Importin α/β Mediates Nuclear Transport of a Mammalian Circadian Clock Component, mCRY2, Together with mPER2, through a Bipartite Nuclear Localization Signal*

Circadian rhythms, which period is approximately one day, are generated by endogenous biological clocks. These clocks are found throughout the animal kingdom, as well as in plants and even in prokaryotes. Molecular mechanisms for circadian rhythms are based on transcriptional oscillation of clock component genes, consisting of interwoven autoregulatory feedback loops. Among the loops, the nuclear transport of clock proteins is a crucial step for transcriptional regulation. In the present study, we showed that the nuclear entry of mCRY2, a mammalian clock component, is mediated by the importin α/β system through a bipartite nuclear localization signal in its carboxyl end. In vitro transport assay using digitonin-permeabilized cells demonstrated that all three importin αs, α1 (Rch1), α3 (Qip-1), and α7 (NPI-2), can mediate mCRY2 import. mCRY2 with the mutant nuclear localization signal failed to transport mPER2 into the nucleus of mammalian cultured cells, indicating that the nuclear localization signal identified in mCRY2 is physiologically significant. These results suggest that the importin α/β system is involved in nuclear entry of mammalian clock components, which is indispensable to transcriptional oscillation of clock genes.

Circadian rhythms, which period is approximately one day, are generated by endogenous biological clocks. These clocks are found throughout the animal kingdom, as well as in plants and even in prokaryotes. Molecular mechanisms for circadian rhythms are based on transcriptional oscillation of clock component genes, consisting of interwoven autoregulatory feedback loops. Among the loops, the nuclear transport of clock proteins is a crucial step for transcriptional regulation. In the present study, we showed that the nuclear entry of mCRY2, a mammalian clock component, is mediated by the importin ␣/␤ system through a bipartite nuclear localization signal in its carboxyl end. In vitro transport assay using digitonin-permeabilized cells demonstrated that all three importin ␣s, ␣1 (Rch1), ␣3 (Qip-1), and ␣7 (NPI-2), can mediate mCRY2 import. mCRY2 with the mutant nuclear localization signal failed to transport mPER2 into the nucleus of mammalian cultured cells, indicating that the nuclear localization signal identified in mCRY2 is physiologically significant. These results suggest that the importin ␣/␤ system is involved in nuclear entry of mammalian clock components, which is indispensable to transcriptional oscillation of clock genes.
Circadian rhythms, periodicities with a near 24-h length, constitute a fundamental physiological function that is seen in nearly all organisms from prokaryotes to humans. The circadian system is generally viewed as consisting of three components: input, oscillator, and output. The input or entraining signal is most often light but can also be other environmental cues, such as temperature, feeding, and social cues. In mammals, the light signals acting on the ganglion cells of the retina are conveyed through the retinohypothalamic tract to the suprachiasmatic nuclei in the anterior hypothalamus, which is a major circadian center. The clock's output drives different kinds of physiological phenomenon including locomotor activity, sleep-wake cycles, and hormonal secretion. The disturbance of circadian rhythms causes, not only sleep-wake disorders, but likely also cardiovascular diseases, psychiatric disorders, cancers, and many other diseases (1), as well as contemporary social problems, such as jet lag, and can be the result of shift work.
Recent findings indicate that mutations of clock genes cause abnormal behaviors in vivo from fly to humans. The first clock mutant, period in Drosophila, was identified as a period gene (2)(3)(4). A missense mutation of human Per2 causes a specific disturbance of the sleep-wake cycle, known as familial advanced sleep phase syndrome (5). Perhaps one of the most surprising results, after the identification of clock genes, is that a molecular clock resides, not only in the suprachiasmatic nuclei in mammals, but also in peripheral tissues and even in the immortalized cells (6,7). It is now generally accepted that molecular mechanisms of the circadian rhythms are based on the interlocked autoregulatory feedback loops of the transcription of the clock genes (3,4,8,9).
