Nuclear Import of Zinc Binuclear Cluster Proteins Proceeds through Multiple, Overlapping Transport Pathways

Strains and growth conditions. A. nidulans (ya2 pabaA1 uaZ11) was used as a host for expression of the full-length AlcR-green fluorescent protein (GFP) fusion whose localization was monitored in the absence or presence of the inducer. The wild-type strain was used as a reference in plate growth tests. A. nidulans strain MF115 (ya2 pabaA1 uaZ11 alc500 palcA::argB2_BglII) served as a recipient for transformation of all other alcR-bearing constructs. Only single-copy transformants integrated at the ectopic uaZ locus were selected for subsequent microscopic studies and transcriptional analysis. For RNA extraction, mycelia were grown under conditions described earlier (38). In plate growth tests, the fungus was grown for 2 days on solid minimal medium containing either 1% glycerol or 1% glycerol plus10 mM allyl alcohol. For fluorescent microscopic studies, samples of A. nidulans mycelia were grown for 16 h at 25°C on coverslips in a droplet of minimal medium with either 1% glucose or 1% ethanol. Nuclei were stained by adding 10 μl of 4,6-diamidino-2-phenylindole (DAPI) solution at concentration of 3 μg/ml.

All yeast strains used in this study were a generous gift of P. A. Silver (Harvard Medical School, Boston, Mass.) and are listed in parentheses in Table 1 according to the original nomenclature. The genotypes of these strains are described elsewhere and are available on request. In microscopic studies, yeast cells were grown to mid-log phase on yeast nitrogen base-dextrose (YNBD) minimal medium at 30°C, unless stated otherwise. The Δkap104 (PSY964) strain was incubated at 23°C, while gsp1-1 (PSY962), srp1-31 (PSY688), rsl1-4 (PSY1103), pse1-1 kap123Δ (PSY1042), xpo1-1 (PSY1105), and mtr10-1 (PSY1133) thermosensitive cells were grown at 25°C followed by a 2-h shift to 37°C. The cse1-1 (PSY1040) strain was cultivated at 30°C and transferred for 12 h at 15°C.

Plasmids.Plasmid pAN52-1:GFP was used to express various AlcR-GFP fusions in A. nidulans. This plasmid, described earlier (38), was modified by insertion of the 2.2-kb ClaI-XhoI fragment of the uaZ gene, which served as a selective marker to confer growth on uric acid. To produce these constructs, DNA fragments coding for different regions of AlcR were amplified by PCR and cloned in frame to the 5′ terminus of the gfp gene in the unique NcoI site of the vector. NcoI sites at both termini of each fragment as well as mutations of the basic residues in regions I to V (except for the region III) as well as cysteines 12 and 15 (see Fig. 3), were introduced during amplification using specific oligonucleotides. Mutations in region III, as well as substitutions within regions IV, V, and IV+V introduced into the entire AlcR protein for in vivo studies, were generated using a QuikChange site-directed mutagenesis kit (Stratagene) and confirmed by DNA sequencing. HindIII-NruI alcR fragments (0.85 kb) containing the desired mutations were cloned into the palcR::uaZ vector described in our previous work (38) to replace the corresponding original sequence. The final plasmids were used for A. nidulans transformation followed by transcriptional analyses. To produce AlcR(1-75)-2xGFP (that is, AlcR aa 1 to 75 plus two GFP tags), Gal4p(1-147)-GFP, Pdr3p(1-100)-GFP, PrnA(1-130)-GFP, IBB(1-60)-GFP, and simian virus 40 (SV40)-GFP fusions, the corresponding coding regions of these transcriptional activators, importin α of S. cerevisiae, and the NLS of SV40 T-antigen, respectively, were amplified by PCR and cloned into the BamHI-SalI sites of the pUG35 reporter construct provided by U. Gueldener and J. H. Hegemann (Institute of Microbiology, Duesseldorf, Germany). Plasmids pET-AlcR(1-821), pGBKT7, pGAD424 (Clontech), pBFG1-PDR3, pFL44L-Kap60 (a gift of C. Jacq, Ecole Normale Supérieure, Paris, France), and pET-PrnA(1-167) (a gift of C. Scazzocchio, Institut de Génétique et Microbiologie, Orsay, France) served as templates for amplification. The sequence of a second egfp was inserted into the pUG35AlcR construct. For complementation of the kap deletions, KAP104, SXM1, and NMD5 were expressed from the centromeric plasmids pRS315 or pRS314 (52) carrying the corresponding karyopherin gene and either LEU2- or TRP-selective markers, depending on the strain used. Plasmids pPS329 (CEN-URA3-KAP104), pPS1575 (CEN-TRP1-SXM1), and pPS2407 (CEN-TRP1-NMD5) containing the aforementioned kap genes, were kindly provided by P. A. Silver (Harvard Medical School). The plasmid expressing AlcR(1-197) tagged with a six-His tag as described previously (37) was used as a template for site-directed mutagenesis and further expression of AlcR proteins mutated in either cysteines 12 and 15 or regions IV+V. The same oligonucleotides as mentioned above were used in the mutagenesis. For the in vitro translation experiments, AlcR(1-821) and AlcR(101-821) were expressed from plasmid pET22b (Novagen). The open reading frames of Kap104p, Sxm1p, and Nmd5p were expressed as glutathione-S-transferase (GST) fusions in pGEX4T-3 (Amersham Pharmacia Biotech), where their coding regions were cloned using appropriate restriction sites. A detailed description of plasmids and the sequence of oligonucleotides used in this study are available on request.

