Title: Importin α/β Mediates Nuclear Transport of a Mammalian Circadian Clock Component, mCRY2, Together with mPER2, through a Bipartite Nuclear Localization Signal
Abstract: 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 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, 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.Hastings M.H. Reddy A.B. Maywood E.S. Nat. Rev. Neurosci. 2003; 4: 649-661Crossref PubMed Scopus (940) Google Scholar), 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.Konopka R.J. Benzer S. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 2112-2116Crossref PubMed Scopus (1631) Google Scholar, 3.Allada R. Emery P. Takahashi J.S. Rosbash M. Annu. Rev. Neurosci. 2001; 24: 1091-1119Crossref PubMed Scopus (262) Google Scholar, 4.Young M.W. Kay S.A. Nat. Rev. Genet. 2001; 2: 702-715Crossref PubMed Scopus (921) Google Scholar). A missense mutation of human Per2 causes a specific disturbance of the sleep-wake cycle, known as familial advanced sleep phase syndrome (5.Toh K.L. Jones C.R. He Y. Eide E.J. Hinz W.A. Virshup D.M. Ptacek L.J. Fu Y.H. Science. 2001; 291: 1040-1043Crossref PubMed Scopus (1165) Google Scholar). 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.Takumi T. Matsubara C. Shigeyoshi Y. Taguchi K. Yagita K. Maebayashi Y. Sakakida Y. Okumura K. Takashima N. Okamura H. Genes Cells. 1998; 3: 167-176Crossref PubMed Scopus (193) Google Scholar, 7.Balsalobre A. Damiola F. Schibler U. Cell. 1998; 93: 929-937Abstract Full Text Full Text PDF PubMed Scopus (1550) Google Scholar). 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.Allada R. Emery P. Takahashi J.S. Rosbash M. Annu. Rev. Neurosci. 2001; 24: 1091-1119Crossref PubMed Scopus (262) Google Scholar, 4.Young M.W. Kay S.A. Nat. Rev. Genet. 2001; 2: 702-715Crossref PubMed Scopus (921) Google Scholar, 8.Dunlap J.C. Cell. 1999; 96: 271-290Abstract Full Text Full Text PDF PubMed Scopus (2341) Google Scholar, 9.Reppert S.M. Weaver D.R. Nature. 2002; 418: 935-941Crossref PubMed Scopus (3334) Google Scholar). 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.Sancar A. Annu. Rev. Biochem. 2000; 69: 31-67Crossref PubMed Scopus (239) Google Scholar, 11.Sancar A. J. Biol. Chem. 2004; 279: 34079-34082Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Mice lacking the mCRY1 or mCRY2 display accelerated and delayed free running periodicity of locomotor activity, respectively (12.Thresher R.J. Vitaterna M.H. Miyamoto Y. Kazantsev A. Hsu D.S. Petit C. Selby C.P. Dawut L. Smithies O. Takahashi J.S. Sancar A. Science. 1998; 282: 1490-1494Crossref PubMed Scopus (331) Google Scholar, 13.van der Horst G.T. Muijtjens M. Kobayashi K. Takano R. Kanno S. Takao M. de Wit J. Verkerk A. Eker A.P. van Leenen D. Buijs R. Bootsma D. Hoeijmakers J.H. Yasui A. Nature. 1999; 398: 627-630Crossref PubMed Scopus (1115) Google Scholar). In the absence of both proteins, an instantaneous and complete loss of free running rhythmicity is observed (13.van der Horst G.T. Muijtjens M. Kobayashi K. Takano R. Kanno S. Takao M. de Wit J. Verkerk A. Eker A.P. van Leenen D. Buijs R. Bootsma D. Hoeijmakers J.H. Yasui A. Nature. 1999; 398: 627-630Crossref PubMed Scopus (1115) Google Scholar, 14.Vitaterna M.H. Selby C.P. Todo T. Niwa H. Thompson C. Fruechte E.M. Hitomi K. Thresher R.J. Ishikawa T. Miyazaki J. Takahashi J.S. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12114-12119Crossref PubMed Scopus (560) Google Scholar), 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.Oster H. Yasui A. van der Horst G.T. Albrecht U. Genes Dev. 2002; 16: 2633-2638Crossref PubMed Scopus (102) Google Scholar). 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.Reppert S.M. Weaver D.R. Nature. 2002; 418: 935-941Crossref PubMed Scopus (3334) Google Scholar, 16.Kume K. Zylka M.J. Sriram S. Shearman L.P. Weaver D.R. Jin X. Maywood E.S. Hastings M.H. Reppert S.M. Cell. 1999; 98: 193-205Abstract Full Text Full Text PDF PubMed Scopus (1296) Google Scholar, 17.Griffin Jr., E.A. Staknis D. Weitz C.J. Science. 1999; 286: 768-771Crossref PubMed Scopus (507) Google Scholar). 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 Iϵ (CKIϵ) 1The abbreviations used are: CKIϵ, casein kinase Iϵ; NLS, nuclear localization signal; GST, glutathione S-transferase; GFP, green fluorescent protein; HA, hemagglutinin; DTT, dithiothreitol; MBP, maltose-binding protein; IBB, importin β binding domain of importin α; WT, wild type; CKI, casein kinase I; Ran, Ras-related nuclear protein; MT, mutant. 1The abbreviations used are: CKIϵ, casein kinase Iϵ; NLS, nuclear localization signal; GST, glutathione S-transferase; GFP, green fluorescent protein; HA, hemagglutinin; DTT, dithiothreitol; MBP, maltose-binding protein; IBB, importin β binding domain of importin α; WT, wild type; CKI, casein kinase I; Ran, Ras-related nuclear protein; MT, mutant. (18.Lee C. Etchegaray J.P. Cagampang F.R. Loudon A.S. Reppert S.M. Cell. 2001; 107: 855-867Abstract Full Text Full Text PDF PubMed Scopus (911) Google Scholar, 19.Shearman L.P. Sriram S. Weaver D.R. Maywood E.S. Chaves I. Zheng B. Kume K. Lee C.C. van der Horst G.T. Hastings M.H. Reppert S.M. Science. 2000; 288: 1013-1019Crossref PubMed Scopus (1112) Google Scholar). Therefore, nuclear entry of the mPER and mCRY proteins is a vital checkpoint for progression of the clockwork cycle (9.Reppert S.M. Weaver D.R. Nature. 2002; 418: 935-941Crossref PubMed Scopus (3334) Google Scholar). 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.Vielhaber E. Eide E. Rivers A. Gao Z.H. Virshup D.M. Mol. Cell. Biol. 2000; 20: 4888-4899Crossref PubMed Scopus (244) Google Scholar, 21.Miyazaki K. Mesaki M. Ishida N. Mol. Cell. Biol. 2001; 21: 6651-6659Crossref PubMed Scopus (71) Google Scholar, 22.Hirayama J. Nakamura H. Ishikawa T. Kobayashi Y. Todo T. J. Biol. Chem. 2003; 278: 35620-35628Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 23.Zhu H. Conte F. Green C.B. Curr. Biol. 2003; 13: 1653-1658Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar); 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.Yoneda Y. Genes Cells. 2000; 5: 777-787Crossref PubMed Scopus (168) Google Scholar). It has been known that there exist many different ways of nuclear transport. One transport pathway is mediated by the importin β-like transport receptor family molecule via importin α family molecules. In contrast to a single gene for importin β in mammals, importin α constitutes a multigene family, and the family can be classified into three distinct subgroups: α1 (Rch1), α3 (Qip-1), and α5 (NPI-1) (25.Kohler M. Ansieau S. Prehn S. Leutz A. Haller H. Hartmann E. FEBS Lett. 1997; 417: 104-108Crossref PubMed Scopus (204) Google Scholar, 26.Tsuji L. Takumi T. Imamoto N. Yoneda Y. FEBS Lett. 1997; 416: 30-34Crossref PubMed Scopus (119) Google Scholar). 