Murine mCRY1 and mCRY2, two mammalian cryptochromes, were originally thought to be the circadian photoreceptor but are now considered as negative elements of mPER/mCRY feedback loops (10,11). Mice lacking the mCRY1 or mCRY2 display accelerated and delayed free running periodicity of locomotor activity, respectively (12,13). In the absence of both proteins, an instantaneous and complete loss of free running rhythmicity is observed (13,14), indicating mCRY1 and mCRY2 are likely core molecules of circadian clocks, as described above. Furthermore, mouse mCRY1 and mCRY2 seem to pull the circadian oscillator in opposing directions (15). Both mammalian mCRY1 and mCRY2 inhibit BMAL1/CLOCK-dependent transcriptional activation through E-boxes in the mPer/mCry promoters by their nuclear entry together with mPER1 and mPER2 (9,16,17). Thus mCry1 and mCry2 are essential components of the negative limb of the circadian clock feedback loops in mammals. Analysis of clock proteins in mCRY-deficient mice showed that mCRYs are necessary for stabilizing phosphorylated mPER2 and for the nuclear accumulation of mPER1, mPER2, and casein kinase I⑀ (CKI⑀) 1 (18,19). Therefore, nuclear entry of the mPER and mCRY proteins is a vital checkpoint for progression of the clockwork cycle (9). Nuclear localization signals (NLSs) in mouse PER1 (mPER1), rat PER2 (rPER2), zebra fish CRY1 (zCRY1), and Xenopus CRY1 (xCRY1) have so far been identified (20 -23); however, it is still unclear whether nuclear entry is achieved via importin/karyopherin binding to NLS within the clock molecules or via other mechanisms. In no species is there detailed information on the nuclear entry of CRY2.
Nuclear transport of proteins occurs through nuclear pore complexes and typically requires a specific NLS (24). It has been known that there exist many different ways of nuclear transport. One transport pathway is mediated by the importin ␤-like transport receptor family molecule via importin ␣ family molecules. In contrast to a single gene for importin ␤ in mammals, importin ␣ constitutes a multigene family, and the family can be classified into three distinct subgroups: ␣1 (Rch1), ␣3 (Qip-1), and ␣5 (NPI-1) (25,26). A small GTPase Ran ensures the direction of nuclear transport by regulating the interaction between the receptors and their cargoes through its GTP/GDP cycle. Alternative nuclear import pathways have been described in which the NLS-containing proteins directly bind one of the nuclear import receptors of the importin-␤ superfamily without interaction with importin ␣ (27)(28)(29). Finally, examples of Ran and energy-or importin-␤-independent nuclear transport mechanisms have been reported (30,31).

EXPERIMENTAL PROCEDURES
Plasmid-The full-length cDNA of mCRY2 was amplified from mouse brain cDNA using primers with the following restriction sites and FLAG tag in the 3Ј end and subcloned into the SmaI and XhoI sites of pGEX-6P-1 (Amersham Biosciences). The sequence of the primers are as follows: Fragments amplified by PCR using the primers above (PKRK-F and PKRK-Rv for mutation from PKRK to PAAA, KRAR-F and KRAR-Rv for mutation from KRAR to AAAA) as a template of mCRY2-FLAG were digested with KpnI and XhoI and subcloned to the XhoI site of the mCRY2-FLAG plasmid. Further, a double mutant (PAAA AAAA) was made using primers KRAR-F and KRAR-Rv as a template of mCRY2-FLAG PKRK mutant. For NLS1 peptide-type mutants, after annealing with each forward and reverse primer (KKVKR(WT)-F and KKVKR(WT)-R, KKVKR(MT)-F and KKVKR(MT)-R, respectively) at 99°C for 2 min, at 72°C for 5 min, and at room temperature for 3 h, the fragments were digested with EcoRI and BamHI and subcloned to pEGFPx3. In the case of NLS2 peptide-type mutants, using primers F5 and R1, amplified fragments of the full-length mutants were used as templates.