Recombinant protein expression and binding assays.Full-length or truncated AlcR protein was produced by in vitro transcription-translation of pET22b-derived plasmids with a TnT kit (Promega) using a rabbit reticulocyte lysate in the presence of [³⁵S]methionine (Amersham) according to the recommendations of the manufacturer. GST fusions of karyopherins were overexpressed in Escherichia coli strain BL21 and purified on glutathione-Sepharose resin using standard techniques. For each binding assay, 20 μl of radioactively labeled reticulocyte lysate reaction was mixed with approximately 2 μg of GST-Kap104p, GST-Sxm1p, GST-Nmd5p, or GST in 150 μl of transport buffer TB containing 20 mM HEPES-KOH, pH 7.5; 110 mM potassium acetate; 2 mM MgCl₂; 0.1% Tween 20; 1 mM dithiothreitol; and protease inhibitor (Complete EDTA-free protease inhibitor mixture tablets; Roche). The mixtures were preincubated for 3 h at 4°C, and then 30 μl of glutathione-Sepharose resin was added. After an additional incubation period of at least 2 h, the beads were washed extensively three times with TB buffer. and the bound fractions were eluted with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, separated by SDS-PAGE, and visualized by fluorography. To estimate the amount of GST-karyopherin fusion protein added into the binding reaction, 1/10 of each sample was tested by Western blot analysis using antibodies against GST in accordance to the recommendations of the supplier (Sigma).

Electrophoretic mobility shift assay.AlcR proteins were expressed and partially purified on Ni²⁺-nitriloacetic acid agarose as described in a previous work (37). Their DNA-binding affinities were tested by gel shift experiments using inverted repeat target b as a probe (37). Equal amounts of protein (∼0.3 μg) were added in each binding reaction mixture.

Northern blot analysis.Total RNAs were isolated from A. nidulans mycelia using the RNA Plus Extraction Solution (Biogen) following the manufacturer's instructions. Approximately 20 μg of total RNA was loaded per each lane and separated on glyoxal agarose gels as described by Sambrook et al. (48). Blots were hybridized with the alcA and actin probes [³²P] radioactively labeled by a random priming kit (Amersham Pharmacia Biotech). The actin probe served as an internal control to normalize the amounts of RNA present on a single blot. The intensities of the signals on autoradiographs were quantified using a PhosphorImager (Molecular Dynamics).

Fluorescence microscopy.All microscopy and image manipulations were performed with a Zeiss Axioplan microscope equipped with a GFP filter set and a 100× digital-camera objective. Images were captured with a charge-coupled device camera (Princeton Instruments) driven by OpenLab imaging software. All images were taken using identical settings and transferred to Adobe Photoshop. The final figures were produced using Adobe Photoshop without further manipulation.

Constitutive nuclear targeting of AlcR.To study the nuclear transport mechanism of AlcR, it was fused at its C terminus to GFP. Driven by the alcR native promoter, the AlcR-GFP fusion protein conferred growth on ethanol to the fungal cells, indicating that the fusion was functional and the GFP moiety does not significantly alter the behavior of the AlcR protein. However, this construct resulted in weak fluorescence observed upon microscopic analysis (results not shown). This can be explained by a low level of expression of the fusion or/and its instability.

As it has been previously established (reviewed in reference 13), alcR gene expression is autoregulated; i.e., its transcription depends on the amount of the active AlcR protein present in the cell. To be active, AlcR absolutely requires an inducer that has been shown to be an intermediate product of ethanol degradation, acetaldehyde (15). Thus, expressed from its native promoter, alcR transcription is turned on only under inducing conditions. Therefore, in such a context it is impossible to determine if a subcellular localization of AlcR is dependent on the presence of the inducer. In order to solve this issue, as well as to enhance the level of transcription, we expressed the AlcR-GFP protein from a strong constitutive gpdA promoter which is widely used for fungal molecular biology studies (44). Cells expressing the fusion protein were grown either in the presence or absence of the inducer, i.e., on ethanol- or glucose-containing medium, respectively. Microscopic examination of the intracellular distribution of GFP revealed that in both cases the fusion protein was targeted exclusively to the nucleus (Fig. 1), whereas without AlcR the GFP carrier alone was distributed uniformly between the nucleus and cytoplasm, as has been previously shown (38). Hence, the presence of the inducer does not affect the nuclear localization of AlcR. It is constitutively addressed to the nucleus after being transcribed in the cytoplasm.

