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Originally published In Press as doi:10.1074/jbc.M700217200 on May 7, 2007

J. Biol. Chem., Vol. 282, Issue 27, 19292-19301, July 6, 2007
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A Novel Conserved Nuclear Localization Signal Is Recognized by a Group of Yeast Importins*Formula

Thomas Fries{ddagger}1, Christian Betz{ddagger}1, Kai Sohn§, Stefanie Caesar{ddagger}, Gabriel Schlenstedt{ddagger}, and Susanne M. Bailer{ddagger}2

From the {ddagger}Universität des Saarlandes, Medizinische Biochemie und Molekularbiologie, Gebaüde 61.4, D-66421 Homburg/Saar, Germany, and the §Fraunhofer Institut für Grenzflächen und Bioverfahrenstechnik, Nobelstrasse 12, D-70569 Stuttgart, Germany

Received for publication, January 8, 2007 , and in revised form, May 3, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Nucleo-cytoplasmic transport of proteins is mostly mediated by specific interaction between transport receptors of the importin β family and signal sequences present in their cargo. While several signal sequences, in particular the classical nuclear localization signal (NLS) recognized by the heterodimeric importin {alpha}/β complex are well known, the signals recognized by other importin β-like transport receptors remain to be characterized in detail. Here we present the systematic analysis of the nuclear import of Saccharomyces cerevisiae Asr1p, a nonessential alcohol-responsive Ring/PHD finger protein that shuttles between nucleus and cytoplasm but accumulates in the nucleus upon alcohol stress. Nuclear import of Asr1p is constitutive and mediated by its C-terminal domain. A short sequence comprising residues 243–280 is sufficient and necessary for active targeting to the nucleus. Moreover, the nuclear import signal is conserved from yeast to mammals. In vitro, the nuclear localization signal of Asr1p directly interacts with the importins Kap114p, Kap95p, Pse1p, Kap123p, or Kap104p, interactions that are sensitive to the presence of RanGTP. In vivo, these importins cooperate in nuclear import. Interestingly, the same importins mediate nuclear transport of histone H2A. Based on mutational analysis and sequence comparison with a region mediating nuclear import of histone H2A, we identified a novel type of NLS with the consensus sequence R/KxxL(x)nV/YxxV/IxK/RxxxK/R that is recognized by five yeast importins and connects them into a highly efficient network for nuclear import of proteins.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Trafficking of proteins and RNAs between cytoplasm and nucleus is essential for the maintenance of cell function and crucially involved in adaptation to altered cellular conditions. Nucleo-cytoplasmic transport of most proteins and some RNAs is mediated by soluble transport receptors of the importin β family (1–3). Directionality of nucleo-cytoplasmic transport is brought about by the small GTPase Ran (Gsp1p in Saccharomyces cerevisiae). The high concentration of RanGTP in the nucleus and of RanGDP in the cytoplasm, called RanGTP gradient, regulates the interaction of importins/exportins with their cargoes. This gradient is generated by the exclusive nuclear localization of the guanine nucleotide exchange factor RCC1/RanGEF (Prp20p in S. cerevisiae) and the nuclear exclusion of the RanGTPase-activating protein RanGAP1 (Rna1p in S. cerevisiae). To initiate nuclear import, an importin forms a dimeric complex with its cytoplasmic cargo. Following docking and translocation through the nuclear pore complex, the importin-cargo complex is dissociated by binding of RanGTP to the importin. Nuclear export starts with formation of a trimeric cargo-exportin-RanGTP complex, which is dissociated in the cytoplasm upon GTP hydrolysis.

The complete genome of S. cerevisiae encodes 14 members of the importin β-like proteins, 10 of which function as importins, while three function as exportins. Msn5p is an exception since it mediates both import and export of proteins. In vertebrates more than 20 transport factors have been identified (1, 4, 5). Most of the transport factors directly interact with their cargoes, while importin β and its yeast counterpart Kap95p interact both in a direct and indirect fashion using importin {alpha} or other proteins as adapters.

Transport factor-cargo interaction is mediated by signals present in the cargoes. To date, only few nuclear localization sequences of proteins have been identified and characterized in detail (6). The leucine-rich nuclear export sequence (NES)3 is recognized by Xpo1p, the main exportin of proteins. The classical SV40 large tumor antigen (SV40TAg)-like nuclear localization signal (NLS), the first nuclear import signal to be identified, consists of a short basic peptide, while the related bipartite NLS contains two stretches of basic residues separated by ~10 linker residues. These NLSs are recognized by importin {alpha} and transported as a trimeric complex with importin β. The M9-like import signal, a signal sequence first identified in the heterogeneous human mRNA-binding protein hnRNP A1, is composed of basic and hydrophobic residues (Ref. 7 and references therein). Additional short sequences mediating direct nuclear import were identified within histone proteins (8, 9) and ribosomal proteins (9). In most cases, however, consensus sequences have not been deduced and prediction of nuclear localization signals other than the classical NLSs using bioinformatic methods remains a challenge.

In general, a single transport factor recognizes and associates with a subset of cargoes. Conversely, several essential proteins among them histones and ribosomal proteins were shown to use a group of nuclear import factors which may guarantee their proper localization (5). Kap114p, Kap95p, Kap123p, Pse1p, and Kap104p recognize import sequences present in histones H2A and H2B (8, 9), while Kap123p, Kap104p, and Pse1p cooperate in import of histones H3 and H4 (9, 10). Kap120p, Kap114p, and Nmd5p mediate import of Rpf1p (11), and several importins function redundantly in nuclear import of ribosomal proteins (12–14). Nevertheless, systematic approaches to determine those transport factors that functionally cooperate by binding to a specific NLS remain rare. The structural basis of transport factor-cargo recognition is also barely understood since, apart from cargo recognition by importin {alpha}, only three crystal structures of importin β-like transport factors in association with a cargo are currently available (3, 15–17).