Antibodies-Rabbit anti-importin ␣3 (Qip-1) polyclonal antibodies were prepared as described previously (33). Goat polyclonal anti-importin ␣1 (Rch1/karyopherin ␣2) and anti-importin ␣5 (NPI-1/karyopherin ␣1), mouse monoclonal anti-importin ␤, murine IgG1 monoclonal anti-FLAG M2, and anti-HA antibody were purchased from Santa Cruz Biotechnology, BD Biosciences, Kodak, and Clontech, respectively. Expression and Purification of Recombinant Proteins-Escherichia coli strain JM109, which had been transformed with pGEX mCRY2-FL, was grown in LB medium containing 100 g/ml ampicillin at 37°C to a density of 0.6 (A 600 ). Expression was induced by adding isopropyl-␤-Dthiogalactopyranoside (to 0.1 mM final concentration) and then incubating for 18 h at 20°C. Cells were harvested by centrifugation and resuspended in high salt buffer (50 mM Tris-HCl, pH7.4, 500 mM NaCl) containing 1 mM phenylmethylsulfonyl fluoride, 2 mM DTT, and protease inhibitor mixture (1 g/ml each aprotinin, leupeptin, and pepstatin), using a 1 ⁄25 volume of the original cell culture. After one freezethaw cycle, the cells were lysed by a French pressure cell press (Thermo Spectronic, 1000 psi) and sonication after adding 10 mM MgATP and 5 mg/ml casein to dissociate GroEL for 20 min at room temperature. After the extract was clarified by centrifugation, the extract was incubated with glutathione-Sepharose (Amersham Biosciences) for 30 min at room temperature. The recombinant protein-bound Sepharose was washed extensively with wash buffer I (20 mM Tris-HCl, pH7.4, 500 mM NaCl) and with buffer II (20 mM Tris-HCl, pH7.4, 50 mM NaCl) and then incubated with Prescission protease (Amersham Biosciences) at 4°C for overnight. The recombinant mCRY2-FLAG cleaved from the GST moiety was collected from the flow-through of a Polyprep chromatography column (Bio-Rad). The flow-through was subjected to chromatography on a HiTrap-SP cation exchange column (1-ml) on a fast protein liquid chromatography system (Amersham Biosciences) at a flow rate of 0.5 ml/min using a linear gradient from 0.05 to 1 M NaCl in 20 mM Tris-HCl, pH 7.5, 1 mM DTT, and protease inhibitor mixture. Peak fractions containing mCRY2-FLAG (eluted between 0.3 and 0.5 mM NaCl) were pooled and concentrated by ultrafiltration using a microconYM-50 column (Millipore). To see purity, the protein concentrated was eluted by boiling in SDS-PAGE sample buffer, separated on a 10% SDS-polyacrylamide gel, and visualized by Coomassie Blue staining.
Transfection and Microinjection-For transfection, NIH3T3 or COS7 cells were transiently transfected using Lipofectamine (Invitrogen) as described previously (37). For microinjection, NIH3T3 cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum and plated on coverslips 36 -48 h before use. Proteins were injected through a glass capillary into the cytoplasm using a micromanipulator (Narishige MMO-202N, Tokyo, Japan). After incubation for 30 min at 37°C or on ice, the cells were fixed with 3.7% formaldehyde in phosphate-buffered saline for 20 min at room temperature. To examine the localization of the injected proteins, fixed cells were permeabilized with 0.5% Triton X-100 in phosphate-buffered saline for 5 min at room temperature, incubated with 3% skim milk in phosphate-buffered saline for 30 min, and then incubated with 30 g/ml monoclonal anti-FLAG M2 antibody for 1 h at room temperature. The mouse antibody was detected with Cy3-labeled goat anti-mouse IgG (Amersham Biosciences). All samples were examined by Axiophot microscopy (Zeiss).
In Vitro Binding Assay-Solution binding assay for recombinant NLS receptors was performed as described previously (27,33). Each GST or GST fusion protein was immobilized on 30 l of glutathione-Sepharose 4B and mixed with 100 pmol of affinity-purified recombinant Rch1, Qip-1, NPI-2, or importin ␤, and total reaction volume was adjusted to 100 l with 25% bovine serum albumin in transport buffer (TB) (20 mM Hepes-KOH, pH 7.3, 110 mM potassium acetate, 2 mM magnesium acetate, 5 mM sodium acetate, and 0.5 mM EGTA). After incubation for 1 h at 4°C, materials bound were washed with TB and eluted with 0.2% SDS in TB for 5 min at 100°C. Immunoblotting with anti-Rch1, anti-NPI-1 (cross-reacted with NPI-2), or anti-Qip-1 antibodies was performed as described previously (33).