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FIG. 1.

Constitutive nuclear accumulation of AlcR. The entire AlcR protein fused to GFP was expressed from the constitutive gpdA promoter in A. nidulans grown in the absence (glucose) or presence (ethanol) of the inducer and visualized in hyphae by fluorescence microscopy. Nuclei of the same filament stained with DAPI are shown on the right.

Mapping and mutational analysis of AlcR NLS.Our previous work led us to suggest that the motif responsible for nuclear accumulation of AlcR resides proximal to its N-terminal part (38). To confirm this hypothesis and more precisely identify the regions involved in nuclear import, we constructed a set of successive AlcR deletion mutants, fused to GFP, whose subcellular localization was monitored by fluorescence microscopy (Fig. 2). Preliminary Western blot analyses with anti-GFP antibodies confirmed that GFP fusion proteins are produced at comparatively equal levels for all the constructions (data not shown). A minimal region sufficient to guide the chimeric protein into the nucleus can be narrowed down to the first 75 aa (Fig. 2A and 3). A further deletion of 19 residues (downstream from aa 56) completely abolished nuclear entry. Likewise, the GFP protein fused to the entire AlcR protein lacking the N-terminal domain (residues 1 to 75) was not accumulated in the nuclear compartment, implying that no other motifs outside the first 75 aa possess nuclear import properties. It is noteworthy that this region entirely comprises the DNA-binding domain of AlcR, which contains a Zn₂Cys₆ binuclear cluster (residues 12 to 49) as well as the Arg6 residue indispensable for recognition of a specific type of targets (38). Several other transcriptional activators of the same family, such as Gal4p (5) and PrnA (43), exhibit a similar pattern of functional organization, where the DNA binding and NLS functions can be attributed to a single domain.

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FIG. 2.

The N-terminal domain of AlcR is necessary for nuclear localization. (A) Schematic drawings of a full-length AlcR and its truncated derivatives fused to the GFP reporter. The numbers indicate the first and the last amino acid residues of the AlcR fragment introduced into each reporter construct. The DNA-binding domain, aa 1 to 60 (DBD), is denoted by a hatched box. Basic clusters within the NLS sequence are shown in black. N and C indicates the nuclear and cytoplasmic localization, respectively. (B) Subcellular localization of the AlcR-GFP fusions presented above in fungal mycelia.

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FIG. 3.

Mutational analysis of the AlcR(1-75) NLS. (A) Schematic representation of the DNA-binding domain of AlcR in a cloverleaf type form. Six cysteines chelating two atoms of Zn as well as arginines and lysines grouped in five clusters (clusters I to V) are shown. Amino acid substitutions introduced during mutational analysis are indicated with arrows. (B) Electrophoretic mobility shift assay of the partially purified AlcR(1-197) proteins with six-His tags with 50 fmol of the radiolabeled probe containing inverted repeat target b as described earlier (37). Aliquots (0.3 μg) of the wild-type (WT) AlcR protein and those mutated in either the Zn binuclear cluster (Cys_12-15) or basic regions IV+V, as noted above the gel, were used in each binding reaction. Two retarded complexes, CI and CII, as well as a free probe, P, are indicated on the right. (C) Cellular distribution of the GFP fusions of the wild-type AlcR(1-75) NLS sequence (WT) and those mutated in the Zn binuclear cluster (Cys_12-15) or basic regions (regions I to V) in A. nidulans mycelia. Not all the nuclei are in the same focal plane.

As far as both functions overlap, it was crucial to know whether AlcR is actively imported into nuclei or is retained via binding to its cognate targets. To discriminate between these two possibilities, we mutagenized two cysteine residues, namely, Cys12 and Cys15, involved in chelating two atoms of Zn (Fig. 3A). This leads to disruption of the three-dimensional structure of the Zn binuclear cluster and, as a consequence, to complete loss of DNA binding (Fig. 3B). As seen in Fig. 3C, this cysteine-mutated AlcR-GFP fusion protein localized to the nucleus even better than its wild-type counterpart. Such an improvement of the nuclear import could be due to a conformational change of the N-terminal domain that unmasks contacts crucial for interaction with a transport receptor. Alternatively, changing the global structure of this region could produce an artificial NLS which is recognized more efficiently by an alternative transport factor. Similar results have been obtained for PrnA, another transcriptional activator of the Zn binuclear cluster family. The nuclear import of PrnA remained intact when a conservative Pro residue essential for correct folding of the DNA-binding domain was mutated (43). It may therefore be concluded that the specific cloverleaf structure of Zn cluster proteins is not required for nuclear accumulation. Moreover, DNA binding of AlcR is not involved in its nuclear targeting.