We recently identified a nonessential S. cerevisiae protein called Asr1p for alcohol-sensitive Ring/PHD finger 1 protein (18). Asr1p constitutively shuttles between nucleus and cytoplasm but exhibits a steady state localization in the cytoplasm. Upon addition of alcohol to the extracellular medium, Asr1p rapidly and reversibly accumulates within the cell nucleus. In search of the mechanism that mediates nuclear import of Asr1p, we identified a short sequence within Asr1p-C that is sufficient and necessary to target a reporter protein to the nucleus and directly and functionally binds to Kap114p, Kap95p, Kap123p, Pse1p, and Kap104p. In vivo analysis confirmed that several transport receptors of the importin β family form a highly versatile system for nuclear import of Asr1p. Moreover, we found that the defined NLS is evolutionarily conserved. Based on mutagenesis of Asr1p243–293 and sequence comparison with a region mediating nuclear import of histone H2A (Ref. 8; research.nhgri.nih.gov/histones/), we identified a consensus NLS recognized by these five importins.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cells, Growth, Microbiological Techniques, and Plasmids— The yeast strains used in this work are listed in supplemental Table I (27–29). The KAP114, SXM1, NMD5, and KAP120 knock-out strains were constructed by replacement of DNA fragments containing the respective coding sequence by selectable markers (19) using double crossover integration. Plasmid fragments containing 5' and 3' regions of the KAP120 or NMD5 gene were used to transform a wild-type strain. In the KAP120 deletion strains, TRP1 or URA3 substitutes for a HindIII fragment of the KAP120 open reading frame. Likewise, the NMD5 null strains contain a replacement within the NMD5 coding sequence by TRP1. The SXM1 null strains contain a replacement within the SXM1 coding sequence by HIS3 and the KAP114 null strains contain a replacement within the KAP114 coding sequence by LEU2. Double or triple knock-outs (GSY519, GSY533, GSY537) were isolated after crossing the SXM1, KAP114, NMD5, and KAP120 null strains and subsequent tetrad dissection of the respective diploids. Yeast cells were grown in minimal SDC or YPD medium. Minimal SDC medium/plates contained all amino acids and nutrients except for those used for selection. HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. The plasmids used are listed in Table II (30).

The coding region of S. cerevisiae Asr1p or fragments thereof were amplified by PCR using specific 5' and 3' primers and genomic DNA isolated from the strain RS453n. To generate the ASR1 internal deletions, two PCR fragments were synthesized and ligated to produce a BamHI site. Chromosomal DNA serving as a template for the PCR amplification of the Candida albicans or Candida glabrata ASR1 homologue ALC1 was isolated from wild-type strains SC5314 (C. albicans) and CBS138 (C. glabrata) according to Hoffman and Winston (20). Briefly, cells were harvested by centrifugation for 3 min at 1.700 x g and washed once with ddH2O. One volume breaking buffer (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 2% Triton X-100, 1% SDS), 1 volume phenol:chloroform:isoamylalcohol (25:24:1; Sigma), and 2 volumes of glass beads were added to the pellets. Samples were vortexed for 3–4 min and then centrifuged for 5 min at high speed. The aqueous phase was recovered, and DNA was precipitated by the addition of EtOH (2.5 volumes) and subsequent centrifugation at high speed for 5 min. DNA was solubilized in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and RNA was degraded by incubation with RNase A for 5 min at 37 °C. DNA was precipitated as described above and solubilized in TE buffer. Specific 5' and 3' primers were used to amplify the 937-bp (C. glabrata) and 1665-bp (C. albicans) fragments by PCR. In general, constructs were verified by DNA sequencing, and expression of the fusion proteins was analyzed by Western blotting.

Fluorescence Microscopy—Expression and localization of GFP, GST-GFP, and GFP-β-galactosidase fusion proteins in yeast was done essentially as described before (21). Specifically, the wild-type W303 yeast strain, strains disrupted for ASR1 or certain importins, strains encoding conditionally temperature-sensitive importin mutants, or the exportin mutant xpo1-1 were transformed with plasmids encoding GFP, GST-GFP, or GFP-β-galactosidase fusion proteins. To analyze the GST-GFP-Asr1p243–293 kinetics of nuclear import, the inducible pGAL1 promoter was cloned upstream of the coding region. The cells were grown at 27 °C to a density of A600 0.5/ml and subsequently supplemented with 2% galactose for 2 h at 27°C to induce expression of the fusion protein. Strains carrying a temperature-sensitive mutation were induced and grown at 35 °C. The localization of the fusion proteins was determined by fluorescence microscopy. Cellular DNA was stained for 10 min by addition of 1 µg/ml 4',6'-diamidino-2-phenylindole (DAPI) to the cell suspension. Images were recorded on a Zeiss Axioskop fluorescence microscope with a Sony 3CCD color video camera using the Axiovision 3 software (Carl Zeiss AG, Oberkochen, Germany) and processed in Adobe Photoshop 4.

Expression, Purification, and Binding of Recombinant Proteins—Purification and subsequent binding assays with immobilized GST and GST fusion proteins were essentially performed as described (22). To solubilize GST-tagged importins or exportins, universal buffer (20 mM Hepes, pH 7.2, 100 mM KAc, 2 mM Mg(Ac)2, 0.1% Tween 20, 10% glycerin) was used. The plasmid encoding maltose-binding protein (MBP)-Asr1p243–280 was generated by an in frame fusion of the coding sequence to the 3' end of the MBP coding sequence and transformed into the ER2508 protease deficient bacterial strain. The cells were grown in LB medium containing 1 mM ampicillin and 2 g glucose/l at 37 °C to a density of A600 0.5/ml. Expression of the fusion protein was induced by addition of 1 mM isopropyl β-D-thiogalactopyranoside and further incubation for 2 h at 37 °C. The cells were harvested by centrifugation at 4,000 x g for 30 min. The pellet was resuspended in lysis buffer (20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 1 mM EDTA) containing Complete EDTA-free protease inhibitor (Roche Diagnostics) and lysed by sonification. The cell lysate was cleared by centrifugation at 10,000 x g for 30 min and subsequently incubated with 0.6 ml amylose resin for 1 h. After washing the resin with lysis buffer, MBP-Asr1p243–280 was eluted in buffer containing 10 mM maltose.

For binding assays, glutathione-Sepharose (GE Healthcare) was equilibrated with universal buffer. 12 µg GST, GST-importins, or GST-exportins were bound to 30 µl of GSH-Sepharose for 40 – 60 min at 4 °C. The total assay volume was 200 µl. The beads were washed with 1 ml of universal buffer and incubated with [35S]methionine labeled H2A, Asr1p, Asr1p-C212–310, or Asr1p-C lacking residues 243–275 (Asr1p-C{Delta}243–275) in vitro translated for 90 min in the TNT · Coupled Reticulocyte Lysate System (Promega). Alternatively, prebound GST or GST-importins were incubated with 12 µg of recombinantly expressed and purified MBP-Asr1p243–280. After 40 – 60 min of incubation at 4 °C the assays were centrifuged at 300 x g for 1 min, the supernatant was discarded, and the beads were washed three times in universal buffer. The functionality of the preformed dimeric complexes was tested by addition of 12 µg of Gsp1pGTP in buffer or of buffer alone and subsequent incubation for 1 h at 4°C. In case of H2A binding to GST-importins, the bound proteins were processed without further incubation. All the samples were washed three times with universal buffer, then 30 µlof2x concentrated Laemmli buffer were added, and incubation proceeded for 5 min at 98 °C. Bound proteins were analyzed by SDS-PAGE, subsequent Coomassie Brilliant Blue staining, and autoradiography or Western blotting using rabbit anti-MBP (New England Biolabs), goat anti-GST (Amersham Biosciences), and rabbit anti-Gsp1p antibodies followed by peroxidase-coupled goat anti-rabbit or rabbit anti-goat secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.; Ref. 23).