In Vitro Transport Assay-Transport assays were performed as described previously (27). Briefly, HeLa cells were plated at a density of 5 ϫ 10 5 cells/ml on an eight-well multitest slide (ICN, Costa Mesa, CA) 36 -48 h before use. The cells grown on slides were rinsed twice in ice-cold TB and permeabilized for 5 min in ice-cold TB containing 40 g/ml digitonin (Nacalai Tesque, Kyoto, Japan; diluted from a 20 mg/ml stock solution in Me 2 SO), 2 mM DTT, and protease inhibitor mixture. After removing the digitonin-containing buffer, the slides were washed and immersed in ice-cold TB containing 2 mM DTT and protease inhibitor mixture for 10 min. The slides were then blotted to remove excess buffer, and 11 l of reaction mixture/single well were applied to the cell. Import reactions were performed by incubating the slides for 30 min at 30°C, unless otherwise described. After incubation, the cells were rinsed twice in TB and fixed with 3.7% formaldehyde in TB for 15 min at room temperature. Each reaction mixture contained an import substrate combined with cytosol or a combination of recombinant transport factors in the presence or absence of an ATP regeneration system (1 mM ATP, 5 mM creatine phosphate, and 20 units/ml creatine phosphokinase). Total cytosol from Ehrlich ascites tumor cells was prepared as described previously (38).
Immunoprecipitation-Immunoprecipitation was performed as described previously (37). At 48 h post-transfection, lysates were prepared at 4°C and dialyzed in radioimmune precipitation assay lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 2 mM DTT, and protease inhibitor mixture). After washing with phosphate-buffered saline three times, 0.4 g of mouse anti-FLAG M2 and protein G-agarose (Roche Applied Science) were added and rotated at 4°C overnight. After rinsing with radioimmune precipitation assay buffer three times, the supernatant was analyzed on 4 -20% SDS-PAGE and detected by immunoblotting with anti-FLAG M2 or anti-HA monoclonal antibody using the Western Lighting Chemiluminescence Reagent Plus (PerkinElmer Life Sciences).

RESULTS
To examine the molecular mechanism for the nucleocytoplasmic shuttling of mCRY2, we purified the recombinant protein of mCRY2-FLAG (Fig. 1A). As shown in Fig. 1B, when mCRY2-FLAG was microinjected into the cell cytoplasm of the NIH3T3 cells, nuclear import of mCRY2 protein was observed and localized in the nucleus (99% in the nucleus, n ϭ 96) 30 min after injection, which is consistent with previous studies (12,39). The nuclear transport of mCRY2 was hindered on ice (97% in the cytoplasm, n ϭ 36) or by co-injection with wheat germ agglutinin (100% in the cytoplasm, n ϭ 36), suggesting that the import appears to be temperature-dependent and sensitive to the lectin wheat germ agglutinin, which inhibits many nuclear transport mechanisms without affecting passive diffusion through the nuclear pore complex by binding to N-acetylglucosamine-modified nucleoporins (40,41). To study further the involvement of small GTPase Ran in the nuclear import of mCRY2, we used a Q69L Ran mutant, which is deficient in GTPase activity and remains in the GTP-bound state, even in the presence of cytoplasmic RanGAP1 (42). The nuclear transport of mCRY2 was dependent on Ran-GDP (99% in the nucleus, n ϭ 97), which is essential for nuclear cytoplasmic transport across the nuclear envelope (43), whereas co-injection with the dominant negative Ran-GTP inhibited the nuclear localization of mCRY2 (86% in the cytoplasm, n ϭ 110), indicating that the nuclear import of mCRY2 is dependent on a gradient of Ran-GTP across the nuclear envelope. Our results suggest that nuclear localization of mCRY2 is not passive but is dependent on the Ran-GTP-motive nuclear trans-location.
