reimann et al., 1999). either directly or with the help of specific adapter proteins Several functions have been attributed to NAC or its subunits. (importin α) and translocate through the driven The entire complex at the ribosome protects nascent by the concentration gradient of -GTP (Wozniak et al., polypeptides from premature interactions with cytosolic 1998; Görlich and Kutay, 1999). Most presently known proteins. The NAC complex also inhibits the binding of substrates bear a so called ‘classical’ nuclear localization ribosomes that translate non-secretory proteins to translocation signal (NLS). These proteins interact with their sites in the of the (Wang et importin β via adapter of the importin α family. al., 1995; Wiedmann et al., 1994; Lauring et al., 1995a; Lauring Other substrates contain a NLS which allows their direct et al., 1995b; Möller et al., 1998; Wiedmann and Prehn, 1999). interaction with one of the various import receptors. Some NAC is associated with the ribosomes and binds to emerging proteins need a dimer of two different for efficient nascent polypeptides before any other cytosolic protein. NAC transport. For example, H1 requires importin β and releases the nascent chain when a binding domain for cytosolic importin 7 (Jäkel et al., 1999). Other proteins can use more proteins such as the signal recognition particle (SRP) or than one import pathway due to their ability to bind to chaperones is completed (Wang et al., 1995). Furthermore, different import receptors. The yeast L25 2642 JOURNAL OF CELL SCIENCE 114 (14) is imported by Yrb4p (also known as Kap123p) and Pse1p In vitro import (also known as Kap121p) (Rout et al., 1997; Schlenstedt et Import assays were performed as described previously (Adam et al., al., 1997; Seedorf and Silver, 1997). The human homologue, 1990; Jäkel and Görlich, 1998, Köhler et al., 1999). Briefly, HeLa L23a, can even be imported by four different receptors, cells were grown on three-well microscopy slides (Roth) to 40-80% namely importin 5, importin 7, importin β, and transportin confluency, washed once in ice-cold PBS, and permeabilized for 8 (Jäkel and Görlich, 1998). We report here, that non-ribosome- minutes in ice-cold 20 mM Hepes-KOH (pH 7.5), 150 mM potassium acetate, 5 mM magnesium acetate, 0.5 mM EGTA, 250 mM sucrose, associated yeast NAC subunits can translocate into the µ nucleus in vivo. Using in vitro assays and in vivo 30 g digitonin (Sigma) per ml. The cells were incubated for 8 minutes on slides with 20 µl of import mixture at room temperature. experiments, we demonstrate that this transport is specific The import reaction was stopped by fixation with 3% and can be mediated by several of the known import factors. paraformaldehyde/PBS. After washing in PBS, the slides were mounted with Vectorshield mounting medium (Vector). Import mixtures contained an energy regenerating system (0.5 mM ATP, 0.5 µ MATERIALS AND METHODS mM GTP, 10 mM creatine phoshate, 50 g creatine /ml), core buffer (2 µg nucleoplasmin core/ml, 20 mM Hepes-KOH (pH 7.5), 150 mM potassium acetate, 5 mM magnesium acetate, 250 mM Plasmids and yeast strains sucrose), 0.5 mM EGTA, 3 µM RanGDP, 0.2 µMRna1p, 0.3 µM Standard yeast methods and media were used. The NAC-knockout RanBP1 and 0.4 µM NTF2. The assay was adjusted to 10% with mutants are based on strain W303, as described previously (Reimann lysate, and 1 µM importin β and 2 µM of importin α were et al., 1999). The ∆kap123 and pse1-1 strains were kindly provided added. by P. Silver (Harvard Medical School, Boston, MA). The srp1-31 and labelling of recombinant proteins was performed with srp1-49 strains were from M. Nomura (University of California, Texas red or fluorescein 5′-maleimide as described previously (Kutay Irvine, CA). et al., 1997). Fluorescence microscopy was performed with a confocal GFP fusion plasmids of EGD1 and EGD2 were constructed by microscope (MRC 1024, BioRAD) equipped with a 63× oil- cloning 500 base pairs upstream of ATG and the coding region in immersion objective (Nikon Diaphot). Images were processed by frame to two C-terminal copies of modified GFP in pRS414 Adobe Photoshop. (Sikorski and Hieter, 1989; Cormack et al., 1997). To yield truncated versions of EGD1 the promoter region of EGD1 was cloned via a Fluorescence microscopy of yeast cells ClaI restriction site to the coding region of EGD1 without its N- Cultures of exponentially growing yeast cells were incubated at 30°C terminal 33 (∆N11-egd1), 42 (∆N14-egd1), 81 (∆N27-egd1) or 132 ∆ (or 25°C for temperature sensitive mutants). To visualize nuclei, ( N44-egd1) base pairs into pRS415, which already contained the