Figure 1
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  		FIGURE 1.
Asr1p is actively imported into the nucleus via its C-terminal domain. A, schematic drawing of the Asr1p constructs GFP-Asr1p-N2–211, GFP-Asr1p-C212–310, and GST-GFP-Asr1p-C212–310. B, fluorescence localization of GFP-Asr1p-N in the asr1{Delta} strain (panel a) and the xpo1-1 strain at the restrictive temperature (panel b), GFP-Asr1p-C (panel c), GST-GFP-Asr1p-C (panel d), and GST-GFP (panel e) in the asr1{Delta} strain.

 
Mutagenesis of Asr1p243–293—To insert mutations into the plasmid pRS425 encoding GST-GFP-Asr1p243–293, the QuikChange XL II mutagenesis kit (Stratagene) was used. In brief, two complementary antiparallel primers were designed, which both carried the desired mutation. The mutated plasmid was created and amplified by PCR. The PCR reaction was treated with DpnI to digest the non-mutated plasmids and subsequently transformed into ultracompetent XL10 gold bacterial cells. The cells were grown, and the plasmids were isolated, verified by DNA sequencing, SDS-PAGE, and Western blotting and transformed into the asr1{Delta} strain.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Asr1p Is Constitutively and Actively Imported into the Nucleus—Previous analysis indicated that under steady state conditions, Asr1p predominantly localizes to the cytoplasm but constitutively shuttles between nucleus and cytoplasm (18). In an xpo1-1 strain or upon alcohol stress, Asr1p accumulates in the nucleus. In search of the mechanism for nuclear import of Asr1p, we first performed a deletion analysis. Individual subdomains of Asr1p, the N-terminal Ring/PHD finger domain (residues 2–211) and the C-terminal domain (residues 212–310) fused to GFP were encoded by a 2µ plasmid (Fig. 1A). Like full-length Asr1p (18), GFP-Asr1p-N localized to the cytoplasm (Fig. 1B, panel a). When expressed in the temperature sensitive xpo1-1 strain, GFP-Asr1p-N containing the nuclear export sequences NES1 and NES2 localized to the cytoplasm at the restrictive temperature indicating that Asr1p-N does not harbor a constitutive nuclear import sequence (Fig. 1B, panel b). In contrast, GFP-Asr1p-C exclusively located to the nucleus (Fig. 1B, panel c). Nuclear import of Asr1p is an active process since fusion of Asr1p-C to GST-GFP resulted in a protein of ~65 kDa that is unable to diffuse through the nuclear pore but still located to the nucleus (Fig. 1B, panel d), while GST-GFP remained in the cytoplasm (Fig. 1B, panel e). Consistent with our previous data (18), we conclude that Asr1p actively and constitutively enters the nucleus via the C-terminal domain and is able to leave it when the N-terminal domain is present.

Asr1p-C Forms Import-competent Complexes with Kap114p, Kap95p, Kap123p, Pse1p, and Kap104p—The active import of GST-GFP-Asr1p-C into the nucleus indicated the presence of an NLS and suggested that it interacts with one or several transport factors of the importin β family. To analyze if this is the case and to identify the importins that interact with Asr1p-C in vitro, [35S]methionine-labeled myc-Asr1p-C212–310 synthesized in rabbit reticulocyte lysate was incubated with each of the GST-tagged importins prebound to GSH-Sepharose. To determine whether the release of Asr1p-C from the importins is Ran-dependent, functional Gsp1pGTP (9) was added to the preformed importin-Asr1p-C complexes. Following incubation, the bound proteins were eluted and analyzed by SDS-PAGE and autoradiography (Fig. 2A). After quantification of the importin-bound Asr1p-C and normalization to Asr1p-C bound to GST-Cse1p, we found that Asr1p-C interacted with a number of different importins. Asr1p-C strongly bound to Kap114p (Fig. 2A, lane 1), Kap95p (lane 3), Kap123p (lane 5), Pse1p (lane 7), and Kap104p (lane 9). Weak binding was observed to Sxm1p (lane 11) and Nmd5p (lane 13), and essentially no binding occurred with the other importins (lanes 15, 17, 19, and 21). Very weak binding of Asr1p-C was observed to the exportin Cse1p (lane 23). Addition of Gsp1pGTP to the preformed importin-Asr1p-C complexes efficiently released Asr1p-C from Kap114p (lane 2), and Kap95p (lane 4), and to a lesser extent from Kap123p (lane 6), Pse1p (lane 8), and Kap104p (lane 10). The same five importins also interact with full-length Asr1p as is shown by incubation of [35S]methionine-labeled Asr1p with GST-tagged Kap114p (Fig. 2B, lane 1), Kap95p (lane 2), Kap123p (lane 3), Pse1p (lane 4), and Kap104p (lane 5). Consistent with NES sequences previously identified in the N-terminal part of Asr1p (18), full-length Asr1p cooperatively interacts with Xpo1p and Gsp1pGTP (Fig. 2B, lanes 7 and 8), while essentially no binding was observed with GST alone (lane 6).

Next we analyzed whether binding of Asr1p to Kap95p is independent of the adapter protein importin {alpha}/Srp1p (Fig. 2C). [35S]Methionine-labeled Asr1p only weakly bound to GST-Srp1p (lane 1) or GST alone (lane 4). Binding of Asr1p to GST-Kap95p is independent of Srp1p (lane 2) and was unaltered by addition of Srp1p (lane 3), which indicates that Asr1p contains an NLS that differs from the lysine-rich SV40 TAg-like NLS. We thus conclude that Asr1p forms specific and import-competent complexes with several transport factors of the importin β family.


