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The HIV-1 Tat Nuclear Localization Sequence Confers Novel Nuclear Import Properties

1998; Elsevier BV; Volume: 273; Issue: 3 Linguagem: Inglês

10.1074/jbc.273.3.1623

1083-351X

Athina Efthymiadis, Lyndall J. Briggs, David A. Jans,

HIV Research and Treatment

The different classes of conventional nuclear localization sequences (NLSs) resemble one another in that NLS-dependent nuclear protein import is energy-dependent and mediated by the cytosolic NLS-binding importin/karyopherin subunits and monomeric GTP-binding protein Ran/TC4. Based on analysis of the nuclear import kinetics mediated by the NLS of the human immunodeficiency virus accessory protein Tat usingin vivo and in vitro nuclear transport assays and confocal laser scanning microscopy, we report a novel nuclear import pathway. We demonstrate that the Tat-NLS, not recognized by importin 58/97 subunits as shown using an enzyme-linked immunosorbent assay-based binding assay, is sufficient to target the 476-kDa heterologous β-galactosidase protein to the nucleus in ATP-dependent but cytosolic factor-independent fashion. Excess SV40 large tumor antigen (T-ag) NLS-containing peptide had no significant effect on the nuclear import kinetics implying that the Tat-NLS was able to confer nuclear accumulation through a pathway distinct from conventional NLS-dependent pathways. Nucleoplasmic accumulation of the Tat-NLS-β-galactosidase fusion protein, in contrast to that of a T-ag-NLS-containing fusion protein, also occurred in the absence of an intact nuclear envelope, implying that the Tat-NLS conferred binding to nuclear components. This is in stark contrast to known NLSs such as those of T-ag which confer nuclear entry rather than retention. Significantly, the ability to accumulate in the nucleus in the absence of an intact nuclear envelope was blocked in the absence of ATP, as well as by nonhydrolyzable ATP and GTP analogs, demonstrating that ATP is required to effect release from a complex with insoluble cytoplasmic components. Taken together, the results demonstrate that, dependent on ATP for release from cytoplasmic retention, the Tat-NLS is able to confer nuclear entry and binding to nuclear components. These unique properties indicate that Tat accumulates in the nucleus through a novel import pathway. The different classes of conventional nuclear localization sequences (NLSs) resemble one another in that NLS-dependent nuclear protein import is energy-dependent and mediated by the cytosolic NLS-binding importin/karyopherin subunits and monomeric GTP-binding protein Ran/TC4. Based on analysis of the nuclear import kinetics mediated by the NLS of the human immunodeficiency virus accessory protein Tat usingin vivo and in vitro nuclear transport assays and confocal laser scanning microscopy, we report a novel nuclear import pathway. We demonstrate that the Tat-NLS, not recognized by importin 58/97 subunits as shown using an enzyme-linked immunosorbent assay-based binding assay, is sufficient to target the 476-kDa heterologous β-galactosidase protein to the nucleus in ATP-dependent but cytosolic factor-independent fashion. Excess SV40 large tumor antigen (T-ag) NLS-containing peptide had no significant effect on the nuclear import kinetics implying that the Tat-NLS was able to confer nuclear accumulation through a pathway distinct from conventional NLS-dependent pathways. Nucleoplasmic accumulation of the Tat-NLS-β-galactosidase fusion protein, in contrast to that of a T-ag-NLS-containing fusion protein, also occurred in the absence of an intact nuclear envelope, implying that the Tat-NLS conferred binding to nuclear components. This is in stark contrast to known NLSs such as those of T-ag which confer nuclear entry rather than retention. Significantly, the ability to accumulate in the nucleus in the absence of an intact nuclear envelope was blocked in the absence of ATP, as well as by nonhydrolyzable ATP and GTP analogs, demonstrating that ATP is required to effect release from a complex with insoluble cytoplasmic components. Taken together, the