Hoechst 33258 (Sigma) was added to a final concentration of 10 µM EGD2 . The new ATG start codon was introduced with for 10 minutes prior to microscopy. Fluorescence microscopy was oligonucleotides that were designed to fuse the promoter with the performed with living cells using a Zeiss Axioplan 1 microscope shortened coding region. These mutated were cloned via SacI equipped with a NEOFLUAR 100×/1.30 oil-immersion objective and EcoRI restriction sites in frame into pRS414-2GFP in order to (Zeiss). GFP fluorescence was obtained using the FITC channel. create GFP fusion proteins. The plasmid pRS414-2GFP was a gift Stained nuclei were visualized in the UV channel. Photographs were of U. Lenk (MDC, Berlin). All constructs were sequenced, and the taken with a CCD camera (PCP computer optics, Kelheim) and the expression of fusion proteins was verified by SDS/PAGE and Axiovision 1 software (Zeiss). Images were processed by Adobe western blot analysis. Photoshop. Recombinant expression of proteins Cell fractionation Coding regions of EGD1 and EGD2 were cloned via PCR into BamHI and HindIII sites of pQE 30 vector (Qiagen). Expression of the His- Cells were grown to middle log-phase on SD, spheroplasted with tagged proteins was induced by IPTG in Escherichia coli M15 at 25°C Zymolyase100T, homogenized in IP-buffer (50 mM HEPES pH 7.5, for 4 hours. HIS-Egd2p was purified under native conditions 150 mM NaCl, 5 mM Mg(OAc)2, 1 mM PMSF, and inhibitor according to the QIAexpress protocol. Briefly, harvested cells of a 1 mix (Sigma)) at about 0.5-1 g fresh weight/ml buffer in a Brown- litre culture were resuspended in 100 mM sodiumphosphate buffer, homogenizer (Brown, Melsungen, Germany). Cell debris, left over pH 7.8, 300 mM NaCl and lysed by freeze/thawing and sonication. cells and nuclei were sedimented by two 15 minute The lysate was centrifuged (30 minutes, 10,000 g) and the supernatant steps at 3,000 g. The supernatant was cleared of mitochondria by a 15 minute centrifugation at 10,000 g. A 30 minute centrifugation (OD600 ~1) was loaded onto a pre-equilibrated Ni-NTA-column (8 ml 50% Ni-NTA-resin). The protein was eluted by 50 mM Hepes, pH 3.5 at 400,000 g yielded a supernatant free of endoplasmic reticulum. and neutralized immediately. His-Egd1p was purified under Ribosomes were separated from cytosol by sedimentation at denaturing conditions according to the QIAexpress protocol. The 400,000 g in a Beckman TL100.4 rotor for 30 minutes. The volume difference to the His-Egd2p purification was that the cells were of fractionation samples was adjusted so that it equalled the original resuspended in 100 mM sodiumphosphate buffer, pH 8.0, 8 M urea. volume and electrophoresis lanes were directly comparable. His-Egd1p was renatured bound to the Ni-NTA-resin by a gradient of 8-0 M urea in 100 mM sodiumphosphate buffer, pH 8.0 and then Co-immunoprecipitation eluted as described above. The 40,000 g supernatant of a cell fractionation was used for Preparation of recombinant import factors occurred as described immunoprecipitation. 2 ml were incubated 2-4 hours with 3 µl earlier: C- terminal His-tagged importin α1/Rch1 and Srp1p (Görlich against Egd1p and/or Egd2p at 6°C on a roller ( et al., 1995), α3, α4, α5/hSrp1 and α7 (Köhler et al., 1999), provided by M. Wiedmann, MSKCC, New York). Antigen-antibody importin β, nucleoplasmin, nucleoplasmin core, human Ran, complexes were collected with 50 µl 50% protein A-sepharose in IP- Schizosaccharomyces pombe Rna1p, murine RanBP1 and NTF2 buffer, washed extensively with IP-buffer, and solubilized by boiling (Kutay et al., 1997). Expression clones for importin β, nucleoplasmin, in 50 µl 2× SDS sample buffer. Proteins were separated on 12% SDS nucleoplasmin core and NTF2 and recombinant protein of human polyacrylamide gels. Western blot detection was performed using the Ran, Schizosaccharomyces pombe Rna1p and murine RanBP1 were ECL system according to the instructions of the manufacturer kindly provided by D. Görlich (ZMBH, Heidelberg). (Amersham Pharmacia Biotech). Transient nuclear passage of NAC 2643

Fig. 1. Intracellular localization of GFP fusion proteins of Egd1p, Egd2p and ∆N11-Egd1p. (A) Expression of Egd2p-2GFP in a ∆egd2 strain (to simulate the wild-type situation) and in the NAC-knockout strain. (B) Expression of Egd1p-2GFP in a ∆egd1 strain (to simulate the wild-type situation) and in the NAC-knockout strain. (C) Expression of ∆N11-Egd1p- 2GFP in the NAC-knockout strain. Cells were grown to logarithmic growth phase at 30°C and stained with Hoechst 33258 to visualize nuclear DNA prior to microscopy. Positions of nuclei are indicated by arrows.