We next targeted the putative NLS in the mCRY2 sequence. In the primary structure of mCRY1, one putative NLS is located in the middle of the sequence. On the other hand, there are two candidate NLS sequences in mCRY2; one is a monopartite type NLS (NLS1) in the middle of the protein (amino acid 292-296), which is conserved between mCRY1 and mCRY2, and another is a bipartite-type NLS (NLS2) in the carboxyl-terminal end (amino acid 558 -578), which is not conserved in mCRY1 ( Fig. 2A). We fused the wild-type or mutant NLSs to GFPx3 and transfected them into NIH3T3 cells to observe the subcellular localization (Fig. 2B). Both the wildtype NLS1 (KKVKR) and mutant NLS1 (AAVAA) did not show clearly different behavior and were seen in both the nucleus and cytoplasm (83 and 84% in the nucleus and cytoplasm, n ϭ 72 and n ϭ 68, respectively), whereas pEGFPx3 (trimer of EGFP), as a negative control, stayed mostly in the cytoplasm (98% in the cytoplasm, n ϭ 123). By contrast, the cellular localization of NLS2 was obviously different between the wild type and mutants. The wild-type NLS2 (PKRK KRAR) was localized to the nucleus (99% in the nucleus, n ϭ 223), whereas the mutant NLS2 (PAAA KRAR or PKRK AAAA) diffused in the whole cell (94% or 95% in the nucleus and cytoplasm, n ϭ 108 and n ϭ 145, respectively), and the double mutant NLS2 (PAAA AAAA) was localized to the cytoplasm (94% in the cytoplasm, n ϭ 142). Moreover, the recombinant mCRY2 proteins, which fused FLAG with wild-type or mutant NLS2s, were purified and microinjected into the cytoplasm of NIH3T3 cells to determine their subcellular localization (Fig. 2C). Consistent with the results of transfection with NLS2-GFPs as described above, mCRY2-FLAG proteins with wild-type NLS2 (PKRK KRAR) were localized to the nucleus (98% in the nucleus, n ϭ 448), whereas those with the mutant NLS2 (PAAA KRAR or PKRK AAAA) diffused in the cell (both 96% in the nucleus and cytoplasm, n ϭ 290 and n ϭ 338, respectively), and those with the double mutant NLS2 (PAAA AAAA) were localized to the cytoplasm (86% in the cytoplasm, n ϭ 408). These findings clearly indicate that NLS2 functionally works as an NLS of mCRY2.
To investigate the requirements of any factors on nuclear transport of mCRY2, we subjected the FLAG-tagged mCRY2 to an in vitro cell-free transport assay using the permeabilized HeLa cells. In this assay, the cytoplasmic membrane is first permeabilized with digitonin, and the soluble endogenous cytosolic factors are depleted. Nuclear entry of a fluorescently labeled protein can be studied in the presence or absence of exogenous nuclear import factors. In a condition where GST alone gave no signals, nuclear accumulation of GST-mCRY2-FLAG was reconstituted in the presence of Ehrlich cytosol and an ATP regenerating system. Nuclear accumulation of GST-mCRY2-FLAG was inhibited by energy depletion, which showed a similar pattern as the control described next (Fig. 3). As a typical basic-type NLS-bearing transport substrate, a chimeric protein consisting of GST fused with SV40 T antigen NLS and GFP (GST-NLS-GFP) was imported with cytosol and ATP and inhibited by energy depletion. Moreover, the mutant mCRY2 with PAAA AAAA in its NLS2 sequence (MT) was not imported even in the presence of Ehrlich cytosol and an ATP regenerating system (Fig. 3). These results demonstrated that the nuclear import of mCRY2 is a facilitated process that is mediated by soluble factors through NLS2 in its carboxylterminal.
The nuclear import machinery of the basic NLS-mediated import constitutes the importin ␣/␤ heterodimer. The results described above showed that mCRY2 containing a bipartite NLS is imported into the nucleus in a temperature-and energydependent manner. Its dependence on Ran and GTP suggested that nuclear entry of mCRY2 most likely occurs through the importin ␤ transport receptor, with importin ␣ as an adaptor protein. To examine which type of importin is involved in this nuclear transport, in vitro binding assays were performed (Fig.  4) using all three kinds of importin ␣s, ␣1 (Rch1/PTAC58), ␣3 (Qip-1), and ␣7 (NPI-2, which is a close family member of ␣5/NPI-1). We used ␣7/NPI-2 as a third representative of importin ␣ instead of ␣5/NPI-1 (which was bound with mCRY2); however, no direct interaction was apparent between importin ␤ and mCRY2. Importin ␣ did not bind to mCRY2 with mutant NLS2 (Fig. 4; a faint band in the case of Qip-1 seems to be a nonspecific one, as explained in the next paragraph). Therefore, the nuclear import of mCRY2 is likely to be mediated by interaction with the importin ␣/␤ heterodimer.