results demonstrate that, dependent on ATP for release from cytoplasmic retention, the Tat-NLS is able to confer nuclear entry and binding to nuclear components. These unique properties indicate that Tat accumulates in the nucleus through a novel import pathway. To enter the eukaryotic cell nucleus, proteins larger than 45 kDa require targeting signals called nuclear localization sequences (NLSs) 1The abbreviations used are: NLS, nuclear localization sequence; T-ag, simian virus SV40 large tumor-antigen; NPC, nuclear pore complex; Tat, human immunodeficiency virus type 1 Tat protein; K D, apparent dissociation constant; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; GST, glutathione S-transferase; CLSM, confocal laser scanning microscopy; HTC, hepatoma tissue culture; ELISA, enzyme-linked immunosorbent assay; AMP-PNP, adenylyl imidodiphosphate; GTPγS, guanosine 5′-3-O-(thio)triphosphate; hnRNP, heterogeneous ribonucleoprotein. defined as the sequences sufficient and necessary for nuclear localization of their respective proteins (1Jans D.A. Hübner S. Physiol. Rev. 1996; 76: 651-685Crossref PubMed Scopus (389) Google Scholar, 2Koepp D.M. Silver P.A. Cell. 1996; 87: 1-4Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). NLSs appear to fall into several classes, including those homologous to the NLS of the simian virus SV40 large tumor-antigen (T-ag) consisting of a single stretch of basic residues (1Jans D.A. Hübner S. Physiol. Rev. 1996; 76: 651-685Crossref PubMed Scopus (389) Google Scholar, 2Koepp D.M. Silver P.A. Cell. 1996; 87: 1-4Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 3Kalderon D. Richardson W.D. Markham A.F. Smith A.E. Nature. 1984; 311: 33-38Crossref PubMed Scopus (910) Google Scholar), those termed bipartite NLSs comprising two clusters of basic amino acids separated by a spacer of 10–12 amino acids (1Jans D.A. Hübner S. Physiol. Rev. 1996; 76: 651-685Crossref PubMed Scopus (389) Google Scholar, 4Robbins J. Dilworth S.M. Laskey R.A. Dingwall C. Cell. 1991; 64: 615-623Abstract Full Text PDF PubMed Scopus (1249) Google Scholar) and those resembling the NLS of the yeast homeodomain protein Matα2 (NKIPIKDLLNPQ13 (5Hall N.M. Hereford L. Herskowitz I. Cell. 1984; 36: 1057-1065Abstract Full Text PDF PubMed Scopus (216) Google Scholar)). All of these types of NLS are similar in terms of the transport process and the cytosolic factors mediating it (see Refs. 1Jans D.A. Hübner S. Physiol. Rev. 1996; 76: 651-685Crossref PubMed Scopus (389) Google Scholar, 2Koepp D.M. Silver P.A. Cell. 1996; 87: 1-4Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, and 6Efthymiadis A. Shao H. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22134-22139Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), whereby NLS-containing proteins are initially bound by a heterodimer consisting of proteins of about 60 and 95 kDa, variously named importin α/β (7Görlich D. Vogel F. Mills A.D. Hartmann E. Laskey R.A. Nature. 1995; 377: 246-248Crossref PubMed Scopus (410) Google Scholar), importin 58/97 (8Imamoto N. Shimamoto T. Takao T. Tachibana T. Kose S. Matsubae M. Sekimoto T. Shimonishi Y. Yoneda Y. EMBO J. 1995; 14: 3617-3626Crossref PubMed Scopus (271) Google Scholar), and karyopherin α/β (9Rexach M. Blobel G. Cell. 1995; 83: 683-692Abstract Full Text PDF PubMed Scopus (665) Google Scholar). The smaller importin/karyopherin subunit binds the NLS specifically, whereas the larger subunit both enhances the affinity of the complex for the NLS (6Efthymiadis A. Shao H. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22134-22139Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 9Rexach M. Blobel G. Cell. 1995; 83: 683-692Abstract Full Text PDF PubMed Scopus (665) Google Scholar, 10Görlich D. Pante N. Kutay U. Aebi U. Bischoff F.R. EMBO J. 1996; 15: 5584-5594Crossref PubMed Scopus (535) Google Scholar, 11Hübner S. Xiao C.