The C-terminal nuclear localization signal of the sex-determining region Y (SRY) high mobility group domain mediates nuclear import through importin beta 1.

The sex-determining factor SRY is a DNA-binding protein that diverts primordial gonads from the ovarian pathway toward male differentiation to form testes. It gains access to the nucleus through two distinct nuclear localization signals (NLSs) that flank the high mobility group (HMG) DNA-binding domain, but the mechanisms through which these NLSs operate have not been studied. In this study, we reconstitute the nuclear import of SRY in vitro, demonstrating a lack of requirement for exogenous factors for nuclear accumulation and a significant reduction in nuclear transport in the presence of antibodies to importin beta but not importin alpha. Using a range of quantitative binding assays including enzyme-linked immunosorbent assay, fluorescence polarization, and native gel mobility electrophoresis, we assess the binding of importins to SRY, demonstrating a high affinity recognition (in the low nm range) by Imp beta independent of Imp alpha. In assessing the contribution of each NLS, we found that the N-terminal NLS was recognized poorly by importins, whereas the C-terminal NLS was bound by importin beta with similar affinity to SRY. We also found that RanGTP, but not RanGDP, could dissociate the SRY-importin beta complex in solution using FP. We describe a novel double-fluorescent label DNA binding assay to demonstrate mutual exclusivity between importin beta recognition and DNA binding on the part of SRY, which may represent an alternative release mechanism upon nuclear entry. This study represents the first characterization of the nuclear import pathway for a HMG domain-containing protein. Importantly, it demonstrates for the first time that recognition of SRY by Imp beta is of comparable affinity to that with which Imp alpha/beta recognizes conventional NLS-containing substrates.

Sex determination and early gonadal differentiation of mammalian embryos take place around week 6 of development. Although the autosomal and X chromosomes carry genes required for basic structural and functional development in both sexes, the Y chromosome carries the specific genes for male sexual development. SRY 1 (sex-determining region Y) is a DNA-binding protein encoded on the Y chromosome and acts as a genetic switch that diverts primordial gonads from the ovarian pathway toward male differentiation to form testes (1)(2)(3). SRY activates transcription at the promoter region of the Mü llerian inhibitor substance (MIS) gene (4,5); the product of MIS is responsible for the regression of the female Mü llerian ducts, the precursor of the uterus, fallopian tubes, and upper vagina, in male embryos.
The DNA binding domain of SRY consists of a single high mobility group (HMG) box located centrally within the protein (6). The HMG box, conserved across the HMG-1/HMG-2 family, consists of around 80 residues and binds within the minor groove of DNA (7)(8)(9). Although HMG-1 and HMG-2 bind DNA with little or no sequence specificity, SRY has been reported to bind specifically to an eight-base pair recognition sequence within the MIS promoter (5,10,11). By binding the minor groove of DNA, SRY induces a large conformational change through helix unwinding, minor groove expansion, and DNA bending (12,13). This is believed to be the molecular switch that allows distantly bound proteins of the transcription machinery to attain close proximity, thereby permitting interaction in a way that can influence transcription (12,13).
SRY nuclear import is believed to be mediated by two distinct nuclear localization signals (NLSs) that flank the HMG box, the N-terminal bipartite NLS (KRPM-NAFIVWSRdqRRK 77 ) and the monopartite C-terminal NLS (RPRRKAK 136 ), both of which are able to act independently to target ␤-galactosidase to the nucleus (14). Protein targeting to the nucleus is mediated by a family of transporters or cytosolic receptor proteins known as importins (karyopherins) (15)(16)(17), which work in concert with the guanine nucleotide-binding protein Ran and other regulatory proteins. In conventional NLS-dependent nuclear protein import, importin ␣ (Imp␣) recognizes the NLS and acts as an adapter to mediate binding of importin ␤1 (Imp␤) (17). The latter then mediates docking of the Imp␣-NLS-containing protein complex to the nuclear pore complex (NPC) followed by energy-dependent translocation through the NPC (18). RanGTP dissociates the complex in the nucleus by binding to Imp␤, and the individual importins are * 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.
recycled back to the cytoplasm (19,20). Direct evidence that transcription factors (TFs) may localize in the nucleus through this NLS-dependent importin ␣/␤-mediated nuclear transport pathway, however, is essentially restricted to inducible TFs such as those of the nuclear factor NF-B and STAT (signal transducer and activator of transcription) families (21)(22)(23).