We addressed the issue of whether importin ␣/␤ mediates the nuclear import of mCRY2 using the in vitro nuclear transport system (Fig. 5). In the presence of three kinds of importin ␣s, ␣1 (Rch1/PTAC58), ␣3 (Qip-1), and ␣7 (NPI-2), and importin ␤ in addition to cytosolic factors, Ran and p10/NTF2 (32), GST-mCRY2-FLAG was targeted to the nucleus of the permeabilized HeLa cells (Fig. 5A). GST-mCRY2-FLAG was not reconstituted in the presence of only importin ␤ (without any importin ␣), which was consistent with the data of in vitro binding (see Fig. 4). Furthermore, an excess amount of MBP-IBB blocked the nuclear import of GST-mCRY2-FLAG (WT) in the presence of each of three kinds of importin ␣s with importin ␤ in permeabilized cells, implying that the nuclear transport is dependent on importin ␣ (for example, see Fig. 5B for Qip-1). MBP-IBB did not affect the in vitro assays for MT with Qip-1 to the contrary (Fig. 5B). The result may explain nonspecific in vitro binding of Qip-1 to mutant mCRY2 (see Fig. 4). These results, combined with the binding assay described above, strongly suggest that the nuclear import of mCRY2 is mediated by importin ␣/␤. mCRY2 is known to directly interact with the mPER proteins and to trans-locate them into the nucleus (16,18). To investigate whether the nuclear import machinery described above is involved in the physiological function of the clock proteins, we examined whether the NLS2 sequence of mCRY2 was required for the nuclear transport of mPER2 (Fig. 6). The transfected wild-type mCRY2 and mPER2 into COS7 cells were localized to the nucleus and cytoplasm, respectively (93% in the nucleus and 100% in the cytoplasm, n ϭ 135 and n ϭ 128, respectively). In contrast, the mutant mCRY2, with PAAA AAAA in its NLS2 sequence, was seen in the cytoplasm (80% in the cytoplasm, n ϭ 134), as shown in Fig. 2. When the wild-type mCRY2 and FIG. 2. A bipartite-type nuclear localization signal (NLS2) in the carboxyl-terminal of mCRY2 is functional. A, scheme for primary structures of mCRY1 and mCRY2. Nuclear localization signals (NLS) are depicted as black boxes. Numbers represent amino acid numbers. Basic amino acid sequences in mCRY2 are indicated in single letter code and underlined. B, the wild-type or mutant NLSs fused to pEGFPx3 was transfected to NIH3T3 cells. Quantitative data are shown in the graphs. Black, lined, and white bars indicate nucleus, cytoplasm, and whole cell, respectively. C, mCRY2-FLAG with wildtype or mutant NLSs was transfected to NIH3T3 cells. The localization was detected by indirect immunofluorescence with anti-FLAG M2 antibody. Quantitative data are shown in the graph. Black, lined, and white bars indicate nucleus, cytoplasm, and whole cell, respectively.

FIG. 3. Nuclear transport of mCRY2 requires soluble factors.