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-β superfamily without interaction with importin α (27.Nagoshi E. Imamoto N. Sato R. Yoneda Y. Mol. Biol. Cell. 1999; 10: 2221-2233Crossref PubMed Scopus (103) Google Scholar, 28.Kurisaki A. Kose S. Yoneda Y. Heldin C.H. Moustakas A. Mol. Biol. Cell. 2001; 12: 1079-1091Crossref PubMed Scopus (152) Google Scholar, 29.Lee S.J. Sekimoto T. Yamashita E. Nagoshi E. Nakagawa A. Imamoto N. Yoshimura M. Sakai H. Chong K.T. Tsukihara T. Yoneda Y. Science. 2003; 302: 1571-1575Crossref PubMed Scopus (171) Google Scholar). Finally, examples of Ran and energy- or importin-β-independent nuclear transport mechanisms have been reported (30.Yokoya F. Imamoto N. Tachibana T. Yoneda Y. Mol. Biol. Cell. 1999; 10: 1119-1131Crossref PubMed Scopus (205) Google Scholar, 31.Miyamoto Y. Hieda M. Harreman M.T. Fukumoto M. Saiwaki T. Hodel A.E. Corbett A.H. Yoneda Y. EMBO J. 2002; 21: 5833-5842Crossref PubMed Scopus (88) Google Scholar). 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 3′ end and subcloned into the SmaI and XhoI sites of pGEX-6P-1 (Amersham Biosciences). The sequence of the primers are as follows: 5′-GG TCA CCC GGG TAT GGC GGC GGC TGC TGT GGT-3′, 5′-GG TCA CTC GAG TCA TCA CTT GTC ATC GTC GTC CTT GTA GTC GGA GTC CTT GCT TGC TGG-3′, respectively. The resultant construct is referred to as pGEX mCRY2-FL. For construction of HA-mPER2 in the mammalian expression vector, the primers used are as follows: HA-mPer2, 5′-ATG CTG TAT CCT TAT GAC GTG CCT GAC TAT GCC AAT GGA TAC GTG GAC TTC TCC CCA AG-3′; and mPer2C, 5′-TTA CGT CTG GGC CTC TAT CCT GGG-3′. The fragment amplified was subcloned to the pTargeT mammalian expression vector (Promega). To generate the GST-NLS-GFP expression vector pGST-NLS-GFP, the oligonucleotide encoding SV40 large T-antigen NLS (PKKKRKVEDP) was ligated into the BamHI-SmaI sites of pGST-GFP (32.Tachibana T. Hieda M. Sekimoto T. Yoneda Y. FEBS Lett. 1996; 397: 177-182Crossref PubMed Scopus (46) Google Scholar). For mutagenesis of NLS in mCRY2, the primers used are as follows: PKRK-F, 5′-TCC AGT GGC CCA GCT TCC CCC GCA GCC GCG CTG GAA GCA GCC GAG-3′; PKRK-Rv, 5′-CTC GGC TGC TTC CAG CGC GGC TGC GGG GGA AGC TGG GCC ACT GGA-3′; KRAR-F, 5′-GGT GAA GAA CTG ACC GCG GCG GCT GCA GTG ACG GAG ATG CCT ACC-3′; KRAR-Rv, 5′-GGT AGG CAT CTC CGT CAC TGC AGC CGC CGC GGT CAG TTC TTC ACC-3′; KKVKR(WT)-F, 5′-A ATT CTC TTC TAC TAC CGC CTG TGG GAC TTG TAC AAG AAG GTG AAG AGG AAC AGC ACA CCC CCC CTC TGA G-3′; KKVKR(WT)-R, 5′-GA TCC TCA GAG GGG GGG TGT GCT GTT CCT CTT CAC CTT CTT GTA CAA GTC CCA CAG GCG GTA GTA GAA GAG-3′; KKVKR(MT)-F, 5′-A ATT CTC TTC TAC TAC CGC CTG TGG GAC TTG TAC GCG GCG GTG GCG GCG AAC AGC ACA CCC CCC CTC TGA G-3′; KKVKR(MT)-R, 5′-GA TCC TCA GAG GGG GGG TGT GCT GTT CGC CGC CAC CGC CGC GTA CAA GTC CCA CAG GCG GTA GTA GAA GAG-3′; F5, 5′-G GTC AGA ATT CGC AAC ACA GGC CCC AGA-3′; R1, 5′-TCA GGA TCC TCA TGC TGG CTC TTG GGT AGG-3′. 