Several proteins have recently been shown to be imported into the nucleus through pathways independent of Imp␣ that are mediated by a range of different Imp␤ homologs. In these pathways, the Imp␤ homolog recognizes the targeting signal of the transport substrate directly (24 -27) as well as performing the NPC docking and Ran binding roles. The Imp␤ (Imp␤1) homologs Imp␤3 (Kap121p/Pse1p) (28,29), Imp␤4 (Yrb4p) (28,30), and Sxm1 (Kap108p) (31), for example, have been shown to mediate the nuclear import of ribosomal proteins. In contrast, Imp␤2 (transportin) appears to be largely responsible for the transport of hnRNPs (heterogeneous nuclear ribonucleoprotein) into and out of the nucleus (25,32,33), whereas Imp7 (RanBP7/Nmd5p/Kap119p) has been reported to mediate nuclear import of the yeast mitogen-activated protein kinase HOG1 (34) and general TF TFIIS (35). An analogous pathway has been described for proteins such as the yeast TF GAL4 (36), the HIV-1 Rev (37),and HTLV-1 Rex (38) proteins, the TCPTP (T-cell protein tyrosine phosphatase) (39), and the polypeptide ligand parathyroid hormone-related protein (PTHrP) (40), where Imp␤1 itself, independent of Imp␣, binds the nuclear import substrates directly. In the case of PTHrP, the ability of Imp␤1 to function in nuclear import independently of Imp␣ has been shown directly by reconstituting nuclear transport in vitro using purified, bacterially expressed components (40).
In this study, we characterize the nuclear import properties of SRY for the first time, demonstrating a requirement for Imp␤ but not Imp␣. Using a range of binding assays including ELISA, fluorescence polarization (FP), and native gel electrophoresis, we report high affinity binding of Imp␤ to SRY(HMG) but only weak association of the conventional NLS receptor Imp␣. We show that nM affinity interaction of SRY with Imp␤ is mediated by the C-terminal NLS and that the association can be disrupted by RanGTP␥S but not by RanGDP. We also use a novel double-fluorescent label gel mobility shift assay to demonstrate mutual exclusivity of DNA-binding and Imp␤ recognition. This study thus represents the first characterization of the nuclear import pathway of an HMG domain-containing TF with the mutual exclusivity of DNA and Imp␤ binding suggesting a possible role for the former in dissociating the transport complex within the nucleus.

Construction, Expression, and Purification of SRY(HMG)-GFP-
DNA encoding the HMG domain of SRY (residues 57-136) was amplified by polymerase chain reaction to include terminal NheI restriction sites and cloned into the unique NheI site of pTRCAgfp (41) to encode a 35-kDa SRY(HMG)-GFP fusion protein. The directionality and fidelity of the insert was verified by sequencing. Host strain BL21 was grown at 28°C until mid-log phase, whereupon SRY(HMG)-GFP was over-expressed by induction with 1 mM isopropyl-1-thio-␤-D-galactopyranoside for 8 h at 28°C. The bacteria were centrifuged and resuspended in His buffer (50 mM phosphate buffer, pH 8.0, 10 mM Tris, pH 8.0, 300 mM NaCl) containing 1 mg/ml lysozyme. Cell debris was removed by centrifugation and the supernatant incubated with 2 ml of pre-washed nickel-nitrilotetraacetic acid-agarose (Qiagen) for 2 h at 4°C. Nonspecific proteins were removed by washing the matrix with His buffer containing 20 mM imidazole, after which the SRY(HMG)-GFP was eluted from the column by incubation with His buffer containing 500 mM imidazole. SRY(HMG)-GFP was then exchanged into phosphatebuffered saline using a PD-10 column (Amersham Pharmacia Biotech), and aliquots were stored at Ϫ70°C.
