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J. Biol. Chem., Vol. 279, Issue 36, 37751-37762, September 3, 2004

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The Bipartite Nuclear Localization Sequence of Rpn2 Is Required for Nuclear Import of Proteasomal Base Complexes via Karyopherin and Proteasome Functions^*

Petra Wendler, Andrea Lehmann, Katharina Janek, Sabine Baumgart, and Cordula Enenkel¶

From the Institut für Biochemie CCM, Charité, Universitätsmedizin Berlin, Monbijoustrasse 2, D-10117 Berlin, Germany

Received for publication, March 31, 2004 , and in revised form, June 11, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
26 S proteasomes fulfill final steps in the ubiquitin-dependent degradation pathway by recognizing and hydrolyzing ubiquitylated proteins. As the 26 S proteasome mainly localizes to the nucleus in yeast, we addressed the question how this 2-MDa multisubunit complex is imported into the nucleus. 26 S proteasomes consist of a 20 S proteolytically active core and 19 S regulatory particles, the latter composed of two subcomplexes, namely the base and lid complexes. We have shown that 20 S core particles are translocated into the nucleus as inactive precursor complexes via the classic karyopherin import pathway. Here, we provide evidence that nuclear import of base and lid complexes also depends on karyopherin . Potential classic nuclear localization sequences (NLSs) of base subunits were analyzed. Rpn2 and Rpt2, a non-ATPase subunit and an ATPase subunit of the base complex, harbor functional NLSs. The Rpt2 NLS deletion yielded wild type localization. However, the deletion of the Rpn2 NLS resulted in improper nuclear proteasome localization and impaired proteasome function. Our data support the model by which nuclear 26 S proteasomes are assembled from subcomplexes imported by karyopherin

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
26 S proteasomes are complex macromolecular assemblies essential for regulated protein turnover in eukaryotic organisms ranging from yeasts to mammals. Substrates are short-lived, misfolded proteins, and most of them are targeted to degradation by covalent attachment of polyubiquitin chains.

26 S proteasomes are composed of proteolytically active complexes, known as 20 S core particles, and regulatory complexes, called 19 S cap complexes. The 19 S cap complex itself is composed of two subcomplexes, the base and lid complexes. The base complex consists of six AAA-ATPases, numbered from Rpt1 to Rpt6, and two high molecular mass non-ATPases, Rpn1 and Rpn2. Rpn10 connects the base and lid complexes, though it is not essential. The lid complex consists of eight Rpn subunits (1).

20 S core particles are only able to degrade peptides and unfolded proteins. Proteolysis of ubiquitylated proteins is ATP-dependent and requires the association of 20 S core with 19 S cap complexes, which regulate substrate recognition and unfolding. Ubiquitin moieties of the substrate are recognized by at least two subunits, namely Rpt5 and Rpn10 (2-4). The lid subunit Rpn11 harbors a deubiquitinating metalloprotease activity, which allows ubiquitin peptide recycling before substrate degradation (5, 6). The base ATPases possess chaperone functions, which promote substrate translocation into the catalytic chamber of the core particle (7). Apart from key roles of 19 S cap complexes in proteasomal proteolysis, base complexes were proposed to serve separate functions in transcription control (8). Despite the importance of the 19 S cap complex, the functions of most of its subunits remain unknown.

In yeasts 80% of 26 S proteasomes are localized inside the nucleus. Core and cap subunits are present in roughly equal stoichiometry with an abundance of 15,000-30,000 molecules/cell, which constitute 1% of the total protein (9).

Immunoelectron microscopy in fission yeast revealed predominant proteasome decorations at the nucleoplasmic side of the NE¹ (10). In our group, we addressed the question how proteasomes are targeted into the nucleus. The NE is gated by nuclear pores. Their dimensions could accommodate assembled 26 S proteasomes. Nevertheless, large cargo proteins do not passively diffuse through nuclear pores but use a signal-mediated transport system. They are carried from the cytoplasm into the nucleus by nuclear transport receptors, called karyopherins or importins. The heterodimer karyopherin was the first described carrier and is referred to as the classic NLS receptor. The subunit recognizes the cargo NLS, and the subunit adapts the cargo complex to the nuclear pore and moves it into the nucleus. Classic NLS are characterized by short stretches of basic amino acid residues. The SV40 T antigen NLS was established as a monopartite NLS prototype, and the nucleoplasmin NLS as a bipartite NLS prototype. The basic amino acid residues of a bipartite NLS are spaced by 10-12 unconserved amino acid residues (11).

With regard to nuclear import of proteasomes, we discovered that 20 S core particles are imported as inactive precursor complexes by karyopherin (12). Karyopherin was confirmed to be associated with core subunits by proteomics organizations that systematically analyzed tandem-affinity-purified protein complexes of yeast (13).

In this work, we investigated nuclear import of 19 S cap complexes. We found that it depends on karyopherin and that Rpn2 confers a crucial NLS for efficient nuclear targeting of base complexes under restrictive conditions.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Yeast Strains, Media, and Plasmids—Saccharomyces cerevisiae strains used in this work are listed in Table I. Standard methods and media were used for yeast growth and manipulation (14). Rpn1 and Rpn11 were C-terminally tagged by GFPHA, CFPHA, or Protein A by homologous recombination of SacIXhoI-cut Rpn1 tag::HIS3::URA3 and Rpn11 tag::HIS3::URA3 into the chromosomal locus, respectively (15). To use LEU2 as an auxotrophic marker in gene replacements, HIS3 and URA3 were excised from the original pBSHU construct as a XbaI-PstI fragment and replaced by the LEU2 gene.