RESULTS

Yeast NAC proteins can be transported into nuclei in vivo Under steady state conditions most of the yeast NAC proteins are in a complex tightly associated with cytosolic ribosomes. However, one cannot exclude the existence of low amounts of free NAC proteins or NAC that is not or not yet ribosome- bound in the cell. Therefore, we sought in vivo conditions that increased the amount of such molecules in the cell to determine their location. Egd2p is associated with the ribosome by Egd1p green fluorescence in addition to a cytosolic . Also, and to a lesser extent by Btt1p. Thus, we expressed a fusion overexpression of Egd2p-2GFP in wild-type yeast caused protein of Egd2p with two copies of the green fluorescence nuclear fluorescence (data not shown) indicating again that free protein (Egd2p-2GFP) in a NAC-knockout strain Egd2p-2GFP can translocate into the nucleus. In each case the (∆egd1∆egd2∆btt1) and looked for the location of that protein. were free of Egd2p-2GFP. Fig. 1A shows the location of Egd2p-2GFP in a ∆egd2 Next, we tested the location of Egd1p-2GFP. As expected background. This strain, which resembles the wild-type from our cell fractionation experiments, full length Egd1p- (Fig. 1A, top two images), was compared with the 2GFP was found exclusively in the cytosol in wild-type cells knockout strain (Fig. 1A, bottom two images). There was no and also in the NAC-knockout strain (Fig. 1B). Therefore, we co-localization of fluorescence with the nuclear staining searched for EGD1 mutants that are deficient in ribosome Hoechst in wildtype. However, when Egd2p-2GFP was the binding and shortened the coding region subsequently at its N- only NAC subunit left in the cell, the nuclei showed an intense terminus. Removal of the first 11 N-terminal amino acids

Fig. 2. of N- terminal truncated Egd1p proteins and co- immunoprecipitation with Egd2p. (A) Logarithmically growing wild-type cells (first two lanes), NAC-knockout cells transformed with plasmids encoding Egd2p plus Egd1p without its N-terminal 11 amino acids (∆N11-EGD1) or 14 amino acids (∆N14-EGD1), and ∆egd1 cells (two lanes on the right) were fractionated and the cytosolic (C) and ribosomal fraction (R) were separated by SDS-PAGE. Egd1p and Egd2p were visualized by western blot analysis. The lower band in the wild-type control is most likely a degradation product. (B) Western blot analysis of immunoprecipitates from wild-type cells and the NAC-knockout strain transformed with the three indicated plasmids (label on top) with an anti-Egd1p antibody. 2644 JOURNAL OF CELL SCIENCE 114 (14) (∆N11-Egd1p-2GFP) abolished the association with ribosomes. Only traces of truncated Egd1p-2GFP were left in the ribosomal fraction (Fig. 2A). The interaction of truncated Egd1p versions with the Egd2p was not affected, as shown by co-immunoprecipitation (Fig. 2B). The N-terminal shortened Egd1p-2GFP accumulated in the nucleus of NAC-knockout cells (compare Fig. 1B,C) and wild-type cells (not shown). In summary, these experiments proved the ability of both proteins (Egd1p, Egd2p) to enter the nucleus. The in vitro import of yeast NAC into nuclei is energy dependent and mediated by different importin α proteins We next investigated the nuclear import of these proteins in more detail. First, we tested whether or not yeast NAC proteins can be transported into the nucleus via the ‘classical’ importin α/importin β-mediated import pathway using an in vitro import assay (Adam et al., 1990; Jäkel and Görlich, 1998, Köhler et al., 1999). Basically, HeLa cells were permeabilized and the cytosol was washed out. Fluorescein labelled recombinant Egd1p or Egd2p and the components necessary for proper import including different human α-importins and the α-importin homologue of yeast Srp1p were added to a standard in vitro import reaction. The fluorescence signal in the nuclei of the samples containing importins α1/Rch1, α3, α7 or yeast α-importin Srp1p demonstrates that both Egd2p and Egd1p were actively transported into nuclei (Fig. 