Expression of SRY(HMG)-The plasmid vector pT7-7 encoding the SRY(HMG) domain was transformed into Escherichia coli BL21(DE3), and expression was induced by isopropyl-1-thio-␤-D-galactopyranoside. Cell pellets were resuspended in 1ϫ HED buffer (50 mM HEPES, 1 mM DDT, 1 mM EDTA, and 50 mM AEBSF), sonicated, and NaCl added to a final concentration of 0.45 M. A 50% slurry of DEAE-Sephadex was added, cell debris was removed by centrifugation, and the supernatant was stored at Ϫ20°C. The supernatant was filtered through an 0.8-mm polyvinylidene difluoride/non-protein-binding filter prior to fast protein liquid chromatography and diluted 2.25-fold, and the sample was purified using an SP-Sepharose column. SRY(HMG) was eluted using a salt gradient at 800 mM NaCl and subsequently stored at Ϫ70°C.
ELISA-based Binding Assay-An ELISA-based binding assay (43)(44)(45)(46) was used to determine the affinity of binding between importins and NLS-containing proteins or peptides. The latter were coated onto 96well microtiter plates and incubated with increasing concentrations of importins, and detection of bound importin-GST was performed using a goat anti-GST primary antibody, an alkaline phosphatase-coupled rabbit anti-goat secondary antibody, and the substrate p-nitrophenyl phosphate. Absorbance measurements were performed over a period of 90 min using a plate reader (Molecular Devices), with values corrected by subtracting absorbance both at 0 min and in wells incubated without importin. Data were fitted to the function (51), where x is the total concentration of probe (free and bound importins), B is the level of importin bound, p is the total concentration of protein, and K d (the apparent dissociation constant) is the concentration of importin resulting in half-maximal binding (52).
Gel Mobility Shift Assay-Probes containing specific DNA-binding recognition sequences (underlined) for SRY were prepared by annealing the complementary oligonucleotides SRY Top, 5Ј-GAT CGC GTC GAC GAA ACA ATG ATC GAT C-3Ј, and SRY Bottom, 5Ј-GAT CGA TCG ATC ATT GTT TCG TCG ACG C-3Ј. The double-stranded oligonucleotides were then purified by electrophoresis, and the concentration estimated from absorbance measurements. Probes were end-labeled using the Texas Red dCTP-Texas Red labeling kit (Bio-Rad), and unincorporated dye was removed by ethanol precipitation. SRY binding interactions with the probe were assessed by incubating 1 M SRY(HMG)-GFP with 0.1 M labeled probe in binding buffer (10 mM Tris, 1 mM MgCl 2 , 0.1% Nonidet P-40, 10 mM dithiothreitol, 0.8 mM EDTA, 3% glycerol, 1.5% sucrose) at room temperature for 15 min before electrophoresis at 4°C on a 5% native polyacrylamide gel. The gel was visualized on a Fuji-Film FLA3000 gel imaging system, and results were quantified using ImageGauge software. Where appropriate, 3 M of importins were pre-incubated with SRY-(HMG)-GFP at room temperature for 15 min before the addition of the DNA probe, or a 10-fold excess of unlabeled DNA was included.
For mobility shift assays involving protein-protein interactions, 1 M SRY(HMG)-GFP was incubated with 3000 -25 nM importins for 20 min prior to electrophoresis as described above. Gels were directly analyzed on the Fuji-Film FLA3000 and quantified using the ImageGauge software.
Peptides-Peptides were synthesized as described previously (40). The NLS residues of SRY comprised residues 57-80 (N-NLS) and 120 -138 (C-NLS); the PTHrP NLS peptide spanned residues 67-94 of Fluorescence Polarization Assay-FP was used to analyze NLS-containing protein-importin interactions in solution (53). When plane polarized light is used to excite a fluorophore, molecules in which the absorption oscillators are orientated parallel to the direction of polarization will be excited preferentially. The polarized components of the emission can be used to calculate an anisotropy value, A ϭ (I ʈ Ϫ I Ќ )/ (I ʈ ϩ 2I Ќ ), which is dependent on the rotational mobility of the fluorophore, which in turn relates directly to its size; therefore, larger fluorophores (with lower rotational mobility) exhibit higher anisotropy values under constant buffer conditions. Fluorescence anisotropy measurements (52, 53) on SRY(HMG)-GFP were carried out using an SLM MC400 fluorimeter fitted with polarization filters. Excitation was at 490 nm, and light emitted from the fluorophore was collected after passage through a 533 nm cut-off filter. For each assay, SRY(HMG)-GFP or CREB-GFP was diluted to a final concentration of 30 nM in phosphate-buffered saline, and anisotropy changes were monitored as aliquots of importin (1-1000 nM), Ran (133 nm), or peptides (1 M) were added successively. Results for changes in anisotropy were fitted to the (51), where x is the total concentration of probe (free and bound importins), p is the total concentration of protein, and K d (the apparent dissociation constant) is the concentration of importin resulting in half-maximal binding.