Endogenous Rpn2 was replaced by the HA-tagged version by homologous recombination techniques using SacI-PvuII-cut Rpn2-HA₂:: HIS3::URA3. Between the last amino acid of Rpn2 and the HA tag a spacer of 15 amino acids (AGSRANSSTLAAVTS) has been inserted. The C-terminal deletion of 161 amino acids of Rpn2 in PWCn2H has been created by using the same integration cassette as for Rpn2 HA. Immunoblot analysis of PWCn2H lysates revealed HA-tagged NLSrpn2 with the predicted molecular mass (88 kDa). However, base complexes purified from PWCn2Hn11P contained NLSrpn2HA and a degradation product NLSrpn2 of 78 kDa.

PWt2H, PWt2G, PWNt2H, and PWNt2G are derived from strain DY62 (16), in which Dp42 was replaced by pRpt2H-16 generating the appropriate shuffle strain PWt2H16. pRpt2H-16 is a pRS316 (URA3, CEN)-based vector containing a 1.8-kb XhoI-BamHI fragment that includes the RPT2 coding sequence plus 482 bp of the promoter region. LEU2-marked CEN plasmids, carrying wild type or truncated versions of Rpt2, were introduced into PWt2H16. Upon FOA reversion, strains were obtained with only the LEU2-marked plasmid (Table II) covering the chromosomal RPT2 deletion. pRpt2H is pRS315 (CEN, LEU2) containing the 1.8-kb XhoI-BamHI RPT2 fragment and a 75-bp BamHIXbaI double HA fragment. Into the unique BamHI site of pRpt2H a fragment encoding GFP without a stop codon was inserted resulting in pRpt2G. pNRpt2H and pNRpt2G were derived from pRpt2H and pRpt2G, respectively. The RPT2 promoter region from -482 bp to the start codon was PCR-amplified as an XhoI-SacI fragment, the coding region from base pair 129 to the last codon as an SacI-XbaI fragment. Both fragments were ligated into pRS315 obtaining plasmids encoding HA- or GFPHA-tagged 1-43 rpt2.

Localization Studies—For in vivo localization studies cells were grown to mid-log phase in synthetic complete medium, transferred to slides, and immediately examined with a Leica DMR microscope (15). Filters for fluorescein isothiocyanate/GFP (495-nm excitation and 525-nm emission) or CFP (436-nm excitation and 480-emission) were used (17). Photographs were taken with a Hamamatsu C5985-10 charge-coupled device camera using the MetaVue Software (Visitron Systems, Puchheim, Germany). Settings were identical for all images in a given experiment. Quantification of Rpn1-GFP and Rpn11-GFP in srp1-49 and SRP1 cells was done with the MetaVue system by selecting a fixed square of approximately the size of one-fifth of a yeast nucleus in the cytoplasm or nucleoplasm, respectively, and calculating the average pixel intensity per area. A minimum of 20 values from different images was averaged. The ratio of average pixel intensity was obtained for each strain by dividing the cytoplasmic intensity by the nucleoplasmic intensity.

Indirect immunofluorescence microscopy was performed on formaldehyde-fixed cells (15). Labeling with mAb12CA5 (1:500 dilution; Roche Applied Science) and mAb414 (1:500 dilution; BabCO) was visualized by Cy3-labeled donkey-anti-mouse IgG (1:100 dilution; Jackson ImmunoResearch Laboratories) using the Cy3 filter. DAPI-stained nuclei were visualized by using the UV filter.

Protein Biochemistry—Yeast cells were grown to mid log phase in YPD medium (yeast extract 1%, peptone 2%, dextrose 2%) and disintegrated by French press in the appropriate buffer. For a 10-40% glycerol gradient, ultracentrifugation buffer B (50 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, pH 7.75, 150 mM NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml DNase I-EP, EDTA free Complete Protease Inhibitor mixture (Roche Applied Science)) was used. 2 mM ATP and 10 mM MgCl₂ were added as indicated. Peptide hydrolysis activity was assayed by using benzyloxycarbonyl-Leu-Leu-Glu--naphthylamide (Bachem) (15).

Protein extracts were subjected to SDS-PAGE followed by immunoblot analysis using polyvinylidene fluoride membrane (polyvinylidene fluoride, Immobilon, Millipore). Ubiquitin was identified by anti-ubiquitin Z0458 antibodies (DAKO), HA by mAb HA.11 (BabCO), proteasomal subunits by polyclonal antibodies raised against Cim3/Rpt6 (18), Pre2/5 (19), and karyopherin by polyclonal antibodies (20).

Affinity Purifications—Standard IgG Sepharose affinity chromatography was performed using buffer PB (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml DNase I-EP) Cells were grown to mid log phase in 1 liter of YPD medium at 28 °C and disintegrated by passing them trough a French pressure cell. After removal of cell debris (14,000 x g, 20 min), the cell extracts were passed twice over a 1/25 lysate volume of IgG-Sepharose beads (Amersham Bioscience) at room temperature. The resin was washed with 50 bed volumes of buffer PB, and bound proteins were eluted with 0.5 M HOAc/NH₄OAc (pH 3.4). Sequential IgG-Sepharose affinity chromatography from PWn11HTP lysates was performed following the standard protocol but in the presence of 2 mM ATP and 10 mM MgCl₂. Proteins were eluted with 4 ml of buffer B (without ATP and MgCl₂), 20 S proteasomes were eluted with 4 ml of buffer B containing 300 mM NaCl, and 19 S base complex was eluted with 4 ml of buffer B containing 600 mM NaCl.

NLS-GFP-Strep-tagged constructs were affinity-purified over Strep-Tactin matrices followed by elution with 5 mM desthiobiotin according to the manufacturer's instructions (Institut für Bioanalytik GmbH, Göttingen, Germany). To eliminate biotinylated proteins from the extract prior to binding to the matrix, it was precleared for 30 min at 4 °C by adding 40 µg of avidin (Sigma) per ml of lysate.