3a,b,d,e,m,n,p,q). The human β-importin alone (Fig. 3s,t) could not mediate transport of yeast NAC proteins. Several lines of evidence indicate that the import of the yeast NAC subunits is due to a specific binding to their import receptors. First, not all α-importins could promote nuclear import of the yeast proteins. Importins α4 and α5 failed to import Egd1p or Egd2p (Fig. 3g,h,j,k) even though they could transport nucleoplasmin, a typical import substrate (Fig. 3i,l). Second, nucleoplasmin competed with the Egd2p for interaction with importin α1 (see Fig. 4), indicating that the import of Egd2p requires an empty NLS-binding site on its import adapter importin α1. Furthermore, there was no transport in the absence of energy (Fig. 4i). Together, these experiments show that yeast NAC subunits in vitro can enter the nuclei of human culture cells with components of the ‘classical’ import pathway. In vivo transport of Egd1p into nuclei is inhibited in nuclear protein import mutants We then asked how yeast NAC proteins are imported in vivo and searched for import mutants that could decrease the intense nuclear accumulation of ∆N11-Egd1p-2GFP. To evaluate the role of the importin α/importin β-mediated Fig. 3. In vitro nuclear import assay of recombinant Egd1p and Egd2p. transport, we looked for localization of ∆N11-Egd1p-2GFP Recombinant Egd1p and Egd2p were labelled with fluorescein-5′- maleimide. Dependency of in vitro nuclear import of Egd1p on different in mutants of SRP1: srp1-31 and srp1-49 (Loeb et al., 1995; α Shulga et al., 1996, Tabb et al., 2000). We observed no importin proteins (left); import of Egd2p (middle); import of the standard substrate nucleoplasmin (NPL) by the different importin α effect in srp1-49 cells but a slight increase of cytosolic proteins (right). The images s, t and u represent the negative controls staining in srp1-31 cells (Fig. 5A) after a 3 hour shift to the (import assay without α importin). HeLa cells were permeabilized, the nonpermissive temperature. The relatively small effect can cytosol was washed out and recombinant import factors, energy mix, and be explained by the already existing strong nuclear labelled substrate were added. After fixation the fluorescence of the cells accumulation of ∆N11-Egd1p-2GFP at permissive was observed by confocal microscopy. temperature. Another possible reason could be that other Transient nuclear passage of NAC 2645 and/or Pse1p. Nuclear accumulation of ∆N14-Egd1p-2GFP compared with ∆N11-Egd1p-2GFP was reduced and the protein localized in the . Removal of 27 amino acids increased this effect slightly (Fig. 6A). Deletion up to 44 amino acids showed the same distribution as that seen in ∆N27- Egd1p-2GFP (data not shown). However, nuclear import was not completely abolished. We always detected truncated Fig. 4. Specificity of in Egd1p-2GFP in nuclei. The remaining nuclear staining of ∆N14-Egd1p-2GFP and ∆N27-Egd1p-2GFP was also vitro import of Egd2p. ∆ Various amounts of observed in kap123 and in the pse1-1 mutant - even at Texas Red-labelled nonpermissive temperature (Fig. 6B). Therefore, we assume nucleoplasmin were that the region responsible for Kap123p/Pse1p-dependent mixed with import is located between amino acids 11 and 27. fluorescein-labelled Egd2p and importin α1/Rch1 and added to DISCUSSION an in vitro import assay using permeabilized HeLa cells (a-f). NAC has been associated with both cytosolic and nuclear Import of Egd2p is functions. Although its cytosolic location is well established, demonstrated in the conflicting results have been reported regarding the presence presence of an of NAC in the nucleus (Yotov et al., 1998; Beatrix et al., 2000). equimolar amount of We show here, for the first time, NAC proteins inside the nucleoplasmin (a), a nucleus in growing yeast. We employed mutant yeast strains threefold excess (c), and demonstrated that yeast NAC subunits may reside in the and a tenfold excess of nucleus when they cannot bind to ribosomes. In the case of nucleoplasmin (e). Egd2p-2GFP, we