Ran-dependent Signal-mediated Nuclear Import Does Not Require GTP Hydrolysis by Ran

Nuclear import of classical nuclear localization sequence-containing proteins involves the assembly of an import complex at the cytoplasmic face of the nuclear pore complex (NPC) followed by movement of this complex through the NPC and release of the import substrate into the nuclear interior. This process has historically been thought to require nucleotide hydrolysis as a source of energy. We found, using hydrolysis-resistant GTP analogs and a mutant Ran unable to hydrolyze GTP, that transport of classical nuclear localization sequence containing substrate through the NPC and release of the substrate into the nucleus did not require hydrolysis of GTP by Ran. In fact, for movement of this type of import substrate into the nuclear interior we did not observe a requirement for hydrolysis of any nucleotide triphosphate. We did, however, find that a pool of free GTP (or its structural equivalent) must be added, probably because the GDP Ran that is added must be converted to GTP Ran during the import process. We found that a requirement for GTP hydrolysis can be restored to an import mixture consisting of recombinant import factors by the addition of RCC1, the Ran guanine nucleotide exchange factor. Nuclear import of classical nuclear localization sequence-containing proteins involves the assembly of an import complex at the cytoplasmic face of the nuclear pore complex (NPC) followed by movement of this complex through the NPC and release of the import substrate into the nuclear interior. This process has historically been thought to require nucleotide hydrolysis as a source of energy. We found, using hydrolysis-resistant GTP analogs and a mutant Ran unable to hydrolyze GTP, that transport of classical nuclear localization sequence containing substrate through the NPC and release of the substrate into the nucleus did not require hydrolysis of GTP by Ran. In fact, for movement of this type of import substrate into the nuclear interior we did not observe a requirement for hydrolysis of any nucleotide triphosphate. We did, however, find that a pool of free GTP (or its structural equivalent) must be added, probably because the GDP Ran that is added must be converted to GTP Ran during the import process. We found that a requirement for GTP hydrolysis can be restored to an import mixture consisting of recombinant import factors by the addition of RCC1, the Ran guanine nucleotide exchange factor. Many nuclear proteins contain a nuclear localization sequence (NLS), 1The abbreviations used are: NLS, nuclear localization signal; NPC, nuclear pore complex; NTP, nucleoside 5′-triphosphate; GEF, guanine nucleotide exchange factor; GAP, GTPase-activating protein; NEM, N-ethylmaleimide; NE, nuclear envelope; BSA, bovine serum albumin; DTT, dithiothreitol; AMP-PNP, adenylyl imidodiphosphate; GMP-PNP, guanylyl imidodiphosphate; GTPγS, guanosine-5′-O-(2-thiodiphosphate); GDPβS, guanosine-5′-O-(2-thiodiphosphate); wt, wild type. the classical type of which consists of either a single or bipartite stretch of primarily basic amino acids. The presence of an NLS on a reporter results in its rapid transport from the cytoplasm through the nuclear pore complex (NPC) and into the nuclear interior. This process can be reconstitutedin vitro using cells that have been treated with a low concentration of digitonin, which permeabilizes the plasma membrane to the passage of macromolecules but leaves the nuclear membrane intact (1Adam S.A. Sterne Marr R. Gerace L. J. Cell Biol. 1990; 111: 807-816Crossref PubMed Scopus (772) Google Scholar). Addition of cytosol as a source of import factors, a nucleoside triphosphate (NTP) as a source of energy, and import substrate to these permeabilized cells results in import of the substrate into the nucleus. From this cytosol, four import factors have been identified that together efficiently support nuclear import of a basic NLS-containing substrate in permeabilized cells in the absence of cytosol. The receptor for the classical or basic NLS (karyopherin/importin α), together with karyopherin/importin β1, targets the import substrate to the NPC (2Adam S.A. Gerace L. Cell. 1991; 66: 837-847Abstract Full Text PDF PubMed Scopus (318) Google Scholar, 3Görlich D. Prehn S. Laskey R.A. Hartmann E. Cell. 1994; 79: 767-778Abstract Full Text PDF PubMed Scopus (602) Google Scholar, 4Moroianu J. Blobel G. Radu A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2008-2011Crossref PubMed Scopus (252) Google Scholar, 5Weis K. Mattaj I.W. Lamond A.I. Scienc e. 1995; 268: 1049-1053Google Scholar, 6Radu A. Blobel G. Moore M.