A close examination of the AlcR N-terminal domain revealed a high percentage of positively charged residues dispersed throughout this region. Nineteen of seventy-five residues are either arginines or lysines (Fig. 3A). Despite the lack of evident sequence similarity, this distribution of basic residues resembles that of nuclear import signals found in human ribosomal proteins rpS6, rpL7a, or rpL23a (22); the IBB domain of importin α (17); snuportin 1 (20); or XRIPα (24) adapter proteins, which are known to interact directly with importin β receptors (Fig. 4). A common feature of these signals is their very basic nature and a greater complexity in comparison to the classical mono- or bipartite NLS. To test whether these basic stretches contribute to nuclear import of AlcR, a series of mutant constructs containing mutations in each basic cluster (regions I to V) was created. Within each region, the positively charged amino acids were changed simultaneously. The introduced mutations are presented in Fig. 3A. Disruption of any of the first two motifs mislocalized AlcR to the cytoplasm, whereas mutagenesis of the following three basic patches (regions III to V) had a less pronounced effect (Fig. 3C). Among them, region V appeared to be less important for nuclear accumulation. Though the GFP fusion was significantly retained in the cytoplasm, the residual nuclear transport domain of AlcR was efficient enough to translocate, at least partially, the protein into the nuclear compartment. Interestingly, simultaneous disruption of the last two motifs, IV and V, completely prevented AlcR nuclear import. These results are consistent with our deletion analysis data shown in Fig. 2. The AlcR N-terminal region, consisting of the first 56 aa residues, which encompasses three basic clusters, was not sufficient for nuclear localization. It is important to note that whether it is truncated in region IV or mutated in both regions IV and V, AlcR(1-197) with a six-His tag is still able to bind DNA with nearly the same affinity as the wild-type protein (Fig. 3B). These data once again demonstrate that nuclear accumulation of AlcR results not from a nuclear retention mechanism but from its active import. Thus, the N-terminal domain of AlcR plays a dual role. It is responsible for transport of the protein into the nucleus, where it binds to its cognate DNA targets via the DNA recognition motif consisting of the Zn cluster, as well as the crucial Arg6 residue. For nuclear entry AlcR absolutely requires at least four basic stretches. Of these, the first two regions are indispensable. Two other motifs can be represented by various combinations of two among the three remaining basic clusters, with a strong preference for regions III and IV, with region V being less important. However, the integrity of all five clusters present within the first 75 aa provides the most efficient nuclear import.

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FIG. 4.

NLSs of several proteins known to interact directly with importin β. Abbreviations: L23 a, rpL23a, human ribosomal protein (22); Snu1, human snuportin 1 (20); IBB α, importin β-binding domain of the importin α subunit from S. cerevisiae (17); RIP α, nuclear import receptor of replication protein A in Xenopus laevis (24); PTHrP, human parathyroid hormone-related protein (27); TCPTP, T-cell protein tyrosine phosphatase (56); Gal4p, transcriptional activator of the galactose metabolic pathway in S. cerevisiae (5). The NLS of PrnA and AlcR, the pathway-specific activators of proline and ethanol gene clusters in A. nidulans (reference 43 and the present study), and a putative NLS sequence of Pdr3p, a transcription factor from S. cerevisiae, are presented separately. For comparison, classical, mono- and bipartite NLSs from the SV40 large tumor antigen and nucleoplasmin (36) are shown underneath the line. Basic amino acid residues are indicated in boldface type.

The contribution of each basic amino acid residue was checked only for region I. As we have recently shown, the Arg6 residue is not directly involved in nuclear transport but rather is involved in DNA target recognition (38). An individual substitution of either Arg5 or Arg7 which, in contrast, do not contribute to the DNA-binding affinity (3, 38), did not result in a drastic accumulation of the GFP fusion in the cytoplasm (results not shown). However, when all three arginines were changed simultaneously, an additive effect was observed, which may reflect a complex network of interactions between the NLS sequence of AlcR and its transport receptors.