removed Egd1p and Btt1p, the two ribosome- Images b, d and f binding proteins. We also overexpressed Egd2p-2GFP. reflect the location of nucleoplasmin in the Ribosomal binding of the Egd1p was abolished when 11 amino same experiment. In g acids of the N-terminus were removed. Whereas truncated and h assays without Egd1p-2GFP accumulated strongly inside the nucleus, Egd2p importin α and in i and was equally distributed between nucleus and cytosol. We did j assays without energy not evaluate the reason for this difference. However, we assume mix are shown. The that the difference is related to folding artifacts in Egd2p-2GFP assay was performed as caused by the C-terminal fusion to GFP or the absence of its described above (see binding partner Egd1p. also Materials and Although the NAC subunits were small enough to enter the Methods). nucleus via diffusion, we found evidence for mechanisms. Our in vitro transport assay revealed that full length Egd2p, a homologue of mammalian αNAC, as well as factors participate in Egd1p nuclear import. NAC proteins are Egd1p, a BTF3 homologue, can be transported into the nucleus similar to ribosomal proteins in size and expression level, and by distinct importin α-proteins, the mammalian α1, α3, α7 and function in alliance with ribosomes in the cytosol. We therefore the yeast importin α Srp1p. The mammalian importins α4 and focused on Kap123p and Pse1p, factors involved in the import α5 failed to import the yeast NAC proteins although they of ribosomal proteins (Rout et al., 1997; Schlenstedt et al., carried other cargos such as nucleoplasmin in the same assay. 1997; Seedorf and Silver, 1997; Stage-Zimmermann et al., Competition of Egd2p-2GFP with nucleoplasmin confirmed 2000). We tested whether or not ∆N11-Egd1p-2GFP can the specificity of the transport process. The weaker accumulate in nuclei of ∆kap123 or pse1-1 mutants, as seen in fluorescence signal found for import with yeast importin α the corresponding wild-type cells (Fig. 5B). Both mutants Srp1p in this assay had also been observed for other cargos showed an enhanced fluorescence in the cytosol and a reduced (Köhler et al., 1999). fluorescence in the nucleus (Fig. 5B, bottom four images) The fact that the NAC proteins do not possess a classical compared with wild-type cells (Fig. 5B, top two images). The NLS as described previously (Makkerh et al., 1996) does not defective import into nuclei of pse1-1 cells was already contradict the observed interaction with importin α proteins. detectable at permissive temperature. We conclude that the Other proteins that lack a classical NLS, such as RanBP3, are ribosome binding subunit of NAC can enter nuclei by several also known to be imported via the importin α/importin β- routes. dependent pathway (Welch et al., 1999). We believe that the result obtained for yeast NAC proteins in vitro is likely to Deletion of the first 27 amino acids of Egd1p reflect the in vivo situation in mammals, since human αNAC abolishes Kap123p and Pse1p dependent nuclear protein can be co-immunoprecipitated from HeLa-cell lysate import using anti-importin α1/Rch1 antibodies (data not shown). We examined the further deleted Egd1p-2GFP to define the Our presumption of an active transport via the importin domain that is responsible for nuclear import by Kap123p α/importin β-dependent pathway was confirmed by in vivo 2646 JOURNAL OF CELL SCIENCE 114 (14)

Fig. 5. In vivo transport of yeast NAC proteins into nuclei is inhibited in mutants for nuclear protein import. (A) Influence of SRP1 on nuclear import of ∆N11-Egd1p-2GFP. srp1-49, srp1- 31 and wild-type cells were transformed with ∆N11-Egd1p- 2GFP. Cells were grown at 25°C (right) and afterwards shifted to 37°C for 3 hours (left). Position of nuclei are indicated by arrows. (B) Influence of KAP123 and PSE1 mutations on nuclear import of ∆N11-Egd1p-2GFP. ∆kap123, pse1- 1 and the corresponding wildtype were transformed with ∆N11- EGD1-2GFP. Cells were grown at 25°C and stained with Hoechst 33258 prior to microscopy.

