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High Levels of the GTPase Ran/TC4 Relieve the Requirement for Nuclear Protein Transport Factor 2

1997; Elsevier BV; Volume: 272; Issue: 34 Linguagem: Inglês

10.1074/jbc.272.34.21534

1083-351X

Bryce M. Paschal, Christian Fritze, Tinglu Guan, Larry Gerace,

Genomics and Chromatin Dynamics

The GTPase Ran/TC4 and the 14-kDa protein nuclear transport factor 2 (NTF2) are two of the cytosolic factors that mediate nuclear protein import in vertebrates. Previous biochemical studies have shown that NTF2 binds directly to the GDP-bound form of Ran/TC4 and to proteins of the nuclear pore complex that contain phenylalanine-glycine repeats. In the present study we have used molecular genetic approaches to study the Saccharomyces cerevisiae homologue of NTF2. The scNTF2 gene encodes a protein that is 44% identical to the human protein. We found that deletion of the scNTF2 gene is lethal and that repression of NTF2p expression by a regulatable promoter results in gross structural distortions of the nuclear envelope. In a screen for high copy number suppressors of a scNTF2 deletion, the only gene we isolated other than scNTF2 itself was GSP1, the S. cerevisiae homologue of Ran/TC4. Furthermore, we found that high levels of Ran/TC4 can relieve the requirement for NTF2 in a mammalian-permeabilized cell assay for nuclear protein import. These data suggest that certain of the nuclear protein import functions of NTF2 and Ran/TC4 are closely linked and that NTF2 may serve to modulate a transport step involving Ran/TC4. The GTPase Ran/TC4 and the 14-kDa protein nuclear transport factor 2 (NTF2) are two of the cytosolic factors that mediate nuclear protein import in vertebrates. Previous biochemical studies have shown that NTF2 binds directly to the GDP-bound form of Ran/TC4 and to proteins of the nuclear pore complex that contain phenylalanine-glycine repeats. In the present study we have used molecular genetic approaches to study the Saccharomyces cerevisiae homologue of NTF2. The scNTF2 gene encodes a protein that is 44% identical to the human protein. We found that deletion of the scNTF2 gene is lethal and that repression of NTF2p expression by a regulatable promoter results in gross structural distortions of the nuclear envelope. In a screen for high copy number suppressors of a scNTF2 deletion, the only gene we isolated other than scNTF2 itself was GSP1, the S. cerevisiae homologue of Ran/TC4. Furthermore, we found that high levels of Ran/TC4 can relieve the requirement for NTF2 in a mammalian-permeabilized cell assay for nuclear protein import. These data suggest that certain of the nuclear protein import functions of NTF2 and Ran/TC4 are closely linked and that NTF2 may serve to modulate a transport step involving Ran/TC4. Exchange of macromolecules between the cytoplasm and nucleus is specified by signals encoded within transported proteins. The signals that specify nuclear protein import, which are called nuclear localization signals (NLSs), 1The abbreviations used are: NLS, nuclear localization signals; NTF2, nuclear transport factor 2; NPC, nuclear pore complex; ORF, open reading frame; PCR, polymerase chain reaction; WT, wild type.usually consist of short stretches of amino acids enriched in basic amino acid residues (1Görlich D. Mattaj I. Science. 