Far Western Analysis—NLS-containing proteins were identified by far Western analysis using Protein A-tagged karyopherin (Srp1) as the binding partner. French press lysates (20 mg/ml protein) of strain CEY1b (20) were prepared in buffer PB with EDTA-free Complete Protease Inhibitor mixture (Roche Applied Science). Proteins were separated by SDS-PAGE, transferred to a polyvinylidene fluoride membrane, and refolded in renaturation buffer (50 mM HEPES, pH 7.3, 100 mM KOAc, 5 mM MgOAc, 0.3% Tween, 0.5% bovine serum albumin) overnight at 4 °C. After blocking for 30 min in 5% dried milk in PBST (MPBST), the filter was incubated in 10 ml of MPBST containing a 1:5 dilution of CEY1b lysate to detect NLS-GFPS proteins and a 1:1 dilution of CEY1b lysate to detect 19 S subcomplexes. As a control, filters were incubated in 10 ml of MPBST containing 10 µg of Protein A. Incubation for 4 h at 4 °C was followed by washing with PBST. The blot was incubated with horseradish peroxidase-coupled rabbit IgG in MPBST for 1 h at 4 °C and processed for enhanced chemiluminescence detection. After ECL detection the blot was stained with Amido Black.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In this work, we set out to investigate the nuclear import of base and lid complexes of proteasome regulatory particles. To study in vivo localizations of both subcomplexes, each complex was functionally tagged with fluorescent proteins. Rpn1 and Rpn11 were chosen as reporter subunits of the base and lid complexes, respectively. By exploiting homologous recombination techniques (12) yeast strains were created in which endogenous Rpn1 and Rpn11 were chromosomally replaced by GFPHA- and CFPHA-tagged variants. To compare the localization of the proteasomal base and lid subunits with the localization of proteins marking the ER/NE network, nucleoporin Nup49 and the ER-resident proteasomal substrate Pdr5^* were co-expressed as GFP-labeled proteins (21, 22). The cyan fluorescence of both proteasomal subunits was predominantly detected in the nucleus and specifically merged with the green fluorescence of Nup49 and Pdr5^* at the NE (Fig. 1A) consistent with previous reports on the localization of Mts4 and Pad1, the fission yeast homologues of budding yeast Rpn1 and Rpn11 (10).

View larger version (42K): [in this window] [in a new window]   FIG. 1. Proteasomal base and lid subunits Rpn1 and Rpn11 are predominantly nuclear in yeast wild type cells. Tagged variants of both proteins are functionally incorporated into 19 S base and lid complexes, respectively. A, direct fluorescence microscopy of S. cerevisiae wild type cells expressing CFPHA-labeled Rpn1 and Rpn11, respectively (strains PWn1C and PWn11C). Either GFP-tagged nucleoporin Nup49 or ER-resident protein Pdr5* were co-expressed from centromere-based plasmids. Fluorescent proteins were visualized with fluorescein isothiocyanate/GFP and CFP filter sets. B, proteasomal 19 S complexes were isolated by IgG-Sepharose affinity chromatography from lysates of cells expressing Protein A-tagged Rpn11 (lane 1) and Rpn1 (lane 2) instead of the endogenous subunits. Proteins tightly bound to the affinity resin were eluted by low pH, subjected to SDS-PAGE, and stained with Coomassie Blue. All proteins identified by fingerprint mass spectroscopy were assigned. Ubp6 was confirmed to be associated with 19 S cap complexes (41).

Based on these findings we concluded that our tagged versions of Rpn1 and Rpn11 are functional, thus suited to report on the in vivo localization of base and lid complexes. To get insight into the import pathway of these complexes, GFPHA-labeled Rpn1 and Rpn11 were expressed in a set of nuclear import mutants. Significant delocalization of both reporter proteins from the nucleus to the cytoplasm was observed in mutants affecting the classic NLS-dependent import pathway, especially in the temperature-sensitive srp1-49 mutant. At restrictive temperatures, GFPHA-labeled Rpn1 and Rpn11 obviously accumulated inside the cytoplasm of the srp1-49 mutant compared with isogenic wild type cells (Fig. 2A). By using the MetaVue system we measured the pixel intensities within the cytoplasm and nucleoplasm of srp1-49 and wild type cells expressing GFPHA-labeled Rpn1 and Rpn11, respectively. The ratio of cytoplasmic to nuclear fluorescence intensity was calculated. At restrictive temperature, the ratio doubled in the srp1-49 mutant, whereas it did not change in wild type (Fig. 2B). One possibility to explain this phenomenon is that the srp1-49 mutation affects nuclear import of base and lid complexes. To ascertain that increased cytoplasmic fluorescence derived from base and lid complexes but not from non-incorporated subunits, lysates from srp1-49 and wild type cells expressing GFPHA-labeled Rpn1 and Rpn11 were fractionated by glycerol gradient ultracentrifugation (Fig. 2C, only shown for Rpn11-GFPHA). All gradient fractions were probed for Rpn11-GFPHA, the base ATPase Rpt6, and the core subunit 5. Rpn11 co-migrated with Rpt6 in fast sedimenting fractions showing that all fusion proteins were incorporated into their proteasomal subcomplexes of srp1-49 as well as of wild type cells (Fig. 2C; panels labeled with Rpn11-GFPHA and Rpt6). Therefore, it is unlikely that single lid and base subunits accumulated inside the cytoplasm of srp1-49.