Furthermore, we delineated the domain of Egd1p responsible for the interaction with importins Kap123p/Pse1p. Whereas deletion of the first 11 amino acids abolished ribosome binding and led to a nuclear accumulation of Egd1p, a further deletion of the next 3 amino acids drastically reduced the Kap123p/Pse1p-dependent import of Egd1p. This region contains a KLXKL motif that is found in all Egd1p homologues in the database. Additional deletion of amino acids 15-27, a region that also bears a highly conserved cluster of basic amino acids, enhanced the import defect only slightly. Both motifs are not present in L25, the TATA-binding protein or Pho4, proteins known to be imported by Pse1p/Kap123p (Schaap et al., 1991; Kaffman et al., 1998; Pemberton et al., 1999). Our finding indicates a close proximity or even partial overlap of the ribosome binding domain and one of the nuclear localization signals in Egd1p. However, nuclear import was not abolished completely when the first 27 amino acids were missing. Therefore, we assume that Egd1p bears several nuclear import signals that trigger nuclear transport by experiments employing temperature sensitive mutants of different pathways. One obvious candidate would be the Srp1p SRP1. It was shown that in the srp1-31 mutant the nuclear (yeast importin α)-dependent pathway. This assumption could import of classical NLS-bearing substrates and of the not be tested because in the available temperature-sensitive ribosomal protein L11b was impaired (Loeb et al., 1995; mutants of SRP1, ∆N14-Egd1p-2GFP is transported into nuclei Shulga et al., 1996; Stage-Zimmermann et al., 2000). ∆N11- efficiently at permissive temperature. Thus, no difference in Egd1p-2GFP accumulated in the nucleus in wild-type cells but location of this protein was detectable after the temperature in srp1-31 cells the truncated protein also localized in the shift before the cells were dead (data not shown). cytoplasm at nonpermissive temperature. In addition, we What is the reason for transport of NAC proteins into the employed the srp1-49 mutant, which exhibits defects in protein nucleus? A possible transcriptional function of yeast Egd1p as degradation and, to a lesser extent, in nuclear transport (Tabb described by other authors had been deduced from DNA gel et al., 2000) and could not detect any significant difference shift experiments and moderately reduced amounts of some compared with the wildtype. We propose two explanations for mRNA species in an EGD1/BTT1-knockout strain (Parthun et the rather moderate effect of the SRP mutations. First, a al., 1992; Hu and Ronne, 1994). However, the simultaneous decrease in nuclear accumulation could not occur because deletion of all NAC subunits in yeast has no additional transport back to the cytosol was not possible. Only the import phenotype (Reimann et al., 1999) and firm direct proof for a of newly synthesized ∆N11-Egd1p-2GFP was blocked. function of Egd1p as is still missing. Second, there may exist alternative import pathways. We tested There are other processes that could demand a transient this hypothesis by employing yeast nuclear import mutants of nuclear localization of NAC subunits. Key components of ribosomal proteins, namely Kap123p and Pse1p. , such as the ribosomal subunits, 5S-RNPs and SRP Transient nuclear passage of NAC 2647 their synthesis in the cytosol to assemble with ribosomal subunits and are exported together with these subunits. In contrast to the SSB proteins, NAC subunits are not exported by the NES ()-dependent factor Crm1p (J.F. et al., unpublished). Interestingly, efficient import of yeast NAC subunits requires both Kap123p and Pse1p, similar to what has been found for ribosomal proteins. We believe that the first efficient binding of NAC to ribosomal subunits occurs in the nucleus. This interpretation would imply that yeast NAC does not leave the ribosome when it releases the nascent polypeptide. The assumption is supported by our observation that ER-bound ribosomes bear NAC and that binding of in vitro translated yeast NAC to ribosomes is inefficient. One cannot exclude the possibility that in the absence of sufficient ribosomal binding sites the nuclear NAC is available for transcriptional processes. However, as all our data are consistent with the idea of a co-regulated NAC assembly with ribosomal subunits, we propose that the subunits of NAC enter the nucleus primarily to bind to their final destination, the ribosome.

We thank U. Lenk for providing us with the GFP-plasmids; D. Görlich for recombinant proteins and expression clones; M. Wiedmann for antibodies; and P. A. Silver and M. Nomura for yeast strains. Thanks to E. Bürger, I. Krenz, B. Nentwig and A. Wittstruck for technical assistance; and to K. Stade, T. Sommer, F. Luft and S. Prehn for comments and advice on experiments and the manuscript. This project was supported by two grants from the Deutsche Forschungsgemeinschaft (DFG) to B.W. and M.K., respectively and a grant from the Fond der Chemischen Industrie to E. H.

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