1996; 271: 1513-1518Crossref PubMed Scopus (1073) Google Scholar). A group of cytosolic factors that mediate nuclear import of proteins containing these basic-type NLSs has been identified. These include the α subunit of the NLS receptor (α importin/α karyopherin; Refs. 2Adam S.A. Gerace L. Cell. 1991; 66: 837-847Abstract Full Text PDF PubMed Scopus (337) Google Scholar, 3Gorlich D. Prehn S. Laskey R.A. Hartmann E. Cell. 1994; 79: 767-778Abstract Full Text PDF PubMed Scopus (607) Google Scholar, 4Moroianu J. Hijikata M. Blobel G. Radu A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6532-6536Crossref PubMed Scopus (252) Google Scholar) and its β subunit (p97/β importin/β karyopherin; Refs. 5Chi N.C. Adam E.J. Adam S.A. J. Cell Biol. 1995; 130: 265-274Crossref PubMed Scopus (249) Google Scholar, 6Görlich 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, 7Radu A. Blobel G. Moore M.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1769-1773Crossref PubMed Scopus (391) Google Scholar), the GTPase Ran/TC4 (8Melchior F. Paschal B. Evans J. Gerace L. J. Cell Biol. 1993; 123: 1649-1659Crossref PubMed Scopus (479) Google Scholar, 9Moore M.S. Blobel G. Nature. 1993; 365: 661-663Crossref PubMed Scopus (653) Google Scholar), and the homodimeric protein nuclear transport factor 2 (NTF2/p10; Refs. 10Paschal B.M. Gerace L. J. Cell Biol. 1995; 129: 925-937Crossref PubMed Scopus (344) Google Scholarand 11Moore M.S. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10212-10216Crossref PubMed Scopus (295) Google Scholar). According to current working models (1Görlich D. Mattaj I. Science. 1996; 271: 1513-1518Crossref PubMed Scopus (1073) Google Scholar, 12Melchior F. Gerace L. Curr. Opin. Cell Biol. 1995; 7: 310-318Crossref PubMed Scopus (227) Google Scholar), the pathway for import of proteins with basic-type NLSs begins in the cytoplasm, where the α subunit of the receptor binds to the NLS of a protein destined for import. This complex then binds to the cytoplasmic surface of the nuclear pore complex (NPC) via the β subunit, an event that is referred to as the initial binding or docking step of transport. Subsequently, the substrate-receptor complex is delivered to the central channel of the NPC and then translocated into the nucleoplasm (events collectively referred to as the translocation step; Ref. 13Panté N. Aebi U. Science. 1996; 273: 1729-1732Crossref PubMed Scopus (116) Google Scholar). The latter processes very likely involves Ran/TC4 and NTF2. A number of binding interactions of Ran/TC4 and NTF2 have been described by in vitro biochemical studies, and these associations can provide a framework for studying the precise functions of these factors in nuclear import. The GTP-bound form of Ran/TC4 can bind to three distinct proteins implicated in nuclear import: the β subunit of the NLS receptor (14Paschal B.M. Delphin C. Gerace L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7679-7683Crossref PubMed Scopus (79) Google Scholar, 15Rexach M. Blobel G. Cell. 1995; 83: 683-692Abstract Full Text PDF PubMed Scopus (668) Google Scholar), RanBP1 (16Bischoff F.R. Kreber H. Smirnova E. Dong W. Pongstingl H. EMBO J. 1995; 14: 705-715Crossref PubMed Scopus (336) Google Scholar), and the NPC protein RanBP2/Nup358 (17Wu J. Matunis M.J. Kraemer D. Blobel G. Coutavas E. J. Biol. Chem. 1995; 270: 14209-14213Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 18Yokoyama N. Hayashi N. Seki T. Panté N. Ohba T. Nishii K. Kuma K. Hayashida T. Miyata T. Aebi U. Fukui M. Nishimoto T. Nature. 1995; 376: 184-188Crossref PubMed Scopus (419) Google Scholar). The GDP form of Ran/TC4 can bind to NTF2 (14Paschal B.M. Delphin C. Gerace L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7679-7683Crossref PubMed Scopus (79) Google Scholar,19Nehrbass U. Blobel G. Science. 1996; 272: 120-122Crossref PubMed Scopus (149) Google Scholar) as well as to a complex containing RanBP1 and the β subunit of the NLS receptor (20Chi N.C. Adam E.J.H. Visser G.D. Adam S.A. J. Cell Biol. 1996; 135: 559-569Crossref PubMed Scopus (158) Google Scholar). NTF2 itself can bind directly to multiple proteins of the NPC containing FXFG repeat motifs, including p62 (10Paschal B.M. Gerace L. J. Cell Biol. 1995; 129: 925-937Crossref PubMed Scopus (344) Google Scholar, 19Nehrbass U. Blobel G. Science. 