Digitonin-permeabilized HeLa cells were incubated with 10 l of a reaction mixture containing 10 pmol of proteins (GST, GST-mCRY2-FLAG, or GST-NLS-GFP) and 4 mg/ml Ehrlich cytosol with or without an ATP regeneration system. The localization was detected with anti-GST antibody. mPER2 were co-transfected, mPER2 was targeted to the nucleus (94% of mCRY2 staining and 93% of mPER2 staining in the nucleus, n ϭ 206 and n ϭ 134, respectively). However, the mutant mCRY2 failed to trans-locate mPER2 into the nucleus (95% of mCRY2 staining and 100% of mPER2 staining in the cytoplasm, n ϭ 152 and n ϭ 129, respectively). This result suggests that NLS2 of mCRY2 may be involved in the nuclear entry of mPER2. To examine the interaction of mCRY2 and mPER2, cell lysates transfected with mCRY2-FLAG and HA-mPER2 were immunoprecipitated with anti-FLAG followed by immunoblotting with anti-HA (Fig. 7). Both the wild-type and mutant mCRY2 showed a binding with mPER2, indicating that the failure of mPER2 to trans-locate into the nucleus is not because of the disability to bind each other physically. Taken together, these results argue that mCRY2 bound with mPER2 is imported to the nucleus through a bipartite NLS2 in the carboxyl-terminal of mCRY2. DISCUSSION This is the first report to show the molecular mechanism for nuclear import of the clock molecules by an in vitro reconstituted system using the semi-intact cells and the purified pro-teins. In general, compared with mCRY1, fewer studies on mCRY2 have appeared so far, despite its unique characteristics among canonical clock genes. Interestingly, mCry2 is the only one among five canonical clock genes in which decreased RNA levels were observed in Clock/Clock animals (16). mCry2 transcript is more abundant than that of mCry1 in the brain; however, mCry2 expression in the suprachiasmatic nuclei is modest (10). In the peripheral tissues, mCry2 transcripts show weaker circadian rhythmicity than those of mPers and Bmal1/ Npas2 (44); however, mCry2 promoter in cultured cells produced circadian oscillation by the in vitro luminescence reporter assays. 2 Despite the central role of mCRY1 and mCRY2 proteins in their nuclear transports with mPERs, only a few reports on nuclear entry of the mammalian CRYs have been reported so far. In the recent study of zebra fish zCRYs, which is a homologue of mCRY1, two regions of the zCRY1a responsible for nuclear trans-localization were identified, and a putative NLS in the middle of its sequence has been reported (22). In Xenopus xCRY1, which is also a homologue of mCRY1, NLS in the carboxyl-terminal tails was shown to be essential for nuclear localization (23). In our present work, the microinjection study using the recombinant mCRY1 proteins showed a possibility for the existence of multiple elements responsible for nuclear entry, which are in the middle and in the carboxyl termini of mCRY1, the results of which are consistent with previous work (22,23). 3 NLS2 of mCRY2 identified here is essential for the nuclear import of mCRY2 bound to mPER2, but the sequence is not conserved in mCRY1 but is unique to mCRY2. The sequence of this bipartite NLS2 is conserved not only in mammals (human, mouse, and rat) but also in another type of vertebrate, the bullfrog. On the other hand, NLS1, the sequence of which is conserved between mCRY2 and mCRY1, was identified as an NLS in mCRY1 (as described above) but does not seem to work in mCRY2. The fact that MT NLS1 (Fig.  2B) and double MT NLS2 (Fig. 2C) are not completely (100%) in the cytoplasm, however, may suggest some involvement of NLS1 in the nuclear import of mCRY2. In fact, the reporter analyses using the Per1 and Per2 promoters showed that transactivating activity (combining CLOCK/BMAL1 with mCRY2 with double MT NLS2) was significantly higher than combining with WT mCRY2, which displays the suppression of transactivating activity of CLOCK/BMAL1 heterodimers (data not shown). However, the activity with double MT NLS2 was lower than the full activation by CLOCK/BMAL1 heterodimers. The lower activity with double MT NLS2 is probably because 14% of cells including the nuclei were not purely in the cytoplasm but in the whole cells (Fig. 2C). In vivo experiments using the knock-out mice revealed the differing tasks of mCRY1 and mCRY2 (15). Similarly, the mechanism of the nuclear transport of mCRY1 and mCRY2 may be different. Although we will have to overcome technical difficulties to purify large proteins such as PERs, further detailed examination using the purified recombinant proteins of clock and nuclear transport machinery components, including a newly found cofactor, Npap60/Nup50 (45), would enable us to understand the difference.
We found that three importin ␣ families are involved in the nuclear import of mCRY2. It has been demonstrated that the expression level and pattern of each importin ␣ family protein differ among different tissues compared with those of importin ␤ (25, 26, 46 -48), suggesting that nuclear import of the classical NLS containing karyophiles may be controlled in a tissuespecific manner by the different expression of importin ␣s. To address whether the mRNA expression level of each importin ␣ is correlated with circadian rhythm, using the quantitative real-time reverse transcription-PCR (37,44), the temporal profile of each importin ␣ (Rch1, Qip-1, and NPI-2) mRNA level was analyzed at the six time points over the third day in constant darkness in mouse peripheral tissues such as heart, liver, and kidney. No robust circadian rhythm of their mRNA expression was observed, however, in these peripheral tissues (data not shown). Provided that molecular clocks reside not only in the central suprachiasmatic nuclei but also in peripheral tissues and that the circadian phase of clock mRNA expression appears to be similar among the different peripheral sites (44), it seems unlikely that nuclear transport of mCRY2 depends on the amount of importin ␣s in each tissue.