RESULTS
For SRY to perform its role as a remodeling factor, it must first gain access to the nucleus. SRY possesses two NLSs at the proximal ends of the HMG-DNA binding domain (14), both of which have been shown to be sufficient for targeting a carrier protein to the nucleus. The import pathways, however, through which these NLSs target SRY to the nucleus have not been characterized. We set out to determine the mechanism by which SRY is targeted to the nucleus.
Nuclear Import of SRY Requires Importin-␤-To examine the nuclear import properties for SRY, we fused the coding sequence of the HMG-DNA binding domain of SRY containing both C-and N-terminal NLSs to that of GFP (see "Material and Methods"). The encoded SRY(HMG)-GFP protein was expressed in bacteria, purified, and confirmed for DNA binding activity in gel mobility shift assays (see also Fig. 7). Nuclear import was assessed using an in vitro nuclear transport assay in conjunction with confocal laser scanning microscopy (42)(43)(44)(45). We found that SRY(HMG)-GFP accumulated in the nucleus in the absence or presence of exogenously added cytosol, indicating a lack of a requirement for the latter (Fig. 1, top  panels). To test the possibility that SRY(HMG)-GFP accumulated in the nucleus simply through its ability to diffuse through the NPC and bind to nuclear components, we performed the nuclear transport assay in the presence of the nuclear envelope-permeabilizing agent, CHAPS (43). Under these conditions, the control Texas Red-labeled Dextran-70 molecule was able to diffuse into the nucleus, but nuclear accumulation of SRY(HMG)-GFP was not observed (Fig. 1), indicating that SRY nuclear accumulation does not occur through binding to nuclear components. In the presence of CHAPS, we noted accumulation at the nuclear envelope, implying that SRY(HMG)-GFP was binding to residual transport factors at the NPC. The lectin wheat germ agglutinin, which impairs NPC function by binding to the nucleoporins critical for translocation through the pore, inhibited SRY(HMG)-GFP nuclear accumulation completely (Fig. 1).
We also assessed the involvement of both importin ␣ and importin ␤ in the accumulation of SRY by using specific antibodies (54). We observed no significant difference in nuclear accumulation in the presence of anti-Imp␣ antibodies, but those against anti-Imp␤ resulted in reduced nuclear accumulation (Fig. 1). Interestingly, SRY(HMG)-GFP was localized at the nuclear envelope in the presence of anti-Imp␤ antibodies, suggesting a direct involvement of Imp␤ in docking SRY at the NPC. As a control for the efficacy of the antibodies, anti-Imp␣ and -Imp␤ antibodies inhibited nuclear accumulation of the conventional Imp␣/␤-recognized NLS-containing protein, Rb-NLS-␤-Gal (43) (data not shown). That SRY(HMG)-GFP nuclear accumulation was dependent on Imp␤ is not inconsistent with the observation that its accumulation appeared to be independent of exogenously added cytosolic factors; it is known that residual Imp␤ remains at the NPC subsequent to the cell perforation procedure and that this can prove sufficient to mediate nuclear import in the case of Imp␤-recognized nuclear import substrates such as insulin growth factor-binding protein 3 (IGFBP-3) (55). Residual Imp␤ is thus most likely responsible for the ability of SRY to accumulate in the absence of cytosolic factors (see "Materials and Methods" and see below).