View larger version (66K): [in this window] [in a new window]   FIG. 2. srp1-49 cells, which are defective in classic NLS import, accumulate GFPHA-labeled Rpn1 and Rpn11 in the cytoplasm at restrictive temperature. A, Rpn1 and Rpn11 were chromosomally replaced by GFPHA-tagged versions in srp1-49 and isogenic SRP1 cells (strains PWn1G1-49, PWn1GSrp, PWn11G1-49, and PWn11GSrp). Cultures were grown for 3 h at 28 °C or 37 °C. The in vivo localization of GFP-labeled Rpn1 and Rpn11 was visualized by direct fluorescence microscopy. B, digital images of at least 20 cells per strain were quantified to yield the average GFP pixel intensity (see "Experimental Procedures"). The ratio of the cytoplasmic to the nuclear pixel intensity is indicated as percent value. All values are shown ± S.D. C, exponentially grown wild-type and srp1-49 cells expressing Rpn11-GFPHA instead of the endogenous protein were shifted for 4 h to 37 °C. Extracts were prepared and run in parallel on 10-40% glycerol gradient ultracentrifugation. Fractions were collected from the top to bottom of the gradient and subjected to SDS-PAGE followed by immunoblot analysis using antibodies against HA epitopes, Rpt6, and 5, respectively. Matured m5 subunits mark the proteasome peak fractions. Immature pro5 subunits indicate 13-16 S precursor complexes (fractions 8-13).

View larger version (141K): [in this window] [in a new window]   FIG. 4. Characterization of mutants expressing NLS rpt2HA and NLS rpn2HA. A, logarithmically grown wild type and NLS deletion strains were diluted to 1 x 107 cells/ml. 4 µl of 10-fold serial dilutions were spotted onto YPD plates to test temperature-sensitive growth and onto minimal media plates supplemented with either 0.6 µg/ml canavanine or 20 mg/ml arginine to test canavanine-sensitive growth. Minimal plates were incubated at 28 °C, and YPD plates were incubated at indicated temperatures for 3 days. B, NLSrpn2HA mutants accumulate polyubiquitylated proteins. Logarithmically grown cells expressing either NLSrpn2HA (lane 1) or Rpn2HA (lane 2) were incubated at 37 °C for 1 h. Extracts from cell equivalents were subjected to SDS-PAGE followed by immunoblotting using anti-ubiquitin, HA, Rpt6, and 5 antibodies. C, NLSrpn2HA is incorporated into the proteasome. Exponentially grown cells either expressing NLSrpn2HA and Rpn2HA instead of the endogenous proteins were shifted for 1 h to 37 °C (strains PWn2H5H and PWCn2H5H). Extracts were prepared in the presence of 150 mM salt and subjected to 10-40% glycerol gradient ultracentrifugation. Fractions were collected from the top to bottom of the gradient and assayed for peptide cleavage activity using the substrate benzyloxycarbonyl-Leu-Leu-Glu--naphthylamide in the absence and presence of ATP. The specific activity is referred to as the maximum value (upper diagram). Protein samples were subjected to SDS-PAGE followed by immunoblot analysis using antibodies against HA epitopes and Rpt6 (lower panels). D, NLSrpt2HA is tightly associated with the base complex, whereas rpn2HA is loosely associated with the base complex. Base complexes from NLSrpt2HA, NLSrpn2HA, and their wild types were isolated by affinity chromatography as follows: Protein A-tagged Rpn11 was chromosomally introduced into cells expressing NLSrpt2HA, NLSrpn2HA, Rpt2HA, and Rpn2HA, respectively. Lysates were passed on IgG-Sepharose beads to bind 26 S proteasomes via the lid subunit Rpn11-ProA. The base and core complexes were eluted by 600 mM salt, while the lid complexes remained bound to the affinity resin. Then, the eluate containing base and core complexes was fractionated by 10-40% glycerol density gradient ultracentrifugation. All fractions were analyzed by SDS-PAGE as shown for Rpt2HA and NLSrpt2HA in the lower panels and for Rpn2HA and NLSrpn2HA in the upper panels. Molecular weight markers indicate the Coomassie Blue-stained fractions. Immunoblots using HA, Rpt6, and 4 antibodies are shown below the Coomassie Blue-stained gels.

View larger version (83K): [in this window] [in a new window]   FIG. 3. Classic NLS of 19 S base subunits are recognized by karyopherin and target non-nuclear proteins into the nucleus. A, 19 S subunits were analyzed for clusters of basic amino acid residues, which resemble NLS-like motifs as highlighted. Potential classic NLS were selected for further investigation as underlined. Amino acid positions are numbered. The monopartite NLS is K(R/K)X(R/K), the bipartite NLS is KRX10-12KRXK (26, 42). B, the NLS-like motifs of Rpt2 and Rpn2, but not of Rpt1, direct GFP chimera into the yeast nucleus as shown by direct fluorescence microscopy. Potential NLS of Rpt1 (panel 3), Rpt2 (panel 4), and Rpn2 (panel 5)as underlined above were fused to GFPS and expressed behind the GAL1 promoter for 4 h at 28 °C (strains PWRpt2NLSG, PWRpt1NLSG, and PWRpn2NLSG). As a control, wild type SV-40 T-antigen NLS 126PKKKRKV132 (panel 1) and the mutated SV-40 T-antigen NLS 126PKTKRV132 (panel 2) were examined as GFPS fusion proteins under the same growth conditions (strains PWNLSG and PWmNLSG). C, the NLS-like motifs of Rpt2 and Rpn2 are recognized by karyopherin as shown by far Western blot analysis. NLS-GFPS chimera were isolated by Streptactin affinity chromatography from yeast cells expressing GFPS-tagged wild type SV40-NLS (lane 1), mutant SV40-NLS (lane 2), Rpt1 NLS (lane 3), Rpt2 NLS (lane 4), and Rpn2 NLS (lane 5) as shown above in B (panels 1-5, respectively). Equal protein amounts were subjected to SDS-PAGE, blotted, and probed for cargoes recognized by Protein A-tagged karyopherin . Reactions with Protein A-tagged karyopherin were detected by horseradish peroxidase-conjugated rabbit IgG and visualized by enhanced chemiluminescence (right panel). Wild type and mutant SV40 NLS-GFPS were assigned with + and -, respectively. The blot was stained with Amido Black (left panel). Degradation products of the Rpt2 NLS fusion protein that do not react in far Western analysis are marked by an asterisk. D, co-immunoprecipitation of karyopherin with NLS-GFPS chimera. NLS-GFPS fusion proteins of wild type SV40 T antigen (lane 1), mutant SV40 T antigen (lane 2), Rpt1 (lane 3), Rpt2 (lane 4), and Rpn2 (lane 5) were precipitated by Streptactin matrices, separated by SDS-PAGE, blotted, and probed for GFP and karyopherin by using specific antibodies as indicated. The asterisk marks cross-reactions.