-Y. Jans D.A. J. Biol. Chem. 1997; 272: 17191-17195Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar) and mediates the docking of the cargo-carrier complex to the nuclear pore complex (NPC). The second, energy-dependent step involves transfer of the cargo-carrier complex to the nucleoplasmic side and requires GTPase activity on the part of the monomeric GTP-binding protein/GTPase Ran/TC4 and other factors such as NTF2 (see Refs. 1Jans D.A. Hübner S. Physiol. Rev. 1996; 76: 651-685Crossref PubMed Scopus (389) Google Scholar, 2Koepp D.M. Silver P.A. Cell. 1996; 87: 1-4Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, and12Melchior F. Paschal B. Evans E. Gerace L. J. Cell Biol. 1993; 123: 1649-1659Crossref PubMed Scopus (472) Google Scholar, 13Moore M.S. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10212-10216Crossref PubMed Scopus (291) Google Scholar, 14Paschal B.M. Gerace L. J. Cell Biol. 1995; 129: 925-937Crossref PubMed Scopus (342) Google Scholar). While the conventional NLSs mentioned above appear to be recognized by importin/karyopherin and transported to the nucleus as outlined above, recent studies have revealed two novel nuclear protein import pathways which are mediated by quite distinct targeting signals and do not appear to involve the importin 58/97 complex (15Pollard V.W. Michael W.M. Nakielny S. Mikiko C.S. Wang F. Dreyfuss G. Cell. 1996; 86: 985-994Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar, 16Fridell R.A. Truant R. Thorne L. Benson R.E. Cullen B.R. J. Cell Sci. 1997; 110: 1325-1331Crossref PubMed Google Scholar, 17Michael W.M. Eder P.S. Dreyfuss G EMBO J. 1997; 16: 3587-3598Crossref PubMed Scopus (330) Google Scholar). Nuclear import of the nuclear-cytoplasmic shuttling hnRNP protein A1 is mediated by an importin-97-homolog transportin (karyopherin β2), which recognizes the A1 "M9" NLS but does not interact with the more conventional NLSs referred to above (15Pollard V.W. Michael W.M. Nakielny S. Mikiko C.S. Wang F. Dreyfuss G. Cell. 1996; 86: 985-994Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar, 16Fridell R.A. Truant R. Thorne L. Benson R.E. Cullen B.R. J. Cell Sci. 1997; 110: 1325-1331Crossref PubMed Google Scholar). In contrast, nuclear import of the shuttling hnRNP K protein through the NPC conferred by the "KNS" NLS-sequence does not appear to require a soluble cytosolic receptor or Ran (17Michael W.M. Eder P.S. Dreyfuss G EMBO J. 1997; 16: 3587-3598Crossref PubMed Scopus (330) Google Scholar). Conventional NLSs, as well as the M9 and KNS NLSs, do not mediate nuclear accumulation by conferring binding to nuclear components, but function exclusively as nuclear entry signals (1Jans D.A. Hübner S. Physiol. Rev. 1996; 76: 651-685Crossref PubMed Scopus (389) Google Scholar, 6Efthymiadis A. Shao H. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22134-22139Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 15Pollard V.W. Michael W.M. Nakielny S. Mikiko C.S. Wang F. Dreyfuss G. Cell. 1996; 86: 985-994Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar). We have been interested for some time in the nuclear import of viral proteins (6Efthymiadis A. Shao H. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22134-22139Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 11Hübner S. Xiao C.-Y. Jans D.A. J. Biol. Chem. 1997; 272: 17191-17195Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar), and in particular the human immunodeficiency virus type 1 (HIV-1) Tat protein, which is a potent activator of viral gene expression and replication (see Ref. 18Luciw P. Fields B.N. Knipe D.M. Howley P.M. Virology. 3rd Ed. Lippincott-Raven, Philadelphia1996Google Scholar). Tat accumulates predominantly in the nucleus and nucleolus (19Hauber J. Malim M. Cullen B. J. Virol. 1989; 63: 601-608Crossref Google Scholar, 20Chin D.J. Selby M.J. Peterlin B.M. J. Virol. 1991; 65: 1758-1764Crossref PubMed Google Scholar, 21Ruben S. Perkins A. Purcell R. Joung K. Sia R. Burgnoff R. Haseltine W.A. Rosen C.A. J. Virol. 1989; 63: 1-8Crossref PubMed Google Scholar) through possession of an amino-terminal stretch of basic amino acid residues purported to be the NLS (GRKKRRQRRRAP59, single-letter amino acid code; basic residues highlighted in bold type) (22Dang C.V. Lee W.M. J. Biol. Chem. 1989; 264: 18019-18023Abstract Full Text PDF PubMed Google Scholar, 23Siomi H. Shida H. Maki M. Hatanaka M. J. Virol. 