SRY Is Recognized by Importin ␤ with High Affinity-The finding that nuclear accumulation of SRY appeared to be dependent on Imp␤ but not Imp␣ led us to test the binding of Imp␣ and Imp␤ to SRY(HMG) in vitro. The interaction of SRY(HMG) with mImp␣, mImp␤, and the mImp␣/␤ heterodimer was initially quantified using an ELISA-based binding assay, previously used to determine the binding affinities of a variety of conventional NLS-containing proteins (43)(44)(45)(46). We observed high affinity binding of mImp␤ and mImp␣/␤ (2.5 and 1.0 nM, respectively) but only weak association with mImp␣ (149 nM) for SRY(HMG) (Fig. 2, left top panel, and Table I), implying that nuclear import of SRY(HMG) is mediated by Imp␤ and not by the conventional NLS receptor, Imp␣. By comparison, the conventional NLS receptor, Imp␣, recognized the T-ag NLS with high affinity (33 nM), showing very low affinity for Imp␤ (441 nM) (Fig. 2, bottom right panel, and Table I).
To confirm the interaction of SRY(HMG) with Imp␤ in solution, we utilized the SRY(HMG)-GFP-tagged fusion protein in FP assays whereby binding of importins to SRY(HMG)-GFP, which results in a significant change in molecular weight, was

FIG. 1. Nuclear import of SRY-(HMG)-GFP in vitro.
Nuclear import was reconstituted in mechanically perforated HTC cells as described under "Materials and Methods." Confocal laser scanning microscopic images of nuclear accumulation of SRY(HMG)-GFP after 20 min (steady state) in the absence or presence of cytosol, CHAPS, wheat germ agglutinin, anti-Imp␣, and anti-Imp␤ as indicated.
measured as a change in anisotropy ( Fig. 3 and Table I). Because the molecular weight of Imp␣ is similar to that of Imp␤ and well in excess of that of SRY(HMG)-GFP, the only small change in anisotropy of SRY(HMG)-GFP, even at high concentrations of Imp␣, was interpreted as indicating that the affinity of binding of Imp␣ to SRY(HMG)-GFP was low (see Ref. 53). In contrast, increasing concentrations of Imp␤ resulted in anisotropy changes consistent with a larger molecular weight complex due to binding of Imp␤ to SRY(HMG)-GFP. The affinity of Imp␤ and Imp␣/␤ binding could be estimated as the concentration giving a half-maximal increase in anisotropy, yielding values of 33 and 35 nM, respectively, consistent with the ELISA results (see above).
Interactions were assessed further using native gel mobility shift analysis, wherein importins were pre-complexed with SRY(HMG)-GFP and analyzed by native gel electrophoresis in conjunction with fluorescence imaging. Although both Imp␣ and Imp␤ induced changes in the electrophoretic mobility of SRY(HMG)-GFP, the fact that these migration differences occurred only at the highest concentrations of Imp␣ and to a much lesser extent than that observed for Imp␤ further demonstrates that Importin-␤ binds SRY(HMG)-GFP with higher affinity than mImp␣ (Fig. 4). Thus, in all binding assays, SRY-(HMG) was clearly recognized by Imp␤ with higher affinity than Imp␣, suggesting that nuclear import of SRY is likely to be mediated through direct interaction with Imp␤ independent of Imp␣.
The C-terminal NLS Mediates Imp␤ Recognition-To determine whether one or both NLSs contributed to the high affinity interaction of Imp␤ for SRY, synthetic peptides comprising the  (53), where x is the total concentration of probe (i.e. free ϩ bound importins), B is the level of importin bound, and p is the total concentration of protein, with the apparent dissociation constants (K d ) representing the importin concentration yielding half-maximal binding. The results are from a single typical experiment performed in triplicate with pooled data shown in Table I. a Data represent the mean Ϯ S.E. (n indicated) for the apparent dissociation constant (K d ) and maximal binding determined by ELISA-based assay as outlined under "Materials and Methods" (see Fig. 2). b B max expressed as percent of binding of mImp␣/␤ to SRY(HMG). c B max expressed as percent of binding of mImp␣/␤ to C-NLS peptide. d B max expressed as percent of binding of mImp␣/␤ to T-ag-NLS peptide. e Data represent the mean Ϯ S.E. (n indicated) for the apparent dissociation constant (K d ) and maximal binding determined by FP as outlined under "Materials and Methods" (see Fig. 3). f B max expressed as percent increase in signal due to mImp␣/␤ binding to SRY(HMG)-GFP.