Next, we searched for direct interactions of karyopherin with our NLS-GFPS fusion proteins. The GFPS fusion proteins were isolated from yeast cell lysates by affinity chromatography on Streptactin matrices and subjected to far Western blots. The blots were probed for classic NLS cargoes by using Protein A-tagged karyopherin . Specific reactions between karyopherin and the NLS chimera were detected by horseradish peroxidase-conjugated rabbit IgG and visualized by enhanced chemiluminescence (Fig. 3C, right panel). As positive and negative controls we established wild type and mutant SV40 NLSGFPS fusion proteins, respectively (lanes 1 and 2, respectively). The putative NLS of Rpt1 did not react with karyopherin (lane 3). However, the NLS of Rpt2 and Rpn2 were recognized by the NLS receptor (lanes 4 and 5) verifying our observations above that the Rpt2 and Rpn2 NLS function in nuclear targeting. To confirm that equal protein amounts were subjected to far Western analysis, the blot was stained with Amido Black (Fig. 3C, left panel). Our results were in line with co-immunoprecipitation experiments using NLS-GFPS chimera as baits. Equal amounts of GFPS fusion proteins were affinity-purified from cells shown in Fig. 3B, separated by SDS-PAGE, blotted, and probed for karyopherin and GFP as loading control. NLS chimera of Rpt2 and Rpn2 were found to be associated with karyopherin like wild type SV40 NLS (Fig. 3D, lanes 1, 4, and 5). NLS chimera of mutant SV40 NLS and Rpt1 were not recognized by karyopherin (lanes 2 and 3).

The next question was whether the N-terminal region of Rpt2 and the C-terminal part of Rpn2 confer crucial NLS to the base complex. Deletion analysis of each NLS should give the answer. Strains were created in which the NLS of Rpt2 and Rpn2 were truncated yielding NLSrpt2HA and NLSrpn2HA mutants, respectively. The short HA epitope at the C terminus of each mutant subunit was introduced to mark the protein. The corresponding isogenic wild type strains expressed Rpt2HA and Rpn2HA instead of the endogenous subunits. Mutations with strong impact on proteasome functions cause sensitivity against elevated temperatures and canavanine, an amino acid analogue of arginine. Both conditions induce an increase of aberrantly synthesized proteins, which are preferred proteasome substrates (1). We tested our NLSrpt2HA and NLSrpn2HA mutants for those phenotypes generally displayed by proteasomal mutants. The NLSrpt2HA mutant showed no canavanine sensitivity, whereas the NLSrpn2HA mutant showed strong canavanine sensitivity compared with the isogenic wild type (Fig. 4A, panels labeled with -can and +can). To test temperature sensitivity, cells were spotted on complete media and grown at 28 °C and 37 °C. The NLSrpt2HA and NLSrpn2HA mutants were strongly temperature-sensitive (Fig. 4A, panels labeled with 28 °C and 37 °C). In the case of the NLSrpt2HA mutant, the corresponding wild type Rpt2HA was already temperature-sensitive suggesting that the C-terminal HA tag interfered with ATPase functions.

Because the NLSrpn2HA mutant displayed both canavanine- and temperature-sensitivity, we anticipated that the NLS deletion of Rpn2 affects proteasome functions. Thus, the NLSrpn2HA mutant was assayed for polyubiquitylated protein accumulation. Equal protein amounts were loaded on SDS-PAGE, blotted, and probed for polyubiquitylated proteins. Increased polyubiquitylation was detected in the NLSrpn2HA mutant (Fig. 4B, lower panel, lane 1) compared with Rpn2HA wild type (lane 2). HA-tagged proteins, Rpt6 and 5, were immunodetected (upper panel).

Before we monitored the subcellular localization of NLSrpt2HA and NLSrpn2HA, we checked the incorporation of NLSrpt2HA and NLSrpn2HA subunits into the base complex, after the cells were grown at restrictive temperatures. Equal protein amounts from lysates of NLSrpt2HA and NLSrpn2HA and their respective wild type cells were subjected to glycerol gradient ultracentrifugation. The fractions were run on SDS-PAGE, blotted, and probed for HA-tagged subunits, Rpt6 and 5. Non-incorporated subunits were found neither in the NLSrpt2HA mutant nor in the NLSrpn2HA mutant (Figs. S2 and 4C, slowly sedimenting fractions 1-7). The HA-tagged proteasomal subunits co-migrated with Rpt6 and matured 5 in fast sedimenting fractions (fractions 11-18). No differences in the peptide cleavage activity of Rpt2HA and NLSrpt2HA proteasomes were detected. Ultracentrifugation fractionations of cells expressing GFPHA-labeled Rpt2 and NLSrpt2 instead of their endogenous subunits yielded similar results (not shown).

In the case of the NLSrpn2HA mutant, two activity peaks arose from 26 S proteasomes in the presence of ATP (Fig. 4C, upper diagram). Compared with the wild type a reduction in proteasome activity of about 50% was determined in the NLSrpn2HA mutant consistent with our finding that the NLSrpn2HA mutant is strongly temperature and canavanine sensitive. As a control we measured the proteolytic activity of the core complexes in the absence of ATP. Under this condition the proteolytic activity did not significantly differ in the mutant and wild type (standard deviations of 15%).