NLS integrity is required for ethanol utilization.If the NLS of AlcR is essential for the nuclear import and function of this transcriptional activator, it would be expected that disruption of this region will give rise to impaired ethanol growth. To assess the physiological relevance of the NLS and investigate any physiological consequences of AlcR mislocalization, we replaced the wild-type copy of the alcR gene with its mutant alleles and tested the ability of A. nidulans to utilize ethanol as a carbon source. Plasmids containing mutated derivatives of the alcR gene were transformed in an alcR-null background. Such a strain was obtained by introducing the alcA gene in the argB2 locus of the alc500 strain, which is has a deletion of the ethanol gene cluster. alcR single-copy transformants were selected. Mutations in AlcR were restricted to regions IV and V because basic residues within clusters I and II have been shown to be involved in direct interactions with DNA (3, 4, 38). Their disruption results in a loss of DNA binding and a concomitant complete loss of transcriptional activation. In this case, any other physiological role that could be attributed to this sequence would be masked. In our in vivo studies, we introduced exactly the same mutations as in the fluorescent microscopic experiments and in the DNA-binding assay. The effects of both individual and combined mutations of regions IV and V were monitored. Toxicity to allyl alcohol was preferred to ethanol growth as a test of AlcR activity as it appears to be more sensitive to the levels of alcohol dehydrogenase (ADHI) activity in the cell and, therefore, is more indicative of the physiological state of the ethanol metabolism system. ADHI oxidizes allyl alcohol into a highly toxic compound, acrolein, resulting in the absence of growth. As seen in Fig. 5A, when grown on plates containing glycerol and 10 mM allyl alcohol, transformants expressing AlcR with alterations in either motif IV or V had exactly the same phenotype as the wild-type or the control alcR nonmutated strain (R), i.e., were unable to grow in the presence of allyl alcohol. In contrast, when both motifs IV and V were mutated, cells were able to survive on such a medium, indicating that the transcription of alcA was impaired. This conclusion was proved by a Northern blot analysis (Fig. 5B). A steady-state level of the alcA transcript observed under induced conditions in transformants containing AlcR modified within the NLS regions IV or V was decreased only two times or less compared to that of the wild-type strain. In contrast, in the strain carrying mutations in both regions IV and V, alcA-induced transcription was severely impaired and under all growth conditions remained at levels obtained for the alcR-null strain. These results are consistent with the absolute inability of the AlcR protein mutated in regions IV and V to enter the nucleus. An unexpectedly elevated basal level of alcA transcription was observed in all transformants as well as in the recipient strain. This could be due to the fact that the alcA gene introduced into the A. nidulans genome lacks several distal CreA-binding sites in the promoter region. Those play a minor role, but their deletion could result in an increase in alcA basal expression which is under the control of the CreA repressor (14). On the other hand, the chromatin structure of the argB locus where alcA was integrated could be more accessible to the transcriptional machinery. Nonetheless, the higher basal level of alcA did not interfere with the growth tests. Taken together, these data show that the growth phenotype and the transcriptional analysis correlate with the nuclear localization of AlcR.

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FIG. 5.

The NLS motif of AlcR is essential for transcriptional activation of the alc system and ethanol utilization by A. nidulans. (A) Growth test of the following A. nidulans strains on 1% glycerol (Gly) and 1% glycerol plus 10 mM of allyl alcohol (Gly+AA): wild type (WT), alcR deletion mutant (ΔR), alcR restored mutant (R), and those with mutations in basic regions (IV, V, and IV+V). Allyl alcohol is oxidized by ADHI into acrolein, which is toxic to the fungal cell and, hence, is indicative of alcA-alcR expression. (B) Northern blot analysis of total RNAs isolated from A. nidulans mycelia pregrown on 0.1% fructose (noninduced [NI]) and induced for 2.5 h with 50 mM ethyl methyl ketone (I) or repressed with 1% glucose in the presence of 50 mM ethyl methyl ketone (IG). For transcriptional analysis, the same A. nidulans strains as those used for growth tests were used. Equal amounts of RNA (20 μg) were loaded per blot, and the blots were subsequently probed with ³²P-labeled alcA and actin gene fragments. Relative amounts of the alcA transcript normalized to the actin signal are given by numbers above each bar presented in a histogram shown below.

AlcR can be targeted to the nuclei by several importin β receptors.Given that the nuclear transport pathway in S. cerevisiae is well characterized at both the genetic and molecular levels (12), we utilized this model organism to investigate the mechanism of nuclear import of AlcR. As a preliminary approach, the functional integrity of the AlcR NLS was tested in yeast. An AlcR-2xGFP fusion protein was expressed from the yeast MET25 promoter in a centromeric vector. A tandem copy of the gfp gene introduced into the construct increased the size of the chimeric protein to avoid its possible nucleocytoplasmic shuttling by passive diffusion, thereby resulting in a reduction of background cytoplasmic fluorescence. The intact NLS sequence guided the GFP fusion predominantly to the nucleus, whereas the mutation in region II led to an aberrant subcellular localization of uniform fluorescence throughout the cell (Fig. 6A). Hence, the nuclear localization of the fusion in the yeast cell is absolutely dependent on the integrity of the AlcR NLS. This sequence is perfectly recognized by the heterologous yeast transport receptors, indicating that the mechanism of the nuclear import is highly conserved between A. nidulans and S. cerevisiae.