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 α3 (Qip-1) polyclonal antibodies were prepared as described previously (33.Miyamoto Y. Imamoto N. Sekimoto T. Tachibana T. Seki T. Tada S. Enomoto T. Yoneda Y. J. Biol. Chem. 1997; 272: 26375-26381Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Goat polyclonal anti-importin α1 (Rch1/karyopherin α2) and anti-importin α5 (NPI-1/karyopherin α1), mouse monoclonal anti-importin β, 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 μg/ml ampicillin at 37 °C to a density of 0.6 (A600). Expression was induced by adding isopropyl-β-d-thiogalactopyranoside (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 μg/ml each aprotinin, leupeptin, and pepstatin), using a 125 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. Expression and purification of mouse importin β (PTAC97) was performed as described previously (34.Kose S. Imamoto N. Tachibana T. Shimamoto T. Yoneda Y. J. Cell Biol. 1997; 139: 841-849Crossref PubMed Scopus (148) Google Scholar), as were the purification of GST-mouse importin α1 (Rch1) (35.Tachibana T. Hieda M. Miyamoto Y. Kose S. Imamoto N. Yoneda Y. Cell Struct. Funct. 2000; 25: 115-123Crossref PubMed Scopus (17) Google Scholar), α3 (Qip-1) (35.Tachibana T. Hieda M. Miyamoto Y. Kose S. Imamoto N. Yoneda Y. Cell Struct. Funct. 2000; 25: 115-123Crossref PubMed Scopus (17) Google Scholar), α7(NPI-2) (35.Tachibana T. Hieda M. Miyamoto Y. Kose S. Imamoto N. Yoneda Y. Cell Struct. Funct. 2000; 25: 115-123Crossref PubMed Scopus (17) Google Scholar), human p10/NTF2 (32.Tachibana T. Hieda M. Sekimoto T. Yoneda Y. FEBS Lett. 1996; 397: 177-182Crossref PubMed Scopus (46) Google Scholar), wild type and Q69L Ran (35.Tachibana T. Hieda M. Miyamoto Y. Kose S. Imamoto N. Yoneda Y. Cell Struct. Funct. 2000; 25: 115-123Crossref PubMed Scopus (17) Google Scholar), and maltose-binding protein-importin β binding domain of importin α (MBP-IBB) (36.Nagoshi E. Yoneda Y. Mol. Cell. Biol. 2001; 21: 2779-2789Crossref PubMed Scopus (56) Google Scholar). Transfection and Microinjection—For transfection, NIH3T3 or COS7 cells were transiently transfected using Lipofectamine (Invitrogen) as described previously (37.Takumi T. Nagamine Y. Miyake S. Matsubara C. Taguchi K. Takekida S. Sakakida Y. Nishikawa K. Kishimoto T. Niwa S. Okumura K. Okamura H. Genes Cells. 1999; 4: 67-75Crossref PubMed Scopus (60) Google Scholar). 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 μg/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.Nagoshi E. Imamoto N. Sato R. Yoneda Y. Mol. Biol. Cell. 1999; 10: 2221-2233Crossref PubMed Scopus (103) Google Scholar, 33.Miyamoto Y. Imamoto N. Sekimoto T. Tachibana T. Seki T. Tada S. Enomoto T. Yoneda Y. J. Biol. Chem. 1997; 272: 26375-26381Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Each GST or GST fusion protein was immobilized on 30 μl of glutathione-Sepharose 4B and mixed with 100 pmol of affinity-purified recombinant Rch1, Qip-1, NPI-2, or importin β, and total reaction volume was adjusted to 100 μl 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.Miyamoto Y. Imamoto N. Sekimoto T. Tachibana T. Seki T. Tada S. Enomoto T. Yoneda Y. J. Biol. Chem. 1997; 272: 26375-26381Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). In Vitro Transport Assay—Transport assays were performed as described previously (27.Nagoshi E. Imamoto N. Sato R. Yoneda Y. Mol. Biol. Cell. 1999; 10: 2221-2233Crossref PubMed Scopus (103) Google Scholar). Briefly, HeLa cells were plated at a density of 5 × 105 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 μg/ml digitonin (Nacalai Tesque, Kyoto, Japan; diluted from a 20 mg/ml stock solution in Me2SO), 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 μl 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.Imamoto N. Tachibana T. Matsubae M. Yoneda Y. J. Biol. Chem. 1995; 270: 8559-8565Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Immunoprecipitation—Immunoprecipitation was performed as described previously (37.Takumi T. Nagamine Y. Miyake S. Matsubara C. Taguchi K. Takekida S. Sakakida Y. Nishikawa K. Kishimoto T. Niwa S. Okumura K. Okamura H. Genes Cells. 1999; 4: 67-75Crossref PubMed Scopus (60) Google Scholar). 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 μg 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). 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 = 96) 30 min after injection, which is consistent with previous studies (12.Thresher R.J. Vitaterna M.H. Miyamoto Y. Kazantsev A. Hsu D.S. Petit C. Selby C.P. Dawut L. Smithies O. Takahashi J.S. Sancar A. Science. 1998; 282: 1490-1494Crossref PubMed Scopus (331) Google Scholar, 39.Kobayashi K. Kanno S. Smit B. van der Horst G.T. Takao M. Yasui A. Nucleic Acids Res. 1998; 26: 5086-5092Crossref PubMed Scopus (81) Google Scholar). The nuclear transport of mCRY2 was hindered on ice (97% in the cytoplasm, n = 36) or by co-injection with wheat germ agglutinin (100% in the cytoplasm, n = 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.Finlay D.R. Newmeyer D.D. Price T.M. Forbes D.J. J. Cell Biol. 1987; 104: 189-200Crossref PubMed Scopus (375) Google Scholar, 41.Yoneda Y. Imamoto-Sonobe N. Yamaizumi M. Uchida T. Exp. Cell Res. 1987; 173: 586-595Crossref PubMed Scopus (176) Google Scholar). 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.Carey K.L. Richards S.A. Lounsbury K.M. Macara I.G. J. Cell Biol. 1996; 133: 985-996Crossref PubMed Scopus (133) Google Scholar). The nuclear transport of mCRY2 was dependent on Ran-GDP (99% in the nucleus, n = 97), which is essential for nuclear cytoplasmic transport across the nuclear envelope (43.Gorlich D. Henklein P. Laskey R.A. Hartmann E. EMBO J. 1996; 15: 1810-1817Crossref PubMed Scopus (361) Google Scholar), whereas co-injection with the dominant negative Ran-GTP inhibited the nuclear localization of mCRY2 (86% in the cytoplasm, n = 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 wild-type 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 = 72 and n = 68, respectively), whereas pEGFPx3 (trimer of EGFP), as a negative control, stayed mostly in the cytoplasm (98% in the cytoplasm, n = 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 = 223), whereas the mutant NLS2 (PAAA KRAR or PKRK AAAA) diffused in the whole cell (94% or 95% in the nucleus and cytoplasm, n = 108 and n = 145, respectively), and the double mutant NLS2 (PAAA AAAA) was localized to the cytoplasm (94% in the cytoplasm, n = 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 = 448), whereas those with the mutant NLS2 (PAAA KRAR or PKRK AAAA) diffused in the cell (both 96% in the nucleus and cytoplasm, n = 290 and n = 338, respectively), and those with the double mutant NLS2 (PAAA AAAA) were localized to the cytoplasm (86% in the cytoplasm, n = 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