N-and C-terminal NLSs were used in ELISA assays. Binding of Imp␤ to the C-terminal NLS peptide was most similar to that of SRY(HMG) (Fig. 2, bottom left panel) with high affinity interaction being observed for mImp␤ and mImp␣/␤ (K d of 8 and 3 nM, respectively). In contrast, the N-terminal NLS exhibited only low affinity binding for mImp␤ and mImp␣/␤ (K d of 271 and 169 nM, respectively; Fig. 2, right top panel).
We confirmed the contribution of each NLS for Imp␤ recognition using FP. SRY(HMG)-GFP binding to mImp␤ was saturated in the presence and absence of the N-or C-terminal NLS peptides. The C-terminal NLS was able to compete with SRY-(HMG)-GFP for mImp␤ binding, whereas the N-terminal NLS exhibited no significant effect (Fig. 5A). Significantly, a peptide incorporating the NLS region of PTHrP (amino acids 67-94), previously shown to be recognized by Imp␤ (40), did not show any significant effect on binding, suggesting that Imp␤ has multiple recognition sites for NLS substrates.
Although our data suggested that SRY and PTHrP do not bind Imp␤ at the same site, we did observe competition of Imp␤ binding to the bZIP-type TF CREB, which we have recently shown to be recognized by Imp␤ with high affinity (56). CREB-(bZIP-NLS)-GFP was saturated with Imp␤ in the presence and absence of the peptides used in the SRY(HMG)-GFP competition experiments (Fig. 5B). We observed similar binding and competition patterns to that of SRY, suggesting that a common site on Imp␤ may exist for SRY and CREB that is distinct from that for PTHrP.
Recognition of SRY Requires the Imp␤ C Terminus-We have previously demonstrated that the C-terminal domain of Imp␤ may be required for NLS recognition of CREB (56). Based on the observation that SRY and CREB were able to compete for Imp␤ binding, we tested whether C-terminally truncated Imp␤ was able to recognize SRY(HMG)-GFP. Using FP, we observed that only full-length hImp␤ was able to induce a change in anisotropy at both high and low concentrations of importins. Because the molecular weight of the truncated isoforms is sufficient to effect a marked change in anisotropy, the lack of a change in anisotropy observed in their presence was attributable to a lack of high affinity binding to SRY(HMG)-GFP. That the C-terminally truncated form (1-643) has previously been shown able to bind PTHrP further supports the idea of distinct NLS binding sites on Imp␤.
Release of Imp␤ from SRY Can Be Effected by RanGTP-It has been established that the nucleotide bound state of Ran regulates the disassembly of transport substrates from importins (19). RanGTP, maintained at high concentration in the nucleus by the nucleotide exchange factor RCC1, binds Imp␤ with high affinity (ϳ1 nM) and dissociates import substrates from importins, whereas RanGDP, which does not bind Imp␤ (19), is generated in the cytoplasm through Ran-mediated GTP hydrolysis facilitated by the Ran-binding protein-2 (RanBP-2) and the GTPase-activating protein, RanGAP1.
We decided to assess whether RanGTP can dissociate the mImp␤-SRY(HMG) complex in vitro using FP. Imp␤ binding to SRY(HMG)-GFP was saturated, and then RanGDP and RanGTP␥S were added successively to the assay. Subsequent to the increase in anisotropy due to saturation binding of Imp␤ to SRY(HMG)-GFP (Fig. 6), the addition of RanGDP did not FIG. 3. Imp␤ binds SRY(HMG)-GFP with higher affinity than Imp␣ as determined by fluorescence polarization. FP measurements for SRY(HMG)-GFP in the presence of increasing concentrations (Conc) of GST-mImp␣, GST-mImp␤, and GST-mImp␣/␤. Data points represent the average of three measurements performed in triplicate, with the S.D. indicated. Curves were fitted for the (51), where x is the total concentration of probe (free and bound importins), p is the total concentration of protein, and K d (the apparent dissociation constant) is the concentration of importin resulting in half-maximal binding. The results are from a single typical experiment performed in triplicate with pooled data shown in Table I. induce a change in anisotropy, indicating a lack of binding to Imp␤. In contrast, the addition of RanGTP␥S shifted the anisotropy back to a basal level representing free (uncomplexed) SRY(HMG)-GFP through dissociation of the Imp␤-SRY(HMG)-GFP complex (Fig. 6). Thus, RanGTP␥S but not RanGDP was able to dissociate Imp␤ from SRY(HMG)-GFP.