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1769-1773Crossref PubMed Scopus (386) Google Scholar, 7Görlich D. Kostka S. Kraft R. Dingwall C. Laskey R.A. Hartmann E. Prehn S. Curr. Biol. 1995; 5: 383-392Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 8Chi N.C. Adam E.J.H. Adam S.A. J. Cell Biol. 1995; 130: 265-274Crossref PubMed Scopus (248) Google Scholar). Thus the addition of these two import factors alone to an in vitro import assay results in binding of import substrate at the nuclear envelope in a characteristic rim pattern. Subsequent passage of import substrate from the NPC into the nuclear interior requires the addition of two other import factors: p10/NTF2 (9Moore M.S. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10212-10216Crossref PubMed Scopus (292) Google Scholar, 10Paschal B.M. Gerace L. J. Cell Biol. 1995; 129: 925-937Crossref PubMed Scopus (343) Google Scholar) and the small GTPase Ran (11Moore M.S. Blobel G. Nature. 1993; 365: 661-663Crossref PubMed Scopus (641) Google Scholar, 12Melchior F. Paschal B. Evans J. Gerace L. J. Cell Biol. 1993; 123: 1649-1659Crossref PubMed Scopus (472) Google Scholar). The exact role of the nuclear import factor p10, which preferentially binds GDP-Ran rather than GTP-Ran, has yet to be defined. Ran, however, has been shown to be a key player not only for the nuclear import of a variety of substrates bearing different types of NLSs, but also for the nuclear export of most RNAs and a number of proteins containing different types of nuclear export signals (13Ren M. Villamarin A. Shih A. Coutavas E. Moore M.S. LoCurcio M. Clarke V. Oppenheim J. Dı́Eustachio P. Rush M.G. Mol. Cell. Biol. 1995; 15: 2117-2124Crossref PubMed Scopus (60) Google Scholar,14Mattaj I.W. Englmeier L. Annu. Rev. Biochem. 1998; 67: 265-306Crossref PubMed Scopus (1013) Google Scholar). Two additional proteins, which are among those retained in large amounts in permeabilized cells, are considered vital to regulation of nuclear import. RCC1, the Ran guanine nucleotide exchange factor (GEF), contains a basic NLS and is localized primarily inside the nucleus bound to chromatin (15Seino H. Hisamoto N. Uzawa S. Sekiguchi T. Nishimoto T. J. Cell Sci. 1992; 102: 393-400Crossref PubMed Google Scholar, 16Bischoff F.R. Ponstingl H. Nature. 1991; 354: 80-82Crossref PubMed Scopus (542) Google Scholar). In contrast, the majority of RanGAP1, the Ran GTPase-activating protein (GAP), is found both free in the cytoplasm and bound on the cytoplasmic filaments of the NPC (17Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (962) Google Scholar, 18Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1012) Google Scholar, 19Bischoff F.R. Klebe C. Kretschmer J. Wittinghofer A. Ponstingl H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2587-2591Crossref PubMed Scopus (420) Google Scholar). The differential localization of these two proteins is predicted to play a role in controlling the direction of nuclear transport by controlling the assembly and disassembly of transport complexes in response to the relative predicted concentrations of GDP-Ran (predominantly cytoplasmic) and GTP-Ran (predominantly nuclear) (20Izaurralde E. Kutay U. von Kobbe C. Mattaj I.W. Görlich D. EMBO J. 1997; 16: 6535-6547Crossref PubMed Scopus (494) Google Scholar). Significantly, the addition of GTP-Ran (instead of GDP-Ran) to the cytoplasmic side of the nuclear envelope is known to inhibit nuclear import, probably due to the ability of GTP-Ran (but not GDP-Ran) to disrupt the karyopherin α-β1 complex and to release