Figure 2
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  		FIGURE 2.
Importins Kap114p, Kap95p, Kap123p, Pse1p, and Kap104p build functional complexes with Asr1p. A,12 µg of the yeast GST-tagged importins Kap114p, Kap95p, Kap123p, Pse1p, Kap104p, Sxm1p, Nmd5p, Msn5p, Pdr6p, Mtr10p, and Kap120p (lanes 1–22) or the exportin Cse1p (lane 23) were immobilized on glutathione-Sepharose and incubated for 45 min at 4 °C with [35S]methionine-labeled ([35S]Met) Asr1p-C synthesized in rabbit reticulocyte lysate (load, lane 24). After washing, the protein complexes were incubated for 45 min at 4 °C with buffer alone (lanes 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23) or with 12 µg of Gsp1pGTP (lanes 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22). The bound [35S]Met Asr1p-C was analyzed by SDS-PAGE and autoradiography, quantified, and normalized to Asr1p-C bound to GST-Cse1p as shown in the diagram (%). The load corresponds to 6% of the proteins used for binding per lane. B,12 µg of GST-tagged importins Kap114p, Kap95p, Kap123p, Pse1p, Kap104p (lanes 1–5), GST alone (lane 6), or the GST-tagged exportin Xpo1p (lanes 7 and 8) were immobilized on glutathione-Sepharose and incubated for 45 min at 4 °C with full-length [35S]Met Asr1p synthesized in rabbit reticulocyte lysate (load, lane 9) or with additional His6-Gsp1pGTP (lane 8). After washing, the bound proteins were analyzed by SDS-PAGE (top panel) and autoradiography (lower panel). The position of the GST band (marked by an asterisk) does not reflect its migration in SDS-PAGE. The load corresponds to 20% of the proteins used for binding per lane. C,12 µg of GST-tagged Srp1p (lane 1), GST-Kap95p (lanes 2 and 3), or GST (lane 4) were immobilized as described in the legend to Fig. 2B. After washing, the proteins were incubated for 45 min at 4 °C with full-length [35S]Met Asr1p alone (lanes 1, 2, and 4) or in addition with 12 µgofHis6-Srp1p (lane 3). The bound Asr1p was analyzed as indicated in Fig. 2B. The position of the GST band (marked by an asterisk) does not reflect its migration in SDS-PAGE.

 
Asr1p-C Harbors a Short Sequence Sufficient and Necessary for Active Nuclear Import—To determine the sequence required for nuclear import of Asr1p-C, plasmids encoding N- and C-terminal deletions of Asr1p-C fused to GST-GFP were expressed in the asr1{Delta} strain (Fig. 3A). GST-GFP-Asr1p-C212–310 and N-terminal deletions thereof up to residue 233 allowed complete nuclear import (Fig. 3B). GST-GFP-Asr1p243–293 or GST-GFP-Asr1p243–280 where additional N- and C-terminal residues were deleted still localized to the nucleus. The identified residues 243–280 are able to mediate active nuclear import of a protein larger than 60 kDa, as was shown by fusion of residues 243–280 to the C-terminal part of GFP-β-galactosidase (GFP-β-gal).

Further C-terminal deletion of GST-GFP-Asr1p243–280 resulting in GST-GFP-Asr1p243–272 or GST-GFP-Asr1p243–254 led to fusion proteins compromised in nuclear import indicating that residues 255–280 are important for efficient nuclear transport. Deletion analysis of the N-terminal part of the import sequence showed that GST-GFP-Asr1p250–280 has residual import activity, while GST-GFP-Asr1p262–280 and GST-GFP-Asr1p265–275 were located in the cytoplasm.

To find out whether the determined nuclear import signal of Asr1p is necessary for its nuclear localization, we deleted residues 243–275 or residues 265–275 in the context of the full-length GFP-Asr1p. As is shown in Fig. 3C, both internal deletion mutants were impaired in nuclear import when expressed in the xpo1-1 mutant at restrictive temperature. Taken together we thus have identified a short sequence within Asr1p-C comprising residues 243–280 that is sufficient and necessary to mediate active nuclear import.

Mutational Analysis of the Asr1p Nuclear Localization Sequence—Comparison of this minimal NLS of Asr1p with corresponding sequences within orthologues of other yeasts revealed two motifs containing a number of conserved charged or hydrophobic residues (Fig. 4A) that in most homologues are separated by a diverse linker consisting of proline and leucine residues. Structural analysis predicts that Asr1p243–293 formstwoshort{alpha} helicesrotatedaroundalinkerregion(Predict-Protein software).


Figure 3
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  		FIGURE 3.
Asr1p243–280 is sufficient and necessary to mediate nuclear import. A, schematic diagram of truncated Asr1p proteins fused to GST-GFP or GFP-β-gal. B, subcellular localization of GST-GFP-Asr1p-C212–310, GST-GFP-Asr1p224–310, GST-GFP-Asr1p233–310, GST-GFP-Asr1p243–293, GST-GFP-Asr1p243–280, GFP-β-gal243–280, GFP-β-gal, GST-GFP-Asr1p243–272, GST-GFP-Asr1p243–254, GST-GFP-Asr1p250–280, GST-GFP-Asr1p262–280, and GST-GFP-Asr1p265–275 in the asr1{Delta} cells grown in liquid media to exponential phase. Subcellular localization of the expressed fusion proteins was analyzed by fluorescence microscopy. C, subcellular localization of GFP-Asr1p{Delta}243–275, GFP-Asr1p{Delta}265–275, and GFP-Asr1p in the xpo1-1 strain at the restrictive temperature of 35 °C.

 
To determine the residues critical for nuclear import of Asr1p243–293, single, double, or triple point mutations were introduced into the plasmid encoding GST-GFP-Asr1p243–293 as described under "Experimental Procedures." GST-GFP-Asr1p243–293 expressed in the asr1{Delta} strain still localized to the nucleus when both isoleucine 243 and glutamine 244 were changed to alanines (Fig. 4B). No mislocalization was observed for the mutants H246A/R248A and L251A/Y254A indicating that although highly conserved, these residues seem not to be critically involved in nuclear import. Mutagenesis of proline 255 in the linker region led to mislocalization of Asr1p243–293 to the plasma membrane, a behavior observed for several Asr1p-C deletion mutants. In contrast, mutant P257A was not impaired in nuclear import. The double proline mutant P255A/P257A showed localization to both the plasma membrane and the nucleus supporting the notion that the presence of these prolines is not critical for nuclear import. Mutational analysis of the second conserved motif showed that the mutant D263A/Y265A/K266A was unaltered in its localization compared with the wild-type peptide. Consistent with the import defect of the deletion mutant 243–272 (Fig. 3B), mutagenesis of residues Ser273/Arg274 or Arg274/Lys275 into alanines led to reduced nuclear import activity indicating that these residues and in particular Arg274 is critical for a functional NLS. Thus Asr1p contains a short NLS characterized by at least one basic residue.