Mutually Exclusive Binding of SRY for Imp␤ and DNA-A previous study characterizing the nuclear import pathway of the yeast TF GAL4 revealed competitive binding between Imp␤ and DNA (36). With SRY demonstrating high affinity recognition by Imp␤, we investigated whether the DNA binding activity of SRY was influenced by Imp␤. First, we demonstrated the ability of SRY(HMG)-GFP to recognize a short Texas Redlabeled oligonucleotide incorporating the recognition sequence of SRY. We found that SRY(HMG)-GFP bound the labeled probe resulting in a mobility shift (Fig. 8, middle panel, lanes 6  and 7). Interestingly, the DNA-bound form of SRY(HMG)-GFP migrated with greater mobility than unbound SRY(HMG)-GFP (Fig. 8, left panel, lanes 1, 6, and 7). This is likely to be the result of a change in charge/mass ratio upon SRY(HMG)-GFP binding the negatively charge oligonucleotide. To confirm that DNA binding was specific, we added unlabeled oligonucleotide in excess to Texas Red-labeled probe to compete for DNA binding (Fig. 8, lane 7). The relatively low affinity binding of SRY gave rise to a smear in the native gel due to DNA bound (faster moving) and unbound (slower moving) SRY forms under the conditions of limiting DNA. Under saturating DNA conditions (Fig. 8, lane 7), most of the SRY(HMG)-GFP fusion protein ran as the faster moving DNA-bound form.
We then carried out electrophoresis in the presence of importin subsequent to the incubation of SRY(HMG)GFP with DNA. Interestingly, we observed that whereas Imp␣ did not affect binding of SRY to its recognition site (Fig. 8, lane 8), Imp␤ and Imp␣/␤ were able to compete for SRY(HMG)-GFP binding to DNA (Fig. 8, lanes 9 and 10). The fact that we observed a prominent band similar to that for saturating levels of DNA is attributable to Imp␤ competing with DNA binding to reduce the amount of SRY(HMG)-GFP that can bind the probe. This increases the ratio of effective probe to DNA-binding protein, similar to that in the presence of high levels of DNA.

DISCUSSION
In this study we demonstrate that nuclear import of SRY is dependent on Imp␤, but not Imp␣, and that Imp␤ is able to recognize SRY directly as demonstrated using a range of assays. Furthermore, we show that the C-terminal NLS of SRY is responsible for mediating the high affinity interaction with Imp␤. We also show that this interaction is sensitive to dissociation by Ran in the GTP-bound form but not in the GDPbound form.
In conventional NLS-dependent nuclear protein import, it is Imp␣ that mediates interaction with the NLS substrate, whereas Imp␤ performs a docking role for the complex at the NPC. Although it is acknowledged that the affinity with which NLS receptors bind their substrate is a critical parameter for nuclear import (24,26), there are very few studies that examine this interaction directly in quantitative fashion, and these have focused mainly on the Imp␣-recognized T-ag, NLS (43,45,46,50,57). The affinity with which Imp␤ binds its nuclear import substrates is clearly of paramount importance, because in the cellular context there is likely to be intense competition between NLS-carrying substrates for NLS receptors (24). Using FP, ELISA-based binding assays, and gel mobility shift analysis, we show that Imp␤ binds strongly to SRY with nM affinity, comparable with the binding observed for Imp␣/␤ to the T-ag or N1N2 NLSs (50). Thus, the initial nuclear translocation event of NLS substrate-importin recognition is likely to be as efficient for SRY-Imp␤ as for conventional NLS-containing substrates (see also Ref. 56).