karyopherin β1 from docking sites on the NPC (21Görlich D. Panté N. Kutay U. Aebi U. Bischoff F.R. EMBO J. 1996; 15: 5584-5594Crossref PubMed Scopus (538) Google Scholar, 22Rexach M. Blobel G. Cell. 1995; 83: 683-692Abstract Full Text PDF PubMed Scopus (666) Google Scholar). Other proteins, for example RanBP1 and components of the NPC, are thought to play a critical role in nuclear import, but do not have to be added to permeabilized cells to achieve efficient import (see below). The role of nucleotide hydrolysis in nuclear transport is unknown. It has been reported recently that certain types of Ran-dependent nuclear export require the presence of GTP Ran (based on microinjection) but do not appear to require GTP hydrolysis by Ran (23Richards S.A. Carey K.L. Macara I.G. Science. 1997; 286: 1842-1844Crossref Scopus (40) Google Scholar). This is in contrast to studies of nuclear import, in which there have been a number of reports, using both hydrolysis-resistant GTP analogs and Ran mutants incapable of GTP hydrolysis (for example, Q69L Ran) (19Bischoff F.R. Klebe C. Kretschmer J. Wittinghofer A. Ponstingl H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2587-2591Crossref PubMed Scopus (420) Google Scholar), that GTP hydrolysis by Ran is required for import (11Moore M.S. Blobel G. Nature. 1993; 365: 661-663Crossref PubMed Scopus (641) Google Scholar, 12Melchior F. Paschal B. Evans J. Gerace L. J. Cell Biol. 1993; 123: 1649-1659Crossref PubMed Scopus (472) Google Scholar, 24Schlenstedt G. Saavedra C. Loeb J.D.J. Cole C.N. Silver P.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 225-229Crossref PubMed Scopus (168) Google Scholar, 25Weis K. Dingwall C. Lamond A.I. EMBO J. 1996; 15: 7120-7128Crossref PubMed Scopus (120) Google Scholar). To understand the role of GTP hydrolysis by Ran in nuclear import, we attempted to identify the critical step(s) that required GTP hydrolysis. We verified that hydrolysis-resistant GTP analogs or Q69L Ran inhibited nuclear import in the presence of cytosol as observed previously. However, we found that GTP hydrolysis was not necessary in assays employing purified recombinant factors instead of cytosol. We found that nuclear import could occur in the presence of both poorly hydrolyzable GTP analogs and Q69L Ran. We hypothesize that earlier results were due to the presence of RCC1 outside of the nucleus during import assays, resulting in the inappropriate generation of GTP Ran in the cytoplasm. Accordingly, we confirmed the existence of RCC1 in cytosolic extracts utilized in many earlier studies. Furthermore, we demonstrate that an apparent hydrolysis requirement can be introduced into recombinant import assays by the addition of recombinant RCC1. The hSRP1ah expression vector was obtained from A. Lamond and the human karyopherin β expression vector was obtained from D. Görlich. Human Ran and Q69L Ran expression plasmids were obtained from M. Rush and E. Fanning, respectively. The human RCC1 expression vector was from T. Nishimoto. The human p10 was produced from anEscherichia coli expression vector obtained from U. Grundmann. When possible, nucleotides were purchased from Boehringer Mannheim. Xanthosine 5′-triphosphate was purchased from Sigma. All recombinant proteins were snap-frozen in the indicated buffer in single use aliquots and stored at −80 °C. The human recombinant karyopherin α2 was expressed in bacteria, purified as described, (5Weis K. Mattaj I.W. Lamond A.I. Scienc e. 1995; 268: 1049-1053Google Scholar) and dialyzed against Buffer A (20 mm Hepes-KOH, pH 7.3, 100 mm potassium acetate, 2 mm DTT). The human karyopherin β1 expression vector was obtained from D. Gàrlich and the protein was purified according to his suggested method (26Görlich D. Vogel F. Mills A.D. Hartmann E. Laskey R.A. Nature. 