The Nuclear Localization Sequence of Asr1p Directly and Functionally Interacts with Several Importins—To determine whether the import factors Kap114p, Kap95p, Kap123p, Pse1p, and Kap104p previously found to interact with Asr1p-C recognize the identified NLS, Asr1p243–280 was expressed in bacteria as a fusion with MBP and purified to homogeneity. The importins Kap114p, Kap95p, Kap123p, Pse1p, Kap104p, the exportin Cse1p fused to GST, or GST alone were prebound to GSH-Sepharose and incubated with MBP-Asr1p243–280. To determine the functionality of the Asr1p243–280-importin complexes, Gsp1pGTP was added to the preformed cargo-importin complexes. Following incubation, the bound proteins were eluted and analyzed by SDS-PAGE and autoradiography (Fig. 5A). MBP-Asr1p243–280 bound to GST-Kap114p (lane 1), -Kap95p (lane 3), -Kap123p (lane 5), -Pse1p (lane 7), and -Kap104p (lane 9), while no binding to GST-Cse1p (lane 11) or GST alone (data not shown) was observed. Binding of MBP-Asr1p243–280 to all GST-tagged importins was sensitive to the addition of Gsp1pGTP (lanes 2, 4, 6, 8, and 10) indicative of a direct and functional interaction of the Asr1p NLS with five different importins.

To analyze whether Asr1p requires this NLS for binding to the importins, [35S]methionine-labeled wild-type Asr1p-C and Asr1p-C where residues 243–275 were deleted were synthesized in rabbit reticulocyte lysate and tested for binding to GST-Kap114p, GST-Kap95p, and GST (Fig. 5B). Consistent with the inability of the NLS deletion mutants Asr1p{Delta}243–275 and Asr1p{Delta}265–275 to enter the nucleus in vivo (Fig. 3C), the mutant lacking the NLS was unable to bind to either importin (Fig. 5B, lanes 2 and 4). This demonstrates that the nuclear import of Asr1p crucially depends on the identified NLS.


Figure 4
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  		FIGURE 4.
Mutagenesis of the Asr1p nuclear localization sequence. A, the determined S. cerevisiae Asr1p nuclear localization sequence (residues 243–280) is highly conserved in the yeast species Saccharomyces bayanus, Saccharomyces mikatae, Saccharomyces pastorianus, Saccharomyces castelli, Saccharomyces kluyveri, Saccharomyces kudriavzevii. B, selected amino acids conserved in other yeasts were replaced by alanine in GST-GFP-Asr1p243–293. Mutated GST-GFP-fusion proteins were expressed in logarithmically growing asr1{Delta} cells, and the subcellular localization was analyzed by fluorescence microscopy (panels a, c, e, g, i, k, m, o, q, and s) or by phase contrast (panels b, d, f, h, j, l, n, p, r, and t).

 
In Vivo, Nuclear Import of Asr1p Is Mediated by Several Transport Receptors of the Importin β Family— To analyze the in vivo requirements for nuclear import of Asr1p, a plasmid encoding GST-GFP-Asr1p-C243–293 under the control of the GAL1 promoter was generated and introduced into the W303 strain. To analyze the galactose-induced expression of GST-GFP-Asr1p243–293, whole cell extracts prepared prior to, or 30, 60, 90, and 180 min after, induction were analyzed by SDS-PAGE and subsequent Western blotting (Fig. 6A). Expression of GST-GFP-Asr1p243–293 could be detected 30 min after galactose addition and reached a maximum between 90 and 180 min of induction. Two hours after galactose addition, GST-GFP-Asr1p243–293 expressed in the W303 strain co-localized with DAPI staining indicative of its constitutive and efficient nuclear import (Fig. 6A). We thus used these conditions for further analysis of nuclear import of GST-GFP-Asr1p243–293.

To determine which members of the importin β family are involved in nuclear import of Asr1p, strains defective in one or several importins were transformed with the plasmid encoding GST-GFP-Asr1p243–293 and tested for import of the fusion protein following 2 h of galactose induction (Fig. 6B). When expressed in the rsl1-1 strain, which encodes a temperature-sensitive mutant of Kap95p, GST-GFP-Asr1p243–293 readily localized to the nucleus at the permissive temperature but was partially mislocalized to the cytoplasm upon shift of the cells to the restrictive temperature of 35 °C. The same was true for the temperature-sensitive mutant of PSE1, pse1-1. Strains harboring a pse1-1 mutant in addition to a deletion of KAP114 or KAP123 showed a slight cytoplasmic retention already at the permissive temperature indicative of the overlap in import activity of Pse1p with Kap114p or Kap123p. Single deletion of KAP114 or KAP123 or double deletion of both genes did not lead to mislocalization of GST-GFP-Asr1p243–293 at 27 °C, whereas single deletion of KAP114 or KAP123 cultured at 35 °C led to weaker nuclear import. In contrast, kap114{Delta}/kap123{Delta} cells grown at 35 °C showed significant mislocalization of GST-GFP-Asr1p243–293 to the cytoplasm suggesting that these importins cooperate in import of Asr1p. Other deletion strains where KAP114 was deleted in conjunction with SXM1, KAP120, or both SXM1 and NMD5, did not show any cytoplasmic mislocalization of GST-GFP-Asr1p243–293, neither did the sxm1{Delta}/nmd5{Delta}/kap120{Delta} strain. Thus, consistent with complex formation of Asr1p, Asr1p-C, and Asr1p243–293 with Kap114p, Kap95p, Kap123p, Pse1p, and Kap104p as well as dissociation of the formed dimeric complexes by Gsp1pGTP, the nonessential Asr1p uses several importins for in vivo localization to the nucleus.

The Mechanism of Asr1p Nuclear Import Is Conserved from Yeast to Human—Two previously undescribed proteins identified in the genome of C. albicans and C. glabrata encoded by the C. albicans open reading frame IPF9048.1 (orf19.1.2229) and C. glabrata open reading frame CATG61534.1 are likely to represent the putative orthologues of S. cerevisiae ASR1. The corresponding proteins will hereafter be called C. albicans or C. glabrata Alc1p for ASR1-like proteins in Candida. Asr1p encoded by YPR093C shows 33.7% homology with the C. albicans Alc1p and 64.4% homology with the C. glabrata Alc1p (ClustalW program and data not shown). A region highly homologous to Asr1p243–293 could be identified in residues 473–534 of C. albicans Alc1p and residues 240–289 of C. glabrata Alc1p (Fig. 4A).