Furthermore, we isolated base complexes from NLSrpt2HA, NLSrpn2HA, and their wild type complexes. For this purpose, Protein A-tagged Rpn11 was chromosomally introduced into the respective strains. Cell lysates were passed on IgG-Sepharose beads to isolate 26 S proteasomes via the lid subunit Rpn11-ProA. The base and core complexes were eluted by 600 mM salt, whereas the lid complexes remained bound to the affinity resin. Then, the eluate containing base and core complexes was fractionated by glycerol density gradient ultracentrifugation. In mutant NLSrpt2HA and wild type Rpt2HA, base complexes migrated in glycerol gradient fractions 8-11. Their protein patterns resembled each other (Fig. 4D, Coomassie Blue-stained gels shown in the lower panels). Western blot analysis confirmed the tight association of NLSrpt2HA and Rpt2HA with base complexes (Fig. 4D, immunoblots labeled with HA and Rpt6 as shown below the Coomassie Blue-stained gels). The core complexes migrated in fractions 11-14 as indicated by the presence of core subunit 4.

In mutant NLSrpn2HA and wild type Rpn2HA, base complexes migrated in fractions 8-11 (Fig. 4D, Coomassie Blue-stained gels shown in the upper left and right panels). However, the fractions of mutant base complexes were slightly shifted to slower sedimenting fractions. The protein pattern of NLSrpn2HA base complexes differed from wild type base complexes. Although Rpn2HA was stably incorporated into the base complex, NLSrpn2HA dissociated from the base components by 600 mM salt and migrated in slow sedimenting fractions 4-6 (Fig. 4D, compare Coomassie Blue-stained gels and HA-labeled blots; Rpn2HA in fractions 8-10, upper right panel; NLSrpn2HA in fractions 4-6, upper left panel). As described above, fractions 11-14 comprise core complexes. These results suggested that NLSrpn2HA is incorporated into base complexes under physiological conditions (Fig. 4C) but not as tightly as the wild type subunit, because NLSrpn2HA is dissociated from base complexes by 600 mM salt (Fig. 4D).

We concluded that under physiological conditions the tagged versions of NLSrpt2 and NLSrpn2 are suited to report on base complex localization. Cells expressing either Rpt2GFPHA or NLSrpt2GFPHA instead of the endogenous proteins were monitored by direct fluorescence microscopy at 28 °C and 37 °C. The base complex harboring NLSrpt2GFPHA localized to the nucleus like wild type base complexes independent of temperature (Fig. 5A; Rpt2GFPHA, lower panels, and NLSrpt2GFPHA, upper panels). In addition, we monitored the localization of Rpn1- and Rpn11-GFPHA in mutant NLSrpt2HA and wild type Rpt2HA cells. Their major localization inside the nucleus remained unchanged even at restrictive temperatures supporting our conclusion that the Rpt2 NLS is not essential for nuclear targeting of base complexes (not shown).

View larger version (48K): [in this window] [in a new window]   FIG. 5. Intracellular localization of base complexes harboring Rpt2 and Rpn2 with NLS deletions. A, cells expressing NLSrpt2GFPHA and Rpt2GFPHA (strains PWNt2G and PWt2G) were grown to mid-log phase at permissive (28 °C) and restrictive temperature (37 °C) for 3 h and monitored by direct fluorescence microscopy. B, cells expressing NLSrpn2HA and Rpn2HA (strains PWCn2H and PWn2H) were grown to mid-log phase at 28 °C and 37 °C for 1 h. Indirect immunofluorescence microscopy was performed on formaldehyde-fixed cells. NLSrpn2HA (upper panels) and Rpn2HA (lower panels) were detected by mAb12CA5 and visualized by Cy3-labeled donkey anti-mouse IgG. Nuclear pore proteins were detected by mAb414 (right panels) and visualized by Cy3-donkey anti-mouse IgG. Nuclear DNA was stained with DAPI.

Finally, we wanted to confirm that base complexes are cargoes of karyopherin , whereas base complexes containing the NLSrpn2HA and NLSrpt2HA subunits, respectively, are not recognized by karyopherin . Wild type and mutant base complexes were purified by using affinity chromatography and stepwise elution of proteasomal subcomplexes with salt as described in Fig. 4D. The base complex fractions were pooled and subjected to far Western blot analysis. Instead of full-length Rpn2HA and Rpt2HA, base complexes isolated from NLS mutant strains harbored the truncated NLS subunits, respectively (Figs. 6, A and B, left panels, compare lanes labeled wt with ). Far Western analysis revealed that karyopherin recognized Rpn2 as cargo protein either tagged with HA (Fig. 6A, middle panel, lane wt) or without tag (Fig. 6B, middle panel, lanes wt and ), whereas NLSrpn2 did not react with karyopherin (Fig. 6A, middle panel, lane ). HA-tagged Rpt2 (Fig. 6B, middle panel, lane wt) as well as endogenous Rpt2 (Fig. 6A, middle panel, lanes wt and ) were recognized by karyopherin , whereas NLSrpt2HA did not react with karyopherin (Fig. 6B, middle panel, lane ). Rpt6, the ATPase of the lowest molecular mass among the base subunits, was additionally recognized by karyopherin (Figs. 6, A and B, middle and lower right panels) suggesting that Rpt6 is additionally involved in nuclear targeting. HA-tagged subunits were identified by immunoblot analysis (Figs. 6, A and B, upper right panels).