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FIG. 6.

Nuclear translocation of AlcR requires Ran/Gsp1p GTPase activity and Kap104p, Sxm1p, and Nmd5p receptors but neither importin α nor importin β. (A) AlcR(1-75) fused to two tandem repeats of GFP (2xGFP) was expressed in S. cerevisiae wild-type and mutated strains carrying temperature-sensitive alleles of GSP1 (gsp1-1), importin α (srp1-31), and importin β (rsl1-4). Cellular distribution of the GFP fusion was monitored by fluorescence microscopy. Cells were grown at 25°C and then shifted for 2 h at 37°C before the images were captured. The AlcR(1-75)-2xGFP fusion mutated in the basic region II (WT/ΔNLS) served as a control to show that the nuclear import of AlcR in yeast wild-type strain (WT) requires the NLS sequence. The coincident DAPI staining of the nuclei is shown. (B) Kap104p, Sxm1p, and Nmd5p mediate the nuclear import of AlcR(1-75). Strains with deletions of KAP104 (Δkap104), SXM1 (Δsxm1), or NMD5 (Δnmd5) mislocalized the AlcR(1-75)-2xGFP fusion to the cytoplasm. Strains were grown at 30°C, except for the KAP104-deficient strain, which was grown at room temperature. The nuclear accumulation of the GFP fusion was restored upon complementation of deletions with the corresponding kap genes (Δkap104 + KAP104; Δsxm1 + SXM1; Δnmd5 + NMD5). Nuclei were visualized by DAPI staining. (C) Control panels showing cellular distribution of the SV40 T-antigen NLS fused to GFP in yeast mutant strains grown under the same conditions as described above. (D) Kap104p and Sxm1p bind directly to the NLS of AlcR. Reticulocyte lysates containing [³⁵S]methionine-labeled either full-length [AlcR(1-821)] or truncated [AlcR(101-821)] AlcR protein were initially incubated with purified GST fusions of Kap104p, Sxm1p, or GST alone, as indicated above the gel, followed by incubation with glutathione-Sepharose. The bound material was separated by SDS-PAGE and analyzed by fluorography. The input lane represents 10% of the reticulocyte reaction used in the binding assay. The amounts of GST and GST-karyopherin fusions used in each binding reaction were tested by Western blot analysis with anti-GST antibodies (panel at right).

We next addressed the mechanism whereby AlcR is targeted to the nucleus. For this purpose, the AlcR-2xGFP fusion was expressed in a panel of S. cerevisiae mutants defective in nuclear transport, and its subcellular localization was examined. With a few exceptions (26), all nuclear import events studied so far require soluble transport factors and energy in the GTP form. To determine whether this is also the case for AlcR, we first examined its localization in a temperature-sensitive gsp1-1 mutant strain with Ran/Gsp1p GTPase impaired at restrictive temperatures (62). In gsp1-1 cells AlcR-2xGFP was found mostly in the cytoplasm after a 2-h shift to the nonpermissive temperature (Fig. 6A). Thus, like other nuclear proteins, AlcR nuclear translocation is dependent on Ran/Gsp1p activity and cannot be due to a passive diffusion mechanism followed by nuclear retention.

It was next important to know if AlcR nuclear import occurs via the classical importin α/β heterodimer or involves another importin β family receptor. To ascertain whether the nuclear import of AlcR requires the importin α subunit in vivo, we observed its localization in srp1-31 cells. This strain carries a conditional allele of importin α and is defective in nuclear translocation of proteins with typical NLSs (29). The localization of the fusion was tested after a 2-h shift to the nonpermissive temperature. The protein remained exclusively nuclear in srp1-31 cells at both permissive and restrictive temperatures, while GFP fused to the classical NLS of SV40 T-antigen was mostly cytoplasmic at 37°C (Fig. 6A and C [only cells at 37°C are shown]). Likewise, a temperature-sensitive mutation of the importin β subunit RSL1/KAP95 (rsl1-4) did not alter the nuclear distribution of AlcR-2xGFP even after a prolonged incubation at the restrictive temperature, whereas cellular distribution of the SV40-GFP fusion was affected (Fig. 6A and C). These data suggest that the import of AlcR into the nucleus proceeds neither via the classical pathway requiring importin α/β heterodimer nor by direct interaction with the importin β receptor.