Figure 5
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  		FIGURE 5.
A, importins Kap114p, Kap95p, Kap123p, Pse1p, and Kap104p form direct and functional complexes with Asr1p243–280. 12 µg of GST-tagged importins Kap114p, Kap95p, Kap123p, Pse1p, Kap104p (lanes 1–10) or the GST-tagged exportin Cse1p (lane 11) were immobilized on glutathione-Sepharose and incubated for 45 min at 4 °C with 12 µg MBP-Asr1p243–280 (lane 12). After washing, the protein complexes were incubated for 45 min at 4 °C with buffer alone (lanes 1, 3, 5, 7, 9, and 11) or with 12 µg of Gsp1pGTP (lanes 2, 4, 6, 8, and 10). The bound proteins were analyzed by SDS-PAGE (top panel) and Western blotting (lower panel) with rabbit anti-MBP antibodies followed by peroxidase-coupled goat anti-rabbit secondary antibodies. The bound MBP-Asr1p243–280 was quantified, normalized to MBP-Asr1p243–280 bound to GST-Cse1p, and shown in the diagram (%). B, the Asr1p NLS is required for binding to the importins Kap114p and Kap95p. 12 µg of GST-Kap114p (lanes 1 and 2), GST-Kap95p (lanes 3 and 4), or GST (lanes 5 and 6) were incubated with wild-type [35S]Met Asr1p-C (lanes 1, 3, and 5) or Asr1p-C lacking residues 243–275 (lanes 2, 4, and 6) synthesized in rabbit reticulocyte lysate (load, lanes 7 and 8, respectively). Incubation and analysis was performed as described in the legend to Fig. 2. The position of the GST band (marked by an asterisk) does not reflect its migration in SDS-PAGE. The load corresponds to 30% of the proteins used for binding per lane.

 
To determine the subcellular localization and the nuclear import sequence of the C. albicans and C. glabrata Alc1p, the sequences encoding the full-length proteins, the C-terminal part, and the regions homologous to Asr1p243–293 were amplified by PCR using C. albicans and C. glabrata genomic DNA as template and cloned in frame to a GST-GFP coding sequence. Heterologous expression of these fusion proteins in the S. cerevisiae asr1{Delta} strain showed that full-length C. albicans Alc1p constitutively localized to the nucleus while C. glabrata Alc1p was found in the cytoplasm (Fig. 7B and data not shown). The C-terminal domain of both proteins and the regions homologous to Asr1p243–293 were exclusively located to the nucleus (Fig. 7B and data not shown). Thus, the nuclear localization sequence of Asr1p is conserved in distantly related fungi.

Next, Asr1p was tested for localization in higher eucaryotes. A plasmid encoding EGFP3-Asr1p-C was generated and transiently expressed in HeLa cells (Fig. 7C). A plasmid encoding EGFP3 was used as control. As is shown in Fig. 7D, EGFP3-Asr1p-C fully localized to the nuclear interior indicating that Asr1p carries a nuclear localization sequence recognized in higher eucaryotic cells.

Histone H2A and Asr1p Share an NLS Bound to the Importins Kap114p, Kap95p, Kap123p, Pse1p, and Kap104p—Previously, it was shown that histone H2A interacts with a similar set of transport factors like Asr1p. One group observed binding of GST-H2A to His6-tagged importins Kap114p, Pse1p, and Kap95p (8), while another group demonstrated additional binding to Kap123p and Kap104p (9). Moreover, in vitro synthesized H2A bound to the GST-tagged importins Kap114p, Kap123p, Pse1p, and Kap104p but failed to interact with Kap95p (9). To reinvestigate this second approach, [35S]methionine-labeled H2A synthesized in rabbit reticulocyte lysate was incubated with GST-tagged importins Kap114p, Kap95p, Kap123p, Pse1p, and Kap104p or GST prebound to GSH-Sepharose. Following incubation, the bound proteins were eluted and analyzed by SDS-PAGE and autoradiography (Fig. 8A). The results show that H2A specifically binds to all five importins that interact with Asr1p.


Figure 6
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  		FIGURE 6.
Yeast importins Kap114p, Kap95p, Kap123p, and Pse1p mediate in vivo nuclear import of Asr1p243–293. A, logarithmically growing W303 cells, carrying a plasmid encoding GST-GFP-Asr1p243–293, were cultured in liquid medium. Expression of the fusion protein was induced by addition of 2% galactose for 0, 30, 60, 90, or 180 min at 27 °C and analyzed by SDS-PAGE and Western blotting using rabbit anti-GFP antibodies followed by goat anti-rabbit peroxidase-coupled antibodies. Localization of the fusion proteins was analyzed before or after galactose induction for 2 h at 27°C by fluorescence microscopy. B, yeast strains deleted for one, two, or three nonessential importins or containing a combination of deleted importins and a temperature-sensitive mutation (ts) in an essential importin were transformed with a plasmid encoding GST-GFP-Asr1p243–293. Expression of the fusion protein was induced by addition of 2% galactose for 2 h either at 27 °C or at 35 °C. Localization of the fusion proteins was analyzed by fluorescence microscopy.

 
Since both H2A and Asr1p-C are able to bind the same transport factors, it is very likely that they contain comparable nuclear localization signals. Previous experiments showed that the N-terminal part of S. cerevisiae H2A harbors an NLS and that the core of this import sequence is located in residues 24–46 (8). To aim at identifying residues important for nuclear import of these proteins, residues 15–46 of S. cerevisiae H2A and homologous regions found in H2A of other organisms were selected (research.nhgri.nih.gov/histones/), aligned, and compared with the alignment of the import sequence identified in S. cerevisiae Asr1p and C. albicans/C. glabrata Alc1p (Fig. 8B). The N-terminal motif of Asr1p243–280 matched well with sequences of related fungal proteins (Fig. 4A) but showed only poor alignment with the N-terminal part of H2A15–46. In contrast, residues 265–274 of Asr1p located in the C-terminal motif of the identified import sequence and residues 29–37 of H2A could be aligned with matching hydrophobic and basic residues. Most strikingly, secondary structure prediction revealed a high probability of both regions to form an {alpha} helix (PredictProtein software), and formation of an {alpha} helix of these H2A residues was confirmed by crystallization (24). Based on mutational analysis (this study and Ref. 8), on sequence alignment, and on secondary structure prediction, we propose the consensus sequence R/KxxL(x)nV/YxxV/IxK/RxxxK/R for a novel NLS recognized by Kap114p, Kap95p, Kap123p, Pse1p, and Kap104p where the core of the import sequence may be composed of a short {alpha} helix.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have identified a novel cargo that is transported into the nucleus by at least five importins, which shows that these importins functionally overlap in nuclear protein import. Our comparative co-precipitation experiments showed that Asr1p binds specifically, directly, independent of an adapter protein, and in a functional manner to Kap114p, Kap95p, Kap123p, Pse1p, and Kap104p. Release of Asr1p from its importins by RanGTP is incomplete and may involve additional factors (9). Strains carrying mutations or deletions in the respective importin genes show defects in nuclear import of Asr1p243–293 in vivo. Strikingly, the identified NLS of Asr1p is evolutionarily conserved and recognized in organisms from yeast to mammals.