View larger version (57K): [in this window] [in a new window]   FIG. 6. Base complexes are cargoes of karyopherin A, base complexes containing either Rpn2HA (lane wt) or NLSrpn2HA (lane ) were purified from yeast (strains PWn2Hn11P and PWC2Hn11P) as described above and subjected to far Western analysis followed by Amido Black staining (left panel). Far Western signals derived from cargo proteins, which were recognized by Protein A-tagged karyopherin . Binding reactions were detected by horseradish peroxidase-coupled rabbit IgG and enhanced chemiluminescence (middle panel). Rpn2HA, NLSrpn2HA, Rpt2, and Rpt6 were assigned. HA-tagged subunits and Rpt6 were identified by specific antibodies (right panels). Proteins marked with an asterisk were cross-reacting Rpn11-ProA fusion proteins, which leaked from the IgG-Sepharose. B, as described above, base complexes from cells expressing Rpt2HA (lane wt; strain PWt2Hn11HTP) and NLSrpt2HA (lane ; strain PWNt2Hn11HTP), respectively, were analyzed by Amido Black staining (left panel) and far Western blotting (middle panel). Rpn2, Rpt2HA, NLSrpt2HA, and Rpt6 were assigned. HA-tagged subunits and Rpt6 were identified by specific antibodies (right panels). C, base complexes interact with karyopherin . IgG-Sepharose beads were loaded with Protein A-tagged karyopherin (right) or mock treated (left). Wild type base complexes were loaded (lane 1). The beads were washed three times (lanes 2-4). Tightly bound proteins were eluted at low pH (lane 5). Fractions were subjected to SDS-PAGE followed by Coomassie Blue staining (CB, upper panels) and immunoblotting. Protein A-tagged karyopherin was probed with rabbit IgG-horseradish peroxidase conjugates (WB, middle panels). Base complexes were detected by antibodies against Rpt1 (lower panels).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
This work provides evidence that 19 S regulatory subcomplexes of 26 S proteasomes are imported into the nucleus via the NLS receptor karyopherin . Our localization studies of temperature-sensitive srp1-49 mutants indicated that the classic NLS-dependent pathway is mainly responsible for nuclear import of lid and base components. The basal transport activity of karyopherin in the srp1-49 mutant suffices to achieve efficient nuclear import of NLS cargoes at permissive temperatures. At restrictive temperatures srp1-49 cells show aberrant nuclear morphologies, defective proteasomal proteolysis, and arrest in mitosis (27-29). An increased fluorescence intensity of GFP-labeled base and lid subunits was measured inside the cytoplasm. Because base and lid subunits were incorporated into complexes of approximately the size of their subcomplexes, we assume that base and lid complexes accumulate as cargoes of karyopherin in the cytoplasm of the srp1-49 mutant. Just an up-regulation of proteasomal gene expression cannot explain this phenomenon. Newly synthesized subcomplexes should not only arise in the cytoplasm but also in the nucleoplasm, if their nuclear import is independent of karyopherin .

Proteasome nuclear trafficking is pivotal for proteasome function and cell cycle progression in yeasts as originally discussed by McDonald and Byers (30), who at first studied the in vivo localization of GFP-labeled base subunits, namely of Rpt4/Pcs1. Nuclear substrates seem to be abundant. Their proteolysis requires proteasomes in the nucleus (30). However, apart from proteolytically active proteasomes, we discovered a considerable amount of inactive precursor complexes in the nucleus. We concluded that nuclear core particles are matured at their place of destination (12, 31). To ensure the constitutive presence of proteasomes in the nucleus, it is plausible that the same transport receptor is used for nuclear import of proteasomal subcomplexes. Base and lid complexes should be synergistically imported with precursor complexes and cooperatively assembled to complete nuclear 26 S proteasome biogenesis. Insufficient amounts of proteasomes in the nucleus may be a main reason for the pleiotropic phenotype of the srp1-49 mutant.

Based on genetic evidence Nomura and coworkers (27) already considered that nuclear import of proteasomal components depends on karyopherin . STS1, a gene essential for nuclear segregation and division, and RPN11 are high copy suppressors of the srp1-49 mutation, which reflected a failure of nuclear import of Sts1, Rpn11, and related proteins by karyopherin (27). Sts1 directly bound to Rpn11 and karyopherin as judged by the yeast two-hybrid analysis. Neither yeast two-hybrid nor in vitro interactions were detected between Rpn11 and karyopherin leading to the conclusion that karyopherin fulfills separate functions in proteasomal proteolysis apart from nuclear import of proteasomal components (27). Indeed, isolated lid complexes were not recognized by karyopherin in far Western blots.² Hence, nuclear import of lid components might be promoted by transiently bound adapter proteins, which render signals for karyopherin recognition. A candidate adapter protein might be Sts1/Cut8, which is essential for nuclear localization of lid complexes (32).

Yin6, a protein regulating cell division and mitotic fidelity via the proteasome, was further discussed to be involved in targeting lid complexes into the nucleus. The deletion of Yin6 resulted in delocalization of lid components to the cytosol, which correlated with proteasome inactivation. One interpretation of these results was that Yin6 accompanies lid complexes into the nucleus, as it contains a presumptive bipartite NLS (33, 34).

To substantiate our conclusion, that 19 S base complexes are cargoes of karyopherin , we analyzed base subunits for functional NLS. Our experimental design facilitated the comparison between in vitro and in vivo data. Fused to GFP-Streptactin each NLS-like peptide could be tested for its potency in nuclear targeting. Localization studies in living cells, co-precipitation, and far Western blot experiments yielded consistent results. The Rpt2 NLS and the Rpn2 NLS turned out to be functional. Both peptides efficiently targeted GFP into the nucleus and were specifically recognized by karyopherin . The Rpn2 NLS fulfills the structural requirements of a bipartite NLS, whereas the Rpt2 NLS consists of two monopartite NLS spaced by 17 amino acid residues, which exceed the common linker length of a bipartite NLS (25).