1990; 64: 1803-1807Crossref PubMed Google Scholar), which is highly conserved among HIV-I isolates. The Tat-NLS resembles similar highly basic amino-terminal sequences of the HIV-1 Rev (RQARRNRRRRWRERQRQ51(24Kubota S. Siomi H. Satoh T. Endo S. Maki M. Hatanaka M. Biochem. Biophys. Res. Commun. 1989; 162: 963-970Crossref PubMed Scopus (111) Google Scholar)) and the HTLV-1 (human T-cell leukemia virus) Rex (MPKTRRRPRRSQRKRPPTP119) proteins, both of which have been shown to constitute functional NLSs (24Kubota S. Siomi H. Satoh T. Endo S. Maki M. Hatanaka M. Biochem. Biophys. Res. Commun. 1989; 162: 963-970Crossref PubMed Scopus (111) Google Scholar, 25Siomi H. Shida H. Nam S.H. Nosaka T. Maki M. Hatanaka M. Cell. 1988; 55: 197-209Abstract Full Text PDF PubMed Scopus (214) Google Scholar, 26Cochrane A.W. Perkins A. Rosen C.A. J. Virol. 1990; 64: 881-885Crossref PubMed Google Scholar, 27Nosaka T. Siomi H. Adachi Y. Ishibashi M. Kubota S. Maki M. Hatanaka M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9798-9802Crossref PubMed Scopus (70) Google Scholar). To gain insight into Tat targeting function as a possible paradigm of this class of viral targeting signal, this study examines the nuclear import kinetics of a β-galactosidase fusion protein carrying Tat amino acids 48–59 in vivo and in vitro at the single cell level, comparing results to those for fusion proteins carrying a classical NLS, that of T-ag (3Kalderon D. Richardson W.D. Markham A.F. Smith A.E. Nature. 1984; 311: 33-38Crossref PubMed Scopus (910) Google Scholar). We find that the Tat-NLS, in contrast to the classical T-ag-NLS, confers nuclear accumulation through an import pathway which appears to require ATP but not cytosolic factors such as importin. Experiments using cells in which the nuclear envelope was permeabilized with CHAPS indicate that Tat fusion proteins can bind to both insoluble cytoplasmic and nuclear factors and that ATP is required to effect release from cytoplasmic retention and relocation to the nucleus. In contrast, the T-ag fusion protein binds neither cytosolic nor nuclear factors under the same conditions. We conclude that the Tat-NLS is able to target β-galactosidase to the nucleus through a novel import pathway. The detergent CHAPS was from Boehringer Mannheim and AMP-PNP from Calbiochem. The bacterial strains for karyopherin α (Kap60) and β (Kap95) fusion protein expression (9Rexach M. Blobel G. Cell. 1995; 83: 683-692Abstract Full Text PDF PubMed Scopus (665) Google Scholar) were provided by Michael Rexach. Other reagents were from the sources previously described (6Efthymiadis A. Shao H. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22134-22139Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 11Hübner S. Xiao C.-Y. Jans D.A. J. Biol. Chem. 1997; 272: 17191-17195Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 28Jans D.A. Ackermann M. Bischoff J.R. Beach D.H. Peters R. J. Cell Biol. 1991; 115: 1203-1212Crossref PubMed Scopus (183) Google Scholar, 29Rihs H.-P. Jans D.A. Fan H. Peters R. EMBO J. 1991; 10: 633-639Crossref PubMed Scopus (302) Google Scholar, 30Jans D.A. Jans P. Briggs L.J. Sutton V. Trapani J.A. J. Biol. Chem. 1996; 272: 30781-30789Abstract Full Text Full Text PDF Scopus (94) Google Scholar, 31Xiao C.-Y. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22191-22198Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Cells of the HTC rat hepatoma tissue culture (a derivative of Morris hepatoma 7288C) line were cultured as described previously (28Jans D.A. Ackermann M. Bischoff J.R. Beach D.H. Peters R. J. Cell Biol. 1991; 115: 1203-1212Crossref PubMed Scopus (183) Google Scholar, 29Rihs H.-P. Jans D.A. Fan H. Peters R. EMBO J. 1991; 10: 633-639Crossref PubMed Scopus (302) Google Scholar). The plasmid expressing the Tat-NLS-β-galactosidase fusion protein Tat-NLS-β-Gal was derived by oligonucleotide insertion into the SmaI restriction endonuclease site of the plasmid vector pPR2 (29Rihs H.