1996; 272: 120-122Crossref PubMed Scopus (149) Google Scholar, 21Hu T.H. Guan T.L. Gerace L. J. Cell Biol. 1996; 134: 589-601Crossref PubMed Scopus (152) Google Scholar). It has become clear that elucidating the mechanism of nuclear protein import will require understanding how the cytosolic transport factors interact with each other and with the NPC at specific transport steps and how these interactions are coupled to the GTPase cycle of Ran/TC4 (1Görlich D. Mattaj I. Science. 1996; 271: 1513-1518Crossref PubMed Scopus (1073) Google Scholar, 12Melchior F. Gerace L. Curr. Opin. Cell Biol. 1995; 7: 310-318Crossref PubMed Scopus (227) Google Scholar). The mechanisms of nuclear protein import are predicted to be conserved between vertebrates and Saccharomyces cerevisiae. Basic-type NLSs within proteins such as the glucocorticoid receptor and the SV40 large T antigen are fully functional in yeast, and the primary structures of the cytosolic transport factors show 40–80% identity when human and yeast proteins are compared. The basic architecture of the NPC appears to be conserved as well (22Panté N. Aebi U. Int. Rev. Cytol. 1995; 162: 225-255Crossref Scopus (49) Google Scholar). We have, therefore, used a molecular genetic approach to characterize NTF2 in the budding yeastS. cerevisiae. We have found that the scNTF2 gene is required for viability and that depletion of scNTF2p leads to nuclear structure defects similar to those observed with mutants of certain nuclear pore complex proteins. We carried out a genetic selection for multicopy suppressors that would compensate for loss of scNTF2p. This analysis revealed that the S. cerevisiaehomologue of Ran/TC4 is a multicopy suppressor of a scNTF2deletion strain, thus providing the first in vivo evidence that certain functions of NTF2 and Ran/TC4 are linked. Furthermore, we have used recombinant proteins to show that Ran/TC4 can relieve the requirement for NTF2 in a permeabilized cell import assay in mammalian cells. Taken together, these data support the hypothesis that NTF2 serves to modulate a transport step(s) involving Ran/TC4. To generate the plasmid used for methionine-regulated expression of scNTF2p, the scNTF2 ORF was amplified by PCR and cloned into the unique BamHI site immediately downstream of the MET3 promoter in the plasmid pRS405-MET3 (generously provided by Drs. Steve Haase and Steve Reed, The Scripps Research Institute). The MET3-scNTF2 fragment was then removed by digestion with NotI andHindIII and cloned into pRS315 (23Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) to generateLEU2-marked construct pMET-scNTF2. The plasmid for galactose-regulated expression of scNTF2 was constructed by PCR amplification of the scNTF2 ORF and subsequent cloning into the unique EcoRI site of yCPG2 (provided by Drs. Steve Haase and S. Reed). All yeast strains used in this study are derivatives of the homozygous diploid strain 15D (MATa/MATα, ura3/ura3, leu2/leu2, trp1/trp1, his2/his2, ade1/ade1, provided by Drs. Steve Haase and Steve Reed). A single copy of thescNTF2 locus was disrupted in 15D via a PCR-based method to generate BPY1 (scNTF2/ΔscNTF2:: URA3) and BPY3 (scNTF2/ΔscNTF2::TRP1). Specifically, to generate BPY1, PCR was used to generate a URA3 fragment flanked by sequences homologous to flanking regions of thescNTF2 gene such that upon integration, the entire ORF ofscNTF2 would be deleted. The oligos used were 5′-TAAGGAACCCAGGTTTTAATACTATTATCTTTATAATGGCTTTTCAATTCAATCATC-3′ and 5′-AAATACATGTTTCTGTGGTGACTTAAAAAATCCTTAAGCAGATTCCCGGGTAATAACTG-3′. After PCR amplification, the fragment was gel-purified and used to transform the diploid strain 15D to Ura+. To confirm correct