That SRY contains two functional NLSs within its DNAbinding HMG domain has important implications for its nuclear import. Although we have described a nuclear import mechanism for SRY mediated by direct, high affinity recognition by Imp␤ of the C-terminal NLS, it is clear that the Nterminal NLS can function independently of the C-terminal NLS to target a carrier protein to the nucleus (14). Our observations here are not inconsistent with this idea, in that anti-Imp␤ antibodies significantly reduce, but do not abolish, SRY nuclear accumulation ( Fig. 1 and data not shown), with the residual transport presumably mediated by the N-terminal NLS. The exact nuclear import pathway utilized by the Nterminal NLS is unclear because it is clearly not recognized by either Imp␤1 or Imp␣/␤, but it is intriguing that it appears to be able to bind calmodulin directly with high affinity (58) and that a calmodulin-dependent, Ran-independent nuclear import pathway has been reported (59).
That Ran is likely to be an essential component of the SRY nuclear import pathway is indicated by the fact that GTPbound Ran was able to dissociate the Imp␤-SRY complex (Fig.  7). Under normal conditions, Ran is maintained at high concentration in its GTP-bound form in the nucleus, with the GDP-bound form predominant in the cytoplasm (27). This gradient of Ran across the nuclear envelope determines the directionality of nuclear transport, RanGTP dissociating the impor-tin-nuclear import substrate complex on the nucleoplasmic side of the NPC upon binding to importin ␤ (27). Our in vitro results that RanGTP can dissociate the SRY-Imp␤ complex imply that SRY is likely to be released from the NPC into the nucleus by a similar mechanism. Although it seems very likely that the release of SRY from Imp␤ at the NPC is effected by RanGTP, the fact that Imp␤ and DNA binding are mutually exclusive is intriguing in this context and parallels observations for GAL4 (36) and the TATA-binding protein, which is specifically recognized by the importin ␤ homolog Kap114 (60). It is known that the promoters of active genes are generally localized very closely to NPCs (8), so that it does not seem unreasonable to speculate that competition for Imp␤ binding by specific promoter sequences in the vicinity may assist in transport substrate release on the nucleoplasmic side of the NPC, possibly as a "fail-safe" mechanism (24,36,58). In the context of competition between NLS-carrying substrates for NLS receptors, this may conceivably be of importance, especially in terms of the competition for transport factors such as Imp␤ and Ran, utilized/required by multiple nuclear import substrates/pathways (24); e.g. in a situation where nuclear RanGTP may be limiting, DNA binding-effected release from the NPC might be critical in overcoming a potential bottleneck in SRY nuclear import. An analogous mechanism appears to hold true for the mRNA-binding protein Npl3, which is only released from its nuclear import receptor, Mtr10, by RanGTP in the presence of RNA (61).
Nuclear translocation of SRY is an important developmental event that diverts primordial gonads from the ovarian pathway toward male differentiation to form testes. If this process is disrupted, for example in XY female sex-reversed patients that contain mutations in the NLS regions of SRY, sexual development reverts to the default female pathway (62). Detailed knowledge of the nuclear import pathway of SRY is critical to understanding the mechanism for sex reversal and the specific role of SRY nuclear translocation therein; intriguingly, sexreversing mutations have been reported to map to the N-and C-terminal NLSs of SRY (14). Future work in this laboratory will be focused on unearthing the role of the SRY Imp␤-recognized C-terminal NLS in SRY nuclear localization, and how this may relate to the function of its novel calmodulin-binding N-terminal NLS. FIG. 8. SRY binding to DNA and Imp␤ are mutually exclusive as demonstrated by a dual fluorescence native gel mobility shift assay. Gel image subsequent to electrophoresis of SRY-(HMG)-GFP (1 M) without (lane 1) or with preincubation (15 min) with GST-Imp␣, GST-Imp␤, and GST-Imp␣/␤ (3 M; lanes 2, 3, and 4, respectively) and in the absence (lanes 1-4) or presence of SRY oligonucleotide labeled with Texas Red (0.1 M; lanes 5-10). The SRY oligonucleotide was incubated in the absence (lane 5) and the presence of SRY(HMG)-GFP (lanes 5-10), which was also incubated in the presence of excess (10-fold) unlabeled probe (lane 7) and importins (lanes 8 -10) as indicated. Binding of SRY(HMG)-GFP to DNA, competable by excess unlabeled DNA (lane 7), was not affected in the presence of Imp␣ (lane 8) but was reduced by Imp␤ (lane 9) and Imp␣/␤ (lane 10). GFP (green) and the Texas Red DNA probe (red) are shown in the left and middle panels, respectively, with the merged image on the right.