1995; 377: 246-248Crossref PubMed Scopus (412) Google Scholar). Transformed BL21(DE3) Rep4 cells were induced atA 600 = 0.8 with 1 mmisopropyl-β-d-thiogalactopyranoside for 4 h at 25 °C. The cells were in mm pH mm mm 1 mm and the was at for β was purified by passage of the a in the buffer followed by with a mm on a in transport buffer (20 mm Hepes-KOH, pH 7.3, mm potassium acetate, 2 mm acetate, 1 mm 2 mm DTT). Ran (13Ren M. Villamarin A. Shih A. Coutavas E. Moore M.S. LoCurcio M. Clarke V. Oppenheim J. Dı́Eustachio P. Rush M.G. Mol. Cell. Biol. 1995; 15: 2117-2124Crossref PubMed Scopus (60) Google Scholar) and Q69L Ran (19Bischoff F.R. Klebe C. Kretschmer J. Wittinghofer A. Ponstingl H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2587-2591Crossref PubMed Scopus (420) Google Scholar) were purified Transformed cells were induced with for 2 h at 25 and in buffer (20 mm Hepes-KOH, pH 7.3, mm potassium acetate, 2 mm acetate, 1 mm containing 1 mm and 100 as Ran was from the by a The was in buffer containing and Ran was by on a in containing Ran were with an of mm Hepes-KOH, pH 7.3, 2 acetate, 1 mm and were a in the buffer containing mm potassium and with the buffer containing potassium were dialyzed against Buffer A containing 2 The human recombinant p10 B. E. J. 1992; PubMed Scopus Google Scholar) was expressed in BL21(DE3) cells, which were to an A of and induced with 1 mm for h at °C. The cells were by in mm Hepes-KOH, pH 7.3, mm 1 mm DTT, 1 mm and as The was with mm Hepes-KOH, pH 7.3, 2 mm DTT, to a in the buffer mm and in a mm were to of the The was in Buffer (20 mm Hepes-KOH, pH 7.3, 100 mm potassium acetate, 1 mm and on a in the buffer to the human RCC1 was purified as M. T. T. Nishimoto T. EMBO J. 1994; PubMed Scopus Google Scholar) and dialyzed against Buffer mm was produced by with recombinant RCC1 followed by of the serum against the RCC1 was with mm for on to the by the addition of to the was added to mm concentration to the addition of mm The concentration of in the RCC1 during was in was The nuclear import assay was as M.S. Blobel G. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar) using a reporter import consisting of to containing the NLS of the Many of these were also using as an import substrate with and M. S. The import assay was in cells permeabilized with the import karyopherin 25 karyopherin β1, Ran, p10, 2 BSA, and 1 concentration of the indicated nucleotide in transport the concentrations were RCC1 at GTP, GMP-PNP, GDPβS, or at 1 All were at for to the import The import assay was in the permeabilized cells for at to and The were observed with a with a and and of the was with and by M. of The nuclear of was as M.S. Blobel G. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar). were at on and cytosol was as M.S. Blobel G. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar). was by cells in cytosol was as in S.A. A of was converted to GDP T. J. Biol. Full Text PDF PubMed Google Scholar) in a containing pH 1 mm and of and 4 h at °C. The was by to for was verified by 25 of Ran was with of in Buffer 2 and mm for at °C. The was by the addition of and a with buffer mm of Ran was in Buffer with 1 GTP and the indicated amounts of RCC1 for at °C. This was by of Buffer C. were with 4 of Buffer and the were in and purified recombinant transport factors α2 and β1, Ran, and nuclear import of in permeabilized cells in the presence of GTP, but not in the presence of GDPβS, AMP-PNP, or in the absence of nucleotide A M.S. Blobel G. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar). also nuclear import as did other nucleoside and 5′-triphosphate; not The of a requirement in this assay is probably due to the presence of nucleoside which is retained in permeabilized cells and of GTP from an added S. Moore and E. D. to reports, we found that the hydrolysis-resistant GTP analogs and also the movement of into the that GTP hydrolysis in fact, not be necessary for import of this type of substrate through the NPC and release into the nucleus A and To that nuclear import could occur in the absence of GTP hydrolysis by Ran, a mutant Ran that is unable to hydrolyze GTP Ran) (19Bischoff F.R. Klebe C. Kretschmer J. Wittinghofer A. Ponstingl H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2587-2591Crossref PubMed Scopus (420) Google Scholar) was for the Ran in the in the Q69L Ran and the Ran were in the to addition to the assay as by protein not