The NLS of Asr1p, sufficient and necessary for complete nuclear import, consists of 38 residues and contains two conserved motifs seperated by a non-conserved linker region. NLS fragments shortened N- and C-terminally are unable to efficiently import a reporter protein and mutagenesis of the C-terminal half of the NLS leads to import defects. This suggests that both halves of the Asr1p NLS are required for efficient nuclear import and that this NLS is bipartite in nature. However, although further N-terminal deletion is not tolerated, neither of the double point mutations located within residues 243–255 impaired nuclear import. Thus, the mutated residues could act redundantly with neighboring residues or their replacement with alanine does not sufficiently interfere with their import function. Alternatively, this N-terminal region may not carry information essential for nuclear import but contribute to correct folding of the more C-terminally located motif of the NLS. Mutagenesis of that region showed that the mutants S273A/R274A and R274A/K275A were impaired in nuclear import, while the mutant D263A/Y265A/K266A was functional. Consistent with the observed import defect of the deletion mutant 243–272, these data hint toward a crucial role of Arg274. Neither of the prolines in the linker region seem to be important for nuclear import. Taken together our data show that Asr1p-C contains a short NLS characterized by at least one positively charged residue.

Several cargoes transported by the importins Kap114p, Kap95p, Kap123p, Pse1p, and Kap104p have been identified so far (5); however, only a few studies showed which of these importins cooperate in transport of a single cargo. Moreover, the information often is incomplete since the sequence within a protein that mediates interaction remains undetermined, and rigorous evidence for a direct interaction between importin and cargo is not provided. The core histones H2A and H2B however seem to use a set of importins similar to Asr1p: Pemberton's group (8) showed that Kap114p, Pse1p, and Kap95p directly interact with the NLSs of both histones H2A and H2B. A comparative approach showed that both H2A and H2B independently interact with Kap114p, Kap123p, Pse1p, Kap104p, and Kap95p (9). We confirmed here that H2A indeed interacts with the same transport factors as Asr1p-C. This suggested that Asr1p and H2A contain similar nuclear transport sequences recognized by all five importins. The NLS of histone H2A is comprised of residues 1–46 (8, 9) with the main nuclear import activity located within residues 24–46. For nuclear import of Asr1p residues 243–280 are sufficient; however, N-terminally truncated versions of the Asr1p NLS at least partially localize to the cell nucleus. Thus both proteins contain a core nuclear import activity that is positively influenced by N-terminally flanking residues. We now searched for matching residues between Asr1p and H2A. At least two conserved residues were found in the N-terminal motif of the NLS, and several hydrophobic and positively charged residues located in the C-terminal motif could be aligned and showed nearly identical spacing. This includes Arg274 of Asr1p that we determined to be crucial for full import activity. Most interestingly, the aligned regions of both proteins show high probability to form an {alpha} helix, and crystallization of H2A revealed an {alpha} helical conformation of this motif (Ref. 24; Protein Data Bank code 1ID3 [PDB] ). Consistent with an import function of the H2A region, this {alpha} helical region is exposed both in the H2A monomer and the nucleosomal core octamer.


Figure 7
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  		FIGURE 7.
The Asr1p243–293 nuclear localization sequence is conserved. A, schematic drawing of GST-GFP fusions to the Asr1p homologue of C. albicans. B, subcellular localization of C. albicans GST-GFP-Alc1p1–548 (panel a), C. albicans GST-GFP-Alc1p316–548 (panel c), and C. albicans GST-GFP-Alc1p473–534 (panel e) expressed in the S. cerevisiae asr1{Delta} cells was analyzed by fluorescence microscopy (panels a, c, and e) or by phase contrast (panels b, d, and f). C, schematic drawing of EGFP3 and EGFP3-Asr1p-C. D, subcellular localization of EGFP3 and EGFP3-Asr1p-C. HeLa cells were transfected with plasmids coding for EGFP3-Asr1p-C (panel d) and incubated for 18 h at 37 °C. For control the plasmid pEGFP3 was used (panel a). The cells were analyzed by fluorescence microscopy for GFP (panels a and d), for DAPI staining (panels b and e), and by phase contrast (panels c and f).

 
Based on the experimental data gained by us and published before (8, 9), we deduced the consensus sequence R/KxxL(x)nV/YxxV/IxK/RxxxK/R that mediates interaction with the transport factors Kap114p, Kap95p, Kap123p, Pse1p, and Kap104p. Previously identified and characterized NLSs include the classical NLS composed of one or two stretches of basic residues, an arginine-rich sequence found in HIV-1 Rev and related proteins (9) and the M9NLS with an overall basic character (Ref. 7 and references therein). The importin β-binding domain of importin {alpha}-like proteins represents a specialized import sequence (3). Structural analysis showed that neither the classical NLS (25) nor the M9-like sequence or the importin β-binding domain are structured (3, 7). In contrast, the major part of both the H2A and the Asr1p NLS is predicted to adopt a short alpha helical conformation (Ref. 24, PredictProtein software; Protein Data Bank code 1ID3 [PDB] ). We thus have identified a novel type of nuclear localization sequence.

In general, the redundancy of nuclear import mechanisms is interpreted to be crucial for essential nuclear cargoes. However, Asr1p is nonessential under normal growth conditions and still uses several importins for nuclear import. This suggests that the reason for evolution of redundancy is not necessarily the function of the cargo. Groups of importins could cooperate because they are structurally related, enabling their binding to similar NLSs. At the same time NLSs could be conformationally flexible. To gain insight into these questions, crystallization of all import factors in conjunction with various cargoes is required.


Figure 8
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  		FIGURE 8.
H2A and Asr1p share a consensus NLS recognized by the importins Kap114p, Kap95p, Kap123p, Pse1p, and Kap104p. A, to determine whether H2A interacts with the importins Kap114p, Kap95p, Kap123p, Pse1p, and Kap104p, 12 µg of GST-tagged importins (lanes 1–5) or 12 µg of GST (lane 6) were immobilized on GSH-Sepharose and incubated for 45 min at 4 °C with [35S]Met H2A synthesized in rabbit reticulocyte lysate. The bound [35S]Met Asr1p-C was analyzed by SDS-PAGE and autoradiography. B, the regions of Homo sapiens, Mus musculus, Danio rerio, Caenorhabditis elegans, Schizosaccharomyces pombe, C. albicans, and C. glabrata H2A homologues to residues 15–46 of S. cerevisiae H2A (HTA1) were retrieved from research.nhgri.nih.gov/histones/ (26) and aligned with the NLS of S. cerevisiae Asr1p, C. albicans Alc1p, and C. glabrata Alc1p. The derived consensus sequence is shown on the bottom.

 
To conclude, this analysis not only increases our knowledge of Asr1p and its subcellular dynamics, but more importantly the identification of a novel cargo and a consensus NLS recognized by five different importin β-like transport factors is an important contribution to a more complete picture of nucleo-cytoplasmic transport in general. Most recent data show that not only the classical NLS but also the M9-like sequence is predictable (7). We therefore believe that our study will promote the in silico prediction of non-classical import sequences.