As described above, mCRYs are known to be repressor proteins against BMAL1/CLOCK heterodimers and to repress BMAL1/CLOCK-induced trans-activation of mPers in the nucleus. Our experiments with mutants indicated that mCRY2 plays a critical role in trans-location of mPER2. Phosphorylation in the cytoplasm also regulates the timing of nuclear entry of specific clock proteins. CKI has been shown to play a role in regulating the nuclear entry of mammalian PERs (20,49). mPER proteins are phosphorylated by CKI␦ and CKI⑀ (18) and would be unstable or degraded unless bound to mCRY (19). The mammalian PERs seem to be dynamic and may be influenced by additional factors (49). At the time of negative feedback, it is mostly phosphorylated forms of mammalian clock proteins that form complexes in the nucleus (18). Collectively, these results suggest that the timing of nuclear entry of mCRY2 is dependent on the amount of partner protein (such as mPER2) present or its phosphorylation level by CKI.
Only one gene coding for importin ␤ has been identified in the organisms analyzed thus far, whereas several isoforms of importin ␣ have been identified in mammals (24 -26). The ␣ family can be classified into three distinct subgroups: importin ␣1 (Rch1), ␣3 (Qip-1), and ␣5 (NPI-1). Since these diverse subgroups of importin ␣ were discovered, the classical nuclear entry through importin receptors has been considered to be composed of multiple cases. Among these pathways using the importin ␣/␤ system, there are several instances where the major three importin ␣ subfamilies (Rch1, Qip-1, and NPI-1) are involved in nuclear transport similar to mCRY2 described here and other cases where the specific importin ␣ is used for nuclear entry. The former, for example, includes hnRNP K (RNA binding protein) (46), P/CAF (stimulator of the Rous sarcoma virus promoter) (46), Duplin (␤-catenin-binding protein) (50), XCTK2 (Xenopus carboxyl-terminal kinesin) (51), and LEDGF/p75 (HIV-1 integrase interactor) (52). There seems to be no common structural homology and no functional similarity among these molecules. However, the number of reports of the latter cases is less. For example, TBP-2 (thioredoxin-binding protein-2/vitamin D 3 -up-regulated protein 1) (53), RCC1 (chromatin-bound protein) (46), and STAT1 (latent cytoplasmic transcription factor) (54) are specifically imported by Rch1, Qip-1, and NPI-1, respectively. This significance of the diversity of nuclear entry mechanisms and its components is largely unknown, although there is significant ongoing interest in this topic. The structural analyses of a complex of importins and their target proteins, especially including NLS and its near region, may provide insights into the basis of this diversity.
Acknowledgments-We thank Gene Block for reviewing the manuscript and acknowledge Yasukazu Nakahata, Setsuko Tsuboi, Taro Tachibana, and Ippei Kotera for their technical assistance. We also thank Takeshi Todo for kindly providing mCRY2 cDNA.
FIG. 6. NLS2 in mCRY2 is necessary for nuclear entry of mPER2. A, mCRY2-FLAG with wild-type NLS, mCRY2-FLAG with mutant NLS2, and HA-mPER2 were transfected to COS7 cells. B, mCRY2-FLAG with wild-type NLS or mCRY2-FLAG with mutant NLS2 is co-transfected with HA-mPER2. mCRY2 and mPER2 were stained with anti-FLAG M2 antibody and anti-HA antibody, respectively. Quantitative data are shown in the graphs. Black, lined, and white bars indicate nucleus, cytoplasm, and whole cell, respectively. FIG. 7. mCRY2 with either wild-type or mutant NLS2 binds to mPER2. mCRY2-FLAG with wild-type NLS or mCRY2-FLAG with mutant NLS2 is co-transfected with HA-mPER2 or pcDNA3 (as a control). The lysates were immunoblotted with anti-HA antibody (upper panel), immunoprecipitated with anti-FLAG and blotted with anti-HA antibody (middle panel), and immunoprecipitated with anti-FLAG and blotted again with anti-FLAG antibody (lower panel).