When added with either GDPβS, or AMP-PNP, Q69L Ran Ran) was unable to support nuclear import A and However, added with either GTP, GMP-PNP, or GTPγS, Q69L Ran was of nuclear import A results indicated that free GTP (or its structural equivalent) was required for but that GTP hydrolysis by Ran was not required for passage of import substrate through the NPC and into the nuclear interior. The of import by Q69L Ran, however, were than that by Ran 1 (see below). we observed that import by cytosol requires nucleotide hydrolysis not import by purified recombinant import factors not in cytosol, and not required for import, the observed in nucleotide hydrolysis the two We that RCC1 Ran nuclear in be in cytosol in low and be for Ran with GTP or its structural or import. that RCC1 in fact, in two different cytosol used to support nuclear vitro 2 1 and as was in a 2 We the addition of recombinant RCC1 to recombinant import factors could the of nuclear import by hydrolysis-resistant GTP RCC1 contains an NLS, was necessary to the RCC1 exchange that be to of nuclear import by on Ran from by for import the two NLS-containing proteins and exchange was found to be by with the 2 RCC1 was added to import assays to apparent hydrolysis in a to cytosol. RCC1 was added as a at the concentration as treated RCC1 The addition of hydrolysis-resistant GTP analogs or and RCC1 to recombinant import factors inhibited nuclear import, the addition of RCC1 exchange the addition of RCC1 to Q69L Ran and GTP also inhibited nuclear import. of these the of import by addition of RCC1. a requirement for GTP hydrolysis by Ran during this type of nuclear import was not observed RCC1 was added to the cytosolic side of the nuclear All of classical Ran-dependent nuclear import have either a GTP hydrolysis requirement or an hydrolysis requirement by a hydrolyzable in the import assay mixture M.S. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). This is the that this type of import can occur in the absence of hydrolysis in and GTP hydrolysis by Ran in The of nuclear import in the presence of hydrolysis-resistant GTP analogs or Q69L Ran as with that with Ran and GTP that GTP hydrolysis by Ran, not required for import, be required for the of of the transport (see below). We also the that the structural Q69L Ran and Ran, and GTPγS, GMP-PNP, and GTP have also been the of nuclear import for to GTP hydrolysis. in the presence of cytosol we observed a requirement for GTP hydrolysis in nuclear import not but not purified transport factors were added was critical to which in cytosol of nuclear import by GTP It has been that the addition of GTP Ran to the cytoplasmic side of the nuclear import, probably because GTP Ran the karyopherin and karyopherin β1 from docking sites on the NPC (21Görlich D. Panté N. Kutay U. Aebi U. Bischoff F.R. EMBO J. 1996; 15: 5584-5594Crossref PubMed Scopus (538) Google Scholar, 22Rexach M. Blobel G. Cell. 1995; 83: 683-692Abstract Full Text PDF PubMed Scopus (666) Google Scholar). Q69L Ran the or Ran bound to either or are unable to hydrolyze bound nucleotide in response to and We that the RCC1 in cytosol could of Ran or Ran on the cytoplasmic side of the and could be for the import observed in the presence of cytosol containing hydrolysis-resistant GTP The in hydrolysis import by recombinant factors RCC1 and import by cytosol this We also find a the requirement for GTP hydrolysis and the presence of RCC1 in earlier studies (9Moore M.S. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10212-10216Crossref PubMed Scopus (292) Google Scholar, M.S. Blobel G. Nature. 1993; 365: 661-663Crossref PubMed Scopus (641) Google Scholar). A M.S. Blobel G. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar), which contains import factors purified from cytosol, also contains RCC1. S. Accordingly, cytosol, hydrolyzable is required for nuclear import in the presence of A (9Moore M.S. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10212-10216Crossref PubMed Scopus (292) Google Scholar, M.S. Blobel G. Nature. 