To further define transport receptors responsible for AlcR nuclear loading, the subcellular distribution of the AlcR-2xGFP fusion was monitored in mutant strains deficient in various members of the importin β family (Table 1). The panel of mutants included those transport receptors that are mainly involved in the nuclear import process, except for the Kap122p/Pdr6p importin (57), as well as Los1p exportin (7, 41) and karyopherin Kap120p, which is implicated in the export of the 60S ribosomal subunit (55). Deletion of either KAP104, SXM1, or NMD5 caused visible defects in AlcR trafficking but did not impair nuclear import of the SV40-GFP fusion (Fig. 6B and C). It has been reported that disruption of the KAP104 gene has a severe impact on growth of the yeast cells (1). Although many of the cells were dead after prolonged culture growth, those with intact nuclei exhibited impaired subcellular localization of AlcR-2xGFP (Fig. 6B). A strong accumulation of the fusion in the cytoplasm was observed in more than 90% of the cells lacking either SXM1 or NMD5, although a residual nuclear fluorescence was observed (Fig. 6B). Interestingly, in all three mutant strains such a mislocalization appeared to be transient. When the rate of protein synthesis was reduced either by decreasing the growth temperature or leaving logarithmically grown cells for a few hours without agitation, the mutant phenotype reversed, resulting in a regular targeting of the GFP fusion to the nuclei (results not shown). This phenomenon may be accounted for by the multiplicity of the transport pathways for AlcR. In the absence of one of the transport receptors, the nuclear import of AlcR is mediated by two other importins, albeit less efficiently, giving rise to temporarily enhanced cytoplasmic fluorescence. The original nuclear localization of AlcR-2xGFP was completely restored when deletions of KAP104, SXM1, and NMD5 were complemented with the corresponding genes (Fig. 6B). These data further confirm direct involvement of the aforementioned importin β receptors in the nuclear import of AlcR.

AlcR interacts directly with two importin β receptors.To examine the mode of AlcR interaction with the corresponding importins β, we tested their ability of direct binding in vitro in a GST pull down assay. The karyopherins Kap104p, Sxm1p, and Nmd5p were expressed as GST fusions in E. coli and purified on glutathione-Sepharose. Full-length AlcR produced and labeled in vitro with [³⁵S]methionine by a coupled transcription-translation rabbit reticulocyte lysate system was initially incubated with recombinant GST-importin β fusions, and interacting complexes were recovered with glutathione-Sepharose beads (Fig. 6D). More than 70% of the AlcR fraction was specifically bound to GST-Sxm1p. A much-reduced binding affinity (about 30% of AlcR was bound) was observed for the GST-Kap104p containing sample. This value, however, significantly exceeds the background level (10%) corresponding to GST alone, which can be ascribed to unspecific binding of proteins present in reticulocyte lysate. Moreover, interactions with both GST-Kap104p and GST-Sxm1p fusions could be specifically attributed to the NLS sequence, since AlcR protein lacking the first 100 aa residues exhibited only a background level of binding (Fig. 6D). Unfortunately, we failed to detect interaction between AlcR and Nmd5p, but we cannot rule out the possibility that the GST-Nmd5p sample could be inactive.

Diversity of the nuclear import pathways of Zn binuclear cluster transcriptional regulators.As shown above, the AlcR NLS overlaps with its DNA-binding domain, itself highly conserved among fungal transcriptional factors belonging to the Zn binuclear cluster protein family. Basic residues that constitute the NLS of AlcR retain their conservative positions in 30% of cases (49). Gal4p and PrnA, two other transactivators of the same class, exhibited a similar type of extended sequence rich in lysines and arginines found within their DNA-binding motifs to deliver the proteins into the nuclei (6, 43). The intriguing question is whether proteins with a similar structural organization have evolved a common mechanism of nuclear import by sharing the same transport receptors. To address this point, Gal4p and PrnA, or more precisely their first 147 and 130 aa residues, respectively, were fused to GFP, and subcellular localization of the fusions was examined in the same set of mutant strains as for AlcR (Table 1). In addition, Pdr3p, another representative of the Zn cluster family whose transport mechanism was unknown but distinct from that of its ortholog Pdr1p (10), was included in the assay. All three proteins are known to be constitutively present in the nuclei (10, 43, 53).