The tertiary structure environment of the NLS peptides within the protein additionally plays an important role in determining their accessibility for karyopherin . By using far Western blot analysis and solution binding assays we verified that the NLS function in full-length Rpt2, Rpn2, and in base complexes. In the case of Rpt2, the NLS comprises the N-terminal amino acid residues 11-37. From the literature, we knew that the truncation of the N-terminal 85 amino acid residues (of S4, the mammalian homologue of Rpt2) abrogates binding to neighboring ATPase subunits. Thus, the N-terminal region of Rpt2 was proposed to be needed for base complex assembly (35). Our data show that at least the N-terminal 43 amino acid residues of Rpt2 can be deleted without interfering with base complex assembly and nuclear localization. Similar results were obtained by McDonald and coworkers (36), who analyzed a putative NLS in the N-terminal region of Rpt4. Virtually all mutations that affected the N-terminal region of Rpt4 containing the putative NLS resulted in wild type phenotype (36).

In the case of Rpn2, the NLS is flanked by charged amino acid residues, namely the KEKE motif (37), which were disrupted in our NLSrpn2 mutant. Because C-terminal amino acid residues adjacent to the NLS and KEKE motif were previously shown to be dispensable for proteasome function (38), we were allowed to conclude that the NLSrpn2 phenotype is caused by the lack of the Rpn2 NLS. Quaternary structure modeling implies that Rpn2 forms a toroid placed on top of the base ATPase ring (39). The hydrophilic region embedding the NLS and KEKE motif protrudes to the protein surface allowing protein interactions such as with karyopherin .

In our previous work we already discussed the redundancy of NLS in subunits of core particles. We found that the deletion of a single NLS could be compensated by the presence of multiple NLS in neighboring subunits (12). With regard to the base complex, the synthetic lethal phenotype of mutants lacking both the Rpt2 and Rpn2 NLS suggested that Rpt2 and Rpn2 contribute the major recognition sites for karyopherin . Moreover, the functions of the base complex NLS might be arranged in a hierarchical order, because the Rpn2 NLS is crucial for proper nuclear localization under restrictive conditions.

Non-classic NLS of proteasomal subunits used for karyopherin binding might still exist, which escaped our approach. STAT1 homodimers and STAT1-STAT2 heterodimers are examples for unconventional cargoes of karyopherin . They harbor nonclassic NLS, which cooperate in STAT dimers and bind to karyopherin apart from the "major" NLS binding groove (40). Presumptive nonclassic NLS in Rpt6 might explain that Rpt6 was detected as a potential cargo of karyopherin in far Western blot analysis.

Taken together, we present evidence that base complexes are direct cargoes of karyopherin . The bipartite NLS of Rpn2 is essential for nuclear localization and proper proteasome functions at least under restrictive conditions. The depletion of nuclear base complexes in our NLSrpn2 mutant correlated with reduced 26 S proteasome activity. The temperature sensitivity of the NLSrpn2HA mutant argues for changes in conformation and dissociation of NLSrpn2HA from the base complex as the cause for impaired nuclear import. Consistent with the latter explanation is the finding that the binding of NLSrpn2HA to the base complex is weakened by salt compared with the wild type subunit (Fig. 4D). However, under physiological conditions NLSrpn2HA was found to be incorporated into the base complex even at restrictive temperatures (Fig. 4C). The question arose as to why removal of the Rpn2 NLS did not block nuclear import at any temperature. Based on our result that Rpn2, Rpt2, and potentially Rpt6 (Fig. 6A) confer NLS to the base complex, we conclude that multiple NLS are present, which might be simultaneously recognized by karyopherin at permissive temperature. At elevated temperatures, conformational changes within the NLSrpn2 base complexes might weaken Rpt2 binding to karyopherin as the cause for insufficient nuclear import of mutant base complexes.

Finally, the accessibility of base complex NLS to karyopherin should be regulated by conformational changes and structural masking. Future tasks are needed to solve the mechanism by which base complexes escape recognition by karyopherin to remain in the cytoplasm.

	FOOTNOTES

 
^* This work was supported in part by the Deutsche Forschungsgemeinschaft (Grant EN 301/4-4 to C. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Figs. S1 and S2.

Present address: School of Crystallography, Birkbeck College, Malet St., London WC1E 7HX, United Kingdom.

Supported by a project to Peter-Michael Kloetzel from the Bundesministerium für Bildung und Forschung.

^¶ To whom correspondence should be addressed. Tel.: 49-30-450-528-158; Fax: 49-30-450-528-916; E-mail: cordula.enenkel{at}charite.de.

¹ The abbreviations used are: NE, nuclear envelope; DAPI, 4,6-dia-midino-2-phenylindole; HA, hemagglutinin epitope; GFP, green fluorescent protein; CFP, cyan fluorescent protein; NLS, nuclear localization signal; S, Streptactin epitope; mAb, monoclonal antibody; PBS, phosphate-buffered saline; PBST, PBS containing 0.1% Tween; MPBST, dried milk in PBST; ER, endoplasmic reticulum; STAT, signal transducers and activators of transcription; FOA, 5-fluororotic acid.

² P. Wendler, A. Lehmann, K. Janek, S. Baumgart, and C. Enenkel, unpublished results.

	ACKNOWLEDGMENTS

 
We thank Dieter H. Wolf, Günter Blobel, Susan Wente, Masayasu Nomura, Gerry Fink, Karl Kuchler, and Carl Mann for sharing yeast strains and antibodies. Due to controversies about srp1-31 and srp1-49 alleles in the literature, we remapped the mutations. Proteasome localization is affected in srp1-49 (E145K). We thank Wolfgang Dubiel for critical reading of the manuscript. Especially, we thank Peter-Michael Kloetzel for discussions and providing the mass spectroscopy facility.

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