   FOOTNOTES
 
* This work was supported by grants (to S. M. B.) from the Deutsche Forschungsgemeinschaft (Ba 1165/3-2), the Forschungsausschuβ (61 CL/TG84), and the HOMFOR program 2005 of the Universität des Saarlandes. 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. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables I and II. Back

1 These two authors contributed equally to this project. Back

2 To whom correspondence should be addressed. Tel.: 49-6841-16-47909; Fax: 49-6841-16-26027; E-mail: dr.susanne.bailer{at}med-rz.uni-saarland.de.

3 The abbreviations used are: NES, nuclear export sequence; NLS, nuclear localization signal; GST, glutathione S-transferase; GFP, green fluorescent protein; DAPI, 4',6'-diamidino-2-phenylindole; β-gal, β-galactosidase; MBP, maltose-binding protein. Back


   ACKNOWLEDGMENTS
 
The vector pEGFP3 was kindly provided by J. Ellenberg, EMBL, Heidelberg, Germany. Many thanks go to the members of the Schlenstedt, the Zimmermann, and the Montenarh laboratories, Universität des Saarlandes, Homburg, Germany, for support and suggestions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Strom, A., and Weis, K. (2001) Genome Biol. 2, 1–9[Medline] [Order article via Infotrieve]
  2. Weis, K. (2002) Curr. Opin. Cell Biol. 14, 328–335[CrossRef][Medline] [Order article via Infotrieve]
  3. Madrid, A., and Weis, K. (2006) Chromosoma (Berl.) 115, 98–109[CrossRef]
  4. Harel, A., and Forbes, D. (2004) Mol. Cell 16, 319–330[Medline] [Order article via Infotrieve]
  5. Mosammaparast, N., and Pemberton, L. (2004) Trends Cell Biol. 14, 547–556[CrossRef][Medline] [Order article via Infotrieve]
  6. Jans, D., Xiao, C., and Lam, M. (2000) Bioessays 22, 532–544[CrossRef][Medline] [Order article via Infotrieve]
  7. Lee, B., Cansizoglu, A., Suel, K., Louis, T., Zhang, Z., and Chook, Y. (2006) Cell 126, 543–558[CrossRef][Medline] [Order article via Infotrieve]
  8. Mosammaparast, N., Jackson, K. R., Guo, Y., Brame, C. J., Shabanowitz, J., Hunt, D. F., and Pemberton, L. F. (2001) J. Cell Biol. 153, 251–262[Abstract/Free Full Text]
  9. Greiner, M., Caesar, S., and Schlenstedt, G. (2004) Eur. J. Cell Biol. 83, 511–520[CrossRef][Medline] [Order article via Infotrieve]
  10. Mosammaparast, N., Guo, Y., Shabanowitz, J., Hunt, D. F., and Pemberton, L. F. (2002) J. Biol. Chem. 277, 862–868[Abstract/Free Full Text]
  11. Caesar, S., Greiner, M., and Schlenstedt, G. (2006) Mol. Cell. Biol. 26, 3170–3180[Abstract/Free Full Text]
  12. Schlenstedt, G., Smirnova, E., Deane, R., Solsbacher, J., Kutay, U., Görlich, D., Ponstingl, H., and Bischoff, F. R. (1997) EMBO J. 16, 6237–6249[CrossRef][Medline] [Order article via Infotrieve]
  13. Rout, M. P., Blobel, G., and Aitchison, J. D. (1997) Cell 89, 715–725[CrossRef][Medline] [Order article via Infotrieve]
  14. Jäkel, S., and Görlich, D. (1998) EMBO J. 17, 4491–4502[CrossRef][Medline] [Order article via Infotrieve]
  15. Cingolani, G., Bednenko, J., Gillespie, M., and Gerace, L. (2002) Mol. Cell 10, 1345–1353[CrossRef][Medline] [Order article via Infotrieve]
  16. Lee, S., Sekimoto, T., Yamashita, E., Nagoshi, E., Nakagawa, A., Imamoto, N., Yoshimura, M., Sakai, H., Chong, K., Tsukihara, T., and Yoneda, Y. (2003) Science 302, 1571–1575[Abstract/Free Full Text]
  17. Matsuura, Y., and Stewart, M. (2004) Nature 432, 872–877[CrossRef][Medline] [Order article via Infotrieve]
  18. Betz, C., Schlenstedt, G., and Bailer, S. M. (2004) J. Biol. Chem. 279, 28174–28181[Abstract/Free Full Text]
  19. Jones, J. S., and Prakash, L. (1990) Yeast 6, 363–666[CrossRef][Medline] [Order article via Infotrieve]
  20. Hoffman, C., and Winston, F. (1987) Gene (Amst.) 57, 267–272[CrossRef][Medline] [Order article via Infotrieve]
  21. Bailer, S. M., Balduf, C., Katahira, J., Podtelejnikov, A., Rollenhagen, C., Mann, M., Pante, N., and Hurt, E. (2000) J. Biol. Chem. 275, 23540–23548[Abstract/Free Full Text]
  22. Solsbacher, J., Maurer, P., Bischoff, F. R., and Schlenstedt, G. (1998) Mol. Cell. Biol. 18, 6805–6815[Abstract/Free Full Text]
  23. Maurer, P., Redd, M., Solsbacher, J., Bischoff, F. R., Greiner, M., Podtelejnikov, A. V., Mann, M., Stade, K., Weis, K., and Schlenstedt, G. (2001) Mol. Biol. Cell 12, 539–549[Abstract/Free Full Text]
  24. White, C. L., Suto, R. K., and Luger, K. (2001) EMBO J. 20, 5207–5218[CrossRef][Medline] [Order article via Infotrieve]
  25. Conti, E., and Kuriyan, J. (2000) Structure (Camb.) 8, 329–338[Medline] [Order article via Infotrieve]
  26. Sullivan, S. A., Aravind, L., Makalowska, U., Baxevanis, A. D., and Landsman, D. (2000) Nucleic Acids Res. 28, 320–322[Abstract/Free Full Text]
  27. Künzler, M., Gerstberger, T., Stutz, F., Bischoff, F. R., and Hurt, E. (2000) Mol. Cell. Biol. 20, 4295–4308[Abstract/Free Full Text]
  28. Seedorf, M., and Silver, P. A. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8590–8595[Abstract/Free Full Text]
  29. Koepp, D. M., Wong, D. H., Corbett, A. H., and Silver, P. A. (1996) J. Cell Biol. 133, 1163–1176[Abstract/Free Full Text]
  30. Daigle, N., Beaudouin, J., Hartnell, L., Imreh, G., Hallberg, E., Lippincott-Schwartz, J., and Ellenberg, J. (2001) J. Cell Biol. 154, 71–84[Abstract/Free Full Text]

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