integration of the construct at the scNTF2 locus, whole cell PCR was performed with flanking primers (primer 1 = 5′-CAAGGTGAGACTTAGGCTGATAAG-3′, primer 2 = 5′-GCCTTATACATCGTTAGCTAAGC-3′) or one flanking primer (primer 1) and one URA3-internal primer, primer 3 = 5′-GCTGACATTGGTAATACAGTC-3′). Isolates in which both PCR reactions produced fragments of the size expected for integration atscNTF2 were judged to be deletion isolates. The same method was applied to replace the scNTF2 ORF with TRP1in BPY3. The primers used to generate the PCR product forTRP1 integration into the scNTF2 locus were 5′-CCTAAGGAACCCAGGTTTTAATACTATTATCTTTATAATGGGCATTGGTGACTATTGAGC-3′ and 5′-AAATACATGTTTCTGTGGTGACTTAAAAAATCCTTAAGCAGGCAAGTGCACAAACAATAC-3′. Strain BPY4 (ΔscNTF2::TRP1containing pMET3-scNTF2) was generated by transforming BPY3 with the pMET3-scNTF2 plasmid followed by sporulation and dissection to yield a Leu+, Trp+ haploid. The mating type of this strain was not determined. A similar procedure was used to generate a ΔscNTF2 haploid carrying the plasmid pGAL10-scNTF2. Propagation of yeast strains was performed using standard media recipes as described in Ref. 24. The screen for multicopy suppressors was carried out in the strain BPY4. Log phase yeast cells were transformed by using polyethylene glycol and lithium acetate (25Gietz R.D. Schiestl R.H. Willems A.R. Woods R.A. Yeast. 1995; 11: 355-360Crossref PubMed Scopus (1734) Google Scholar) with a S. cerevisiae genomic library constructed in YEp24 (26Carlson M. Botstein D. Cell. 1982; 28: 145-154Abstract Full Text PDF PubMed Scopus (1001) Google Scholar) and plated onto synthetic medium containing 5 mm methionine and lacking uracil. We plated 1% of the transformation onto media lacking methionine and determined that this procedure yielded a total of ∼108 Ura+ transformants. The 480 Ura+ colonies that grew under the condition of scNTF2p repression were replated onto methionine-containing media. Whole cell PCR was used to screen the 480 colonies for the presence of the library plasmids that contained the scNTF2 gene. Log phase yeast cells were harvested up to 7 h after the addition of methionine and processed for electron microscopy essentially as described by Byers and Goetsch (27Byers B. Goetsch L. Methods Enzymol. 1991; 194: 602-608Crossref PubMed Scopus (83) Google Scholar). We used both Spurr and Epon resins, the latter of which yielded slightly better preservation of membranes in our experiments. The permeabilized cell assay used to quantitate nuclear protein import in HeLa cells was described previously (10Paschal B.M. Gerace L. J. Cell Biol. 1995; 129: 925-937Crossref PubMed Scopus (344) Google Scholar). The reporter molecule for nuclear protein import was fluorescein isothiocyanate-labeled bovine serum albumin-conjugated with the SV40 large T antigen NLS (10Paschal B.M. Gerace L. J. Cell Biol. 1995; 129: 925-937Crossref PubMed Scopus (344) Google Scholar). Pretreatment of HeLa cell cytosol with p62-Sepharose and preparation of recombinant NTF2 and Ran/TC4 have also been described previously (8Melchior F. Paschal B. Evans J. Gerace L. J. Cell Biol. 1993; 123: 1649-1659Crossref PubMed Scopus (479) Google Scholar, 10Paschal B.M. Gerace L. J. Cell Biol. 1995; 129: 925-937Crossref PubMed Scopus (344) Google Scholar). The antibody to recombinant human NTF2 was generated in rabbits, affinity purified on NTF2-Sepharose, and used at a final concentration of 2 μg/ml. Anti-serum to the GSP1 protein (28Belhumeur P. Lee A. Tam R. DiPaolo T. Fortin N. Clark M.W. Mol. Cell. Biol. 1993; 13: 2152-2161Crossref PubMed Scopus (123) Google Scholar) was used at a dilution of 1:3000 and was kindly provided by Dr. Pierre Belhumeur (University of Montreal). Immunoblots were developed with peroxidase-labeled