A temperature-sensitive rsl1-4 mutant that has defects in yeast importin β/Kap95p partially mislocalized Gal4p(1-147)-GFP to the cytoplasm of 25 to 30% cells after a shift to the restrictive temperature (Fig. 7A). Similarly, the IBB(1-60) domain of importin α known to interact directly with Kap95p was retained in the cytoplasm as judged by GFP fluorescence in rsl1-4 cells (Fig. 7B). On the other hand, mutation in the importin α subunit, represented by the conditional allele srp1-31, had no significant effect on the nuclear accumulation of Gal4p (Table 1). Such patterns of Gal4p(1-147)-GFP distribution correlate with importin-α-independent, direct in vitro binding established between Gal4p and importin β by Chan et al. (6). However, it is important to note that this mode of nuclear entry via a direct loading of the importin β receptor with Gal4p dimers probably plays a marginal role in living cells. Rather, the principal transport pathway for Gal4p could be mediated by a nonessential karyopherin, Nmd5p. As for AlcR, deletion of NMD5 resulted in a drastic accumulation of Gal4p(1-147)-GFP in the cytoplasm of more than 90% of cells (Fig. 7A). This aberrant distribution of the fusion was totally restored to the wild-type pattern of localization into the nuclei when a functional copy of the NMD5 gene was introduced back into the yeast cell. Thus, AlcR and Gal4p share the same Nmd5p receptor for their nuclear import, whereas neither Kap104p, nor Sxm1p, nor any other importin β homolog altered the nuclear localization of Gal4p (Table 1).

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FIG. 7.

Nuclear import of Zn binuclear cluster proteins Gal4p, Pdr3p, and PrnA in S. cerevisiae kap mutants. (A) Cellular localization of the Gal4p(1-147)-GFP fusion in a thermosensitive strain of importin β (rsl1-4) and a Δnmd5 strain. After a 2-h shift from a permissive (25°C) to restrictive (37°C) temperature, Gal4p(1-147)-GFP was partially mislocalized to the cytoplasm (left panel). Mislocalized in the Δnmd5 strain, the fusion was targeted back to the nuclei after a complementation with the NMD5 gene (Δnmd5 + NMD5). (B) Cellular distribution of the IBB(1-60) domain of importin α from S. cerevisiae fused to GFP in the importin β mutant strain (rsl1-4) was impaired upon a shift to 37°C. (C) Nuclear accumulation of Pdr3p(1-100) fused to GFP was affected in rsl1-4 cells (rsl1-4) both at 25 and 37°C and was restored when an exogenous copy of KAP95/RSL1 was expressed (rsl1-4 + RSL1) in the yeast cell. (D) Nuclear localization of PrnA(1-130)-GFP remains intact in rsl1-4 cells at both permissive (25°C) and restrictive (37°C) temperatures. The coincident DAPI staining is shown below each panel.

The NLS responsible for the nuclear transport of Pdr3p lies within the N-terminal 100 aa residues, since this sequence is sufficient for the nuclear targeting of GFP as efficiently as the full-length protein (results not shown). As in AlcR and Gal4p, this region comprises the DNA-binding domain and is rich in basic amino acid residues. This distinguishes Pdr3p from its functional homolog Pdr1p, whose NLS enriched in Ser/Tyr residues maps closer to the C terminus (10). Interestingly, none of the importin β-like receptors that supported the transport of AlcR appeared to be operative on the Pdr3p NLS (Table 1). In contrast, importin β/Kap95p seems to be involved in the import process. In rsl1-4 cells, a Pdr3p(1-100)-GFP fusion was slightly accumulated in the cytoplasm even at the permissive temperature (Fig. 7C). The phenomenon was much more pronounced at 37°C. Expression of an extragenic copy of the RSL1/KAP95 gene restored exclusive nuclear compartmentalization of Pdr3p(1-100)-GFP and confirms involvement of the importin β subunit (Fig. 7C). Like Gal4p, Pdr3p does not require the importin α adapter for nuclear delivery, since in srp1-31 cells its transport was not affected (Table 1). In this respect, both proteins share a common transport pathway, provided that for Pdr3p importin β is the sole transport receptor among those tested. The possibility of a direct interaction between Pdr3p and Kap95p was not checked experimentally.

Surprisingly, none of the receptors listed in Table 1 abolished the nuclear localization of PrnA(1-130)-GFP. A typical pattern of nuclear fluorescence exemplified by the one observed in rsl1-4 cells is present in Fig. 7D. We did not verify the localization of the fusion in the Δkap122/pdr6 strain, which lacks one of the members of the importin β family. This strain has been shown to import the large and small subunits of the general transcription factor TFIIA (57). Two other receptors, Los1p and Kap120p, not included in these assays, are probably not involved in nuclear import. Rather, they have been shown to export tRNAs and 60S ribosomal subunits, respectively (41, 55). Although a model of bidirectional function for a specified karyopherin does not meet any theoretical obstacles, it has been thus far demonstrated only for yeast transporter Msn5p (63) or human importin 13 (31). However, in the MSN5-deficient strain, the nuclear localization of PrnA(1-130)-GFP remained intact (Table1). Further studies are required to highlight the nuclear import mechanism of PrnA.