-P. Jans D.A. Fan H. Peters R. EMBO J. 1991; 10: 633-639Crossref PubMed Scopus (302) Google Scholar). The resultant fusion protein comprises Tat amino acids 48–59 (GRKKRRQRRRAP59) fused amino-terminal to theEscherichia coli β-galactosidase enzyme sequence (amino acids 9–1023). The T-ag β-galactosidase fusion protein (T-ag-CcN-β-Gal) used in the comparative studies contains T-ag amino acids 111–135, including the CcN motif (comprising protein kinaseCK2 and cdk phosphorylation sites and theNLS) fused amino-terminal to β-galactosidase amino acids 9–1023 (28Jans D.A. Ackermann M. Bischoff J.R. Beach D.H. Peters R. J. Cell Biol. 1991; 115: 1203-1212Crossref PubMed Scopus (183) Google Scholar, 29Rihs H.-P. Jans D.A. Fan H. Peters R. EMBO J. 1991; 10: 633-639Crossref PubMed Scopus (302) Google Scholar). 1 mm isopropyl-β-thiogalactoside was used to induce expression of fusion proteins in E. coli. They were purified by affinity chromatography and labeled using the sulfhydryl labeling reagent 5-iodoacetamidofluorescein (Molecular Probes) as described (29Rihs H.-P. Jans D.A. Fan H. Peters R. EMBO J. 1991; 10: 633-639Crossref PubMed Scopus (302) Google Scholar). Nuclear import kinetics at the single cell level were measured using either microinjected (in vivo) or mechanically perforated (in vitro) HTC cells in conjunction with confocal laser scanning microscopy (CLSM) (6Efthymiadis A. Shao H. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22134-22139Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 11Hübner S. Xiao C.-Y. Jans D.A. J. Biol. Chem. 1997; 272: 17191-17195Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar,28Jans D.A. Ackermann M. Bischoff J.R. Beach D.H. Peters R. J. Cell Biol. 1991; 115: 1203-1212Crossref PubMed Scopus (183) Google Scholar, 29Rihs H.-P. Jans D.A. Fan H. Peters R. EMBO J. 1991; 10: 633-639Crossref PubMed Scopus (302) Google Scholar, 30Jans D.A. Jans P. Briggs L.J. Sutton V. Trapani J.A. J. Biol. Chem. 1996; 272: 30781-30789Abstract Full Text Full Text PDF Scopus (94) Google Scholar, 31Xiao C.-Y. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22191-22198Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). In the case of microinjection, HTC cells were fused with polyethylene glycol about 1 h prior to microinjection to produce polykaryons (6Efthymiadis A. Shao H. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22134-22139Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 11Hübner S. Xiao C.-Y. Jans D.A. J. Biol. Chem. 1997; 272: 17191-17195Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 28Jans D.A. Ackermann M. Bischoff J.R. Beach D.H. Peters R. J. Cell Biol. 1991; 115: 1203-1212Crossref PubMed Scopus (183) Google Scholar, 29Rihs H.-P. Jans D.A. Fan H. Peters R. EMBO J. 1991; 10: 633-639Crossref PubMed Scopus (302) Google Scholar, 31Xiao C.-Y. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22191-22198Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Reticulocyte lysate (Promega) was used as the source of cytosol for the in vitro assay (6Efthymiadis A. Shao H. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22134-22139Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 28Jans D.A. Ackermann M. Bischoff J.R. Beach D.H. Peters R. J. Cell Biol. 1991; 115: 1203-1212Crossref PubMed Scopus (183) Google Scholar, 30Jans D.A. Jans P. Briggs L.J. Sutton V. Trapani J.A. J. Biol. Chem. 1996; 272: 30781-30789Abstract Full Text Full Text PDF Scopus (94) Google Scholar,31Xiao C.-Y. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22191-22198Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Image analysis of CLSM files using the NIH Image public domain software and curve-fitting were performed as described (6Efthymiadis A. Shao H. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22134-22139Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 11Hübner S. Xiao C.-Y. Jans D.A. J. Biol. Chem. 1997; 272: 17191-17195Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 30Jans D.A. Jans P. Briggs L.J. Sutton V. Trapani J.A. J. Biol. Chem. 1996; 272: 30781-30789Abstract Full Text Full Text PDF Scopus (94) Google Scholar,31Xiao C.-Y. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22191-22198Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). In in vitro experiments where the ATP dependence of transport was tested, apyrase pretreatment was used to hydrolyze endogenous ATP in both cytosol (10 min at room temperature with 800 units/ml) and perforated cells (15 min at 37 °C with 0.2 unit/ml) (6Efthymiadis A. Shao H. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22134-22139Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 30Jans D.A. Jans P. Briggs L.J. Sutton V. Trapani J.A. J. Biol. Chem. 1996; 272: 30781-30789Abstract Full Text Full Text PDF Scopus (94) Google Scholar, 32Newmeyer D.D. Forbes D.J. Cell. 1988; 52: 641-653Abstract Full Text PDF PubMed Scopus (371) Google Scholar), and transport assays were performed in the absence of the ATP regenerating system (28Jans D.A. Ackermann M. Bischoff J.R. Beach D.H. Peters R. J. Cell Biol. 1991; 115: 1203-1212Crossref PubMed Scopus (183) Google Scholar, 30Jans D.A. Jans P. Briggs L.J. Sutton V. Trapani J.A. J. Biol. Chem. 1996; 272: 30781-30789Abstract Full Text Full Text PDF Scopus (94) Google Scholar) which was otherwise used. Where the dependence on the GTP-binding protein Ran was tested, cytosolic extract was treated with 860 μm GTPγS (nonhydrolyzable GTP analog) for 5 min at room temperature, prior to use in the in vitro assay (final concentration of 300 μm) (6Efthymiadis A. Shao H. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22134-22139Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 12Melchior F. Paschal B. Evans E. Gerace L. J. Cell Biol. 1993; 123: 1649-1659Crossref PubMed Scopus (472) Google Scholar,13Moore M.S. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10212-10216Crossref PubMed Scopus (291) Google Scholar, 30Jans D.A. Jans P. Briggs L.J. Sutton V. Trapani J.A. J. Biol. Chem. 1996; 272: 30781-30789Abstract Full Text Full Text PDF Scopus (94) Google Scholar). The ability of GTP to substitute for ATP in the transport assay was assessed by replacing the ATP-regenerating system with 2 mm GTP/2 mm GDP. In competition experiments, peptides P101 (CGPGSDDEAAADAQHAAPPKKKRKVGY, including T-ag amino acids 111–132) and P101T (identical to P101, but containing the NLS-inactivating Lys128 to Thr substitution) (3Kalderon D. Richardson W.D. Markham A.F. Smith A.E. Nature. 1984; 311: 33-38Crossref PubMed Scopus (910) Google Scholar, 11Hübner S. Xiao C.-Y. Jans D.A. J. Biol. Chem. 1997; 272: 17191-17195Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 29Rihs H.-P. Jans D.A. Fan H. Peters R. EMBO J. 1991; 10: 633-639Crossref PubMed Scopus (302) Google Scholar, 33Akhlynina T.V. Jans D.A. Statsyuk N.V. Balashova I.Y. Toth G. Pavo I. Rosenkranz A.A. Rubin A.B. Sobolev A.S. J. Biol. Chem. 1997; 272: 20328-20331Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) were used at final molar concentrations 200-fold those of the Tat and T-ag fusion proteins (4.2 × 10−7m). Nuclear accumulation was also examined in vitro in the presence of 1% glycerol and 0.025% CHAPS which results in permeabilization of the nuclear envelope; accumulation under these conditions results solely from binding to nuclear components such as lamins, chromatin etc. (6Efthymiadis A. Shao H. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22134-22139Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). An ELISA-based binding assay (6Efthymiadis A. Shao H. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22134-22139Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar,11Hübner S. Xiao C.-Y. Jans D.A. J. Biol. Chem. 1997; 272: 17191-17195Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 31Xiao C.-Y. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22191-22198Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar) was used to examine the binding affinity between importin subunits (mouse importin 58 and 97 glutathione S-transferase (GST)- fusion proteins, expressed as described (8Imamoto N. Shimamoto T. Takao T. Tachibana T. Kose S. Matsubae M. Sekimoto T. Shimonishi Y. Yoneda Y. EMBO J. 1995; 14: 3617-3626Crossref PubMed Scopus (271) Google Scholar, 11Hübner S. Xiao C.