1993; 365: 661-663Crossref PubMed Scopus (641) Google Scholar). the presence of RCC1 the ability of GTP analogs to inhibit import in vitro in the presence of cytosol, in certain studies these analogs were also reported to inhibit import in the presence of recombinant import factors K. Dingwall C. Lamond A.I. EMBO J. 1996; 15: 7120-7128Crossref PubMed Scopus (120) Google Scholar). It is that in in different amounts of RCC1 on the cytoplasmic side of the The (1Adam S.A. Sterne Marr R. Gerace L. J. Cell Biol. 1990; 111: 807-816Crossref PubMed Scopus (772) Google Scholar) for import assays with cells bound to that were and for import. The in the utilized cells that were permeabilized and in in the two in the to by and could be for and release of RCC1 from In not in the many using this permeabilized cells for use D. Prehn S. Laskey R.A. Hartmann E. Cell. 1994; 79: 767-778Abstract Full Text PDF PubMed Scopus (602) Google Scholar). This could also be to the release of RCC1 from the shown in these in the concentration of RCC1 have on apparent nucleotide was added instead of GTP, the ability of RCC1 to inhibit import was these we that there are of GTP from added and GDP by nucleoside of Ran by RCC1 with GDP or GTP, and hydrolysis of GTP by Ran by of nuclear import has been reported the addition of Ran in the on the cytoplasmic side of the NE, but the of the Ran in the required to this has not been (20Izaurralde E. Kutay U. von Kobbe C. Mattaj I.W. Görlich D. EMBO J. 1997; 16: 6535-6547Crossref PubMed Scopus (494) Google Scholar, D. Panté N. Kutay U. Aebi U. Bischoff F.R. EMBO J. 1996; 15: 5584-5594Crossref PubMed Scopus (538) Google Scholar, 22Rexach M. Blobel G. Cell. 1995; 83: 683-692Abstract Full Text PDF PubMed Scopus (666) Google Scholar). this of GTP Ran at a GTP has to be produced from added as to 1 mm GTP is added to the import which that nuclear import can be obtained the GTP Ran to treated with RCC1 and hydrolysis-resistant GTP analogs the rim in containing GDPβS, or AMP-PNP, that the GTP Ran these the docking of substrate at the cytoplasmic face of the that the addition of RCC1 and hydrolysis-resistant GTP together GTP Ran) to the cytoplasmic side of the nuclear import is with results that of GTP Ran on the cytoplasmic side of the inhibit nuclear import (20Izaurralde E. Kutay U. von Kobbe C. Mattaj I.W. Görlich D. EMBO J. 1997; 16: 6535-6547Crossref PubMed Scopus (494) Google Scholar, D. Panté N. Kutay U. Aebi U. Bischoff F.R. EMBO J. 1996; 15: 5584-5594Crossref PubMed Scopus (538) Google Scholar). import requires both GDP Ran and free GTP (or its structural GTP Ran must be at during substrate transport through the NPC or release into the This has been predicted to occur an import complex the nuclear GTP Ran, which the import substrate from the also that this release of the NLS substrate not require this not demonstrate that the NLS substrate has from of the components of the import that the Ran-dependent movement of transport complexes through the NPC by rather than by GTP hydrolysis the for movement through the NPC or release into the Ran mutants unable to hydrolyze GTP are in we a requirement for GTP hydrolysis by Ran In import factors have to be to and can support of import, and of the GTPase could example, GTP hydrolysis by Ran be required for the of karyopherin β from Ran. In the presence of hydrolysis-resistant this complex to the cytoplasmic face of the NPC, but be unable to in response to GTP hydrolysis by However, we have been unable to demonstrate in the of known import factors in response to different nucleotide analogs by in permeabilized A of these is in Nuclear import of an NLS-containing substrate requires movement of protein against its concentration The of transport of nuclear import is to the of the NLS-containing which must require energy. However, we that nucleotide hydrolysis by Ran during the import of the is not the source of this energy. these that be required either during of import factors or in of the GDP Ran Thus the for nuclear transport be a of the of nuclear import. We T. A. M. E. Fanning, D. and U. for E. B. for the and P. for on the and of We also and for and C. for many and a critical of the