secondary antibodies and enhanced chemiluminescence. Sequence from the S. cerevisiae genome project revealed an open reading frame on chromosome V (cosmid 9537) that is highly related to the sequence of NTF2, a cytosolic factor that facilitates protein transport into the nucleus in mammalian cells (10Paschal B.M. Gerace L. J. Cell Biol. 1995; 129: 925-937Crossref PubMed Scopus (344) Google Scholar). The predicted ORF encodes a 125-amino acid protein that is 44.4% identical and 61.5% similar to the 127-amino acid human NTF2 protein (Fig. 1 A). The central region of the protein (Phe-12–Pro-76) is 58% identical, whereas the primary structures of the amino and carboxyl termini are unrelated except for residues Asn-116–Leu-121. The sequence relationship together with the similarities in size and isoelectric point suggest the yeast ORF encodes the S. cerevisiae homologue of NTF2 and is hereafter referred to as scNTF2p. Evidence that these proteins are functional homologues was obtained in recent work showing that the human cDNA encoding NTF2 can substitute for the yeast gene (29Corbett A.H. Silver P.A. J. Biol. Chem. 1996; 271: 18477-18484Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). We assessed the requirement for the scNTF2p in cell viability by standard gene replacement methods in the yeast strain 15D. To generate anscNTF2 deletion allele, we first used PCR primers that included sequences complementary to the 5′ and 3′ regions of thescNTF2 gene to amplify the URA3 gene (Fig. 1 B). The diploid strain 15D was then transformed with the linear PCR product. Stable Ura+ transformants were screened for integration of the URA3 gene by PCR using oligonucleotides that flank the scNTF2 locus (primers 1 and 2; Fig. 1 C). The DNA template from one Ura+transformant yielded PCR products with sizes of ∼560 and ∼1300 base pairs. The smaller product is the size expected from amplification of the scNTF2 locus, whereas the larger product is consistent with the replacement of the scNTF2 ORF with theURA3 gene. This was confirmed by showing that a PCR reaction with a primer that flanks scNTF2 (primer 1) and a primer within the URA3 gene (primer 3) produces a ∼700-base pair fragment. The heterozygote deletion strain for scNTF2(scNTF2/ ΔscNTF2::URA3, denoted BPY1) displayed no obvious defects in growth, morphology, or thermosensitivity (data not shown). Sporulation of the BPY1 strain and subsequent analysis of >40 tetrads revealed a 2:0 segregation (live:dead), demonstrating that the scNTF2 gene is required for viability (Fig. 1 D). We confirmed that viable segregants were Ura− by their failure to grow in the absence of uracil. These data are in agreement with those obtained using other strains of S. cerevisiae (PSY853, Ref. 29Corbett A.H. Silver P.A. J. Biol. Chem. 1996; 271: 18477-18484Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar; W303, data not shown, Ref. 19Nehrbass U. Blobel G. Science. 1996; 272: 120-122Crossref PubMed Scopus (149) Google Scholar). As a first step toward analyzing the function of scNTF2p in vivo, we examined the effects of scNTF2p depletion in haploid deletion strains containing plasmids with the scNTF2 gene under the control of different regulatable promoters. Initial experiments were carried out with scNTF2p expression regulated by the GAL10 promoter. We observed that colony growth in haploid deletion strains carrying theGAL10 plasmid was slowed substantially but not completely inhibited (data not shown), suggesting that expression from this plasmid is leaky. We chose the MET3 promoter as an alternative for achieving repressible scNTF2p expression. Transcription driven by this promoter can be strongly repressed by growing cells in the presence of millimolar concentrations of methionine (30Amon A. Irniger S. Nasmyth K. Cell. 1994; 77: 1037-1050Abstract Full Text PDF PubMed Scopus (404) Google Scholar). In a haploid deletion strain (BPY4) containing scNTF2 under the control of the MET3 promoter, transcription from the scNTF2 plasmid in the absence of methionine restored growth of this strain to the same level as the WT strain (Fig. 2,−Methionine). In contrast, substantial growth inhibition resulted from including 5 mm methionine in the media, an effect that was manifest as an approximately 100-fold difference in viability as compared with the WT strain (Fig. 2,+Methionine). The BPY4 colonies that did survive selection in the presence of methionine were also visibly smaller than colonies composed of WT cells. Methionine-repression of the scNTF2 gene in the BPY4 strain resulted in distortion and apparent fragmentation of the nucleus such that the nuclear DNA appeared as small 4′,6-diamidino-2-phenylindole dihydrochloride staining domains within these cells (data not shown). Surprisingly, the distorted nuclei present in the mutant cells accumulated a NLS-containing version of green fluorescent protein. 2D. Goldfarb, personal communication. However, it was not possible to quantititively compare the nuclear import rate in these mutant cells to the rate in wild type cells, due to the altered nuclear morphology of the mutant cells. We note that two mutant alleles ofscNTF2 have been isolated that show a temperature-sensitive defect in protein import (29Corbett A.H. Silver P.A. J. Biol. Chem. 1996; 271: 18477-18484Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), providing direct evidence that scNT2p is involved in nuclear import. To further characterize the altered nuclear morphology associated with depletion of the scNTF2p, we grew the WT and BPY4 strains in the presence of methionine for 7 h and processed the cells for thin section electron microscopy. Cells from the BPY4 strain (Fig. 3 B) displayed grossly distorted nuclear envelopes that often took the form of large intranuclear invaginations. These structures were never observed in WT cells (Fig. 3 A). Some of the mutant cells contained areas with apparent discontinuities or holes in the nuclear envelope (arrowheads, Fig. 3 B). We also observed the juxtaposition of two double pore-containing membranes within a single nucleus and the alignment of NPCs (arrows, Fig. 3 D). Interestingly, similar nuclear morphology phenotypes have been reported for yeast strains deficient for certain NPC proteins. For example, repression or overexpression of the nucleoporin Nup170p in a POM152 null background results in structural alterations of the nuclear envelope (29Corbett A.H. Silver P.A. J. Biol. Chem. 1996; 271: 18477-18484Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) that are nearly identical to the changes observed by simple repression of scNTF2p expression reported here. These observations suggest that scNTF2p is involved in the ongoing maintenance of nuclear structure. This could be indirectly due to the need for scNTF2p in nuclear protein import or could reflect a direct involvement of scNTF2p in some feature of nuclear architecture. We carried out a multicopy suppressor screen to identify components that allow cells to grow in the absence of NTF2p, with the goal of identifying functionally linked components in the nuclear protein import pathway. The screen relied on the efficient repression of scNTF2p expression obtained with the MET3 promoter (30Amon A. Irniger S. Nasmyth K. Cell. 1994; 77: 1037-1050Abstract Full Text PDF PubMed Scopus (404) Google Scholar). The relatively small number and size of colonies that grew in the presence of methionine (Fig. 2) suggested that the selection scheme would have a background of 96% of cytosolic NTF2 as determined by quantitative