-Y. Jans D.A. J. Biol. Chem. 1997; 272: 17191-17195Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar)) and Tat or T-ag fusion proteins. This involved coating 96-well microtiter plates with β-galactosidase fusion proteins, hybridization with increasing concentrations of importin subunits, and detection of bound importin-GST using goat anti-GST primary, and alkaline phosphatase-coupled rabbit anti-goat secondary antibodies, and the substrate p-nitrophenyl phosphate (6Efthymiadis A. Shao H. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22134-22139Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 11Hübner S. Xiao C.-Y. Jans D.A. J. Biol. Chem. 1997; 272: 17191-17195Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). Absorbance measurements were performed over 90 min using a plate reader (Molecular Devices), and values were corrected by subtracting absorbance both at 0 min and in wells incubated without importin (6Efthymiadis A. Shao H. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22134-22139Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 11Hübner S. Xiao C.-Y. Jans D.A. J. Biol. Chem. 1997; 272: 17191-17195Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 31Xiao C.-Y. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22191-22198Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). To quantitate importin binding specifically to the NLSs, quantitation was performed in identical fashion for β-galactosidase itself, and the values were subtracted from those for the respective fusion proteins (6Efthymiadis A. Shao H. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22134-22139Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 11Hübner S. Xiao C.-Y. Jans D.A. J. Biol. Chem. 1997; 272: 17191-17195Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 31Xiao C.-Y. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22191-22198Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Fusion proteins were also subjected to a parallel β-galactosidase ELISA (see Refs. 6Efthymiadis A. Shao H. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22134-22139Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 11Hübner S. Xiao C.-Y. Jans D.A. J. Biol. 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To examine the ability of the HIV-1 Tat basic region to target a large (476-kDa) heterologous protein (β-galactosidase from E. coli) to the nucleus, a plasmid was derived expressing fusion protein Tat-NLS-β-Gal containing Tat amino acids 48–59 (see "Materials and Methods") fused amino-terminal to β-galactosidase amino acids 9–1023. Its nuclear import kinetics were measured in vivo and in vitro using microinjected cells of the HTC line (6Efthymiadis A. Shao H. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22134-22139Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 11Hübner S. Xiao C.-Y. Jans D.A. J. Biol. Chem. 1997; 272: 17191-17195Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 28Jans D.A. Ackermann M. Bischoff J.R. Beach D.H. Peters R. J. Cell Biol. 1991; 115: 1203-1212Crossref PubMed Scopus (183) Google Scholar, 29Rihs H.-P. Jans D.A. Fan H. Peters R. EMBO J. 1991; 10: 633-639Crossref PubMed Scopus (302) Google Scholar,31Xiao C.-Y. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22191-22198Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar) and mechanically perforated HTC cells (6Efthymiadis A. Shao H. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22134-22139Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 28Jans D.A. Ackermann M. Bischoff J.R. Beach D.H. Peters R. J. Cell Biol. 1991; 115: 1203-1212Crossref PubMed Scopus (183) Google Scholar, 30Jans D.A. Jans P. Briggs L.J. Sutton V. Trapani J.A. J. Biol. Chem. 1996; 272: 30781-30789Abstract Full Text Full Text PDF Scopus (94) Google Scholar, 31Xiao C.-Y. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22191-22198Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), respectively, and compared with those for a fusion protein (T-ag-CcN-β-Gal) carrying the T-ag NLS and β-galactosidase itself. The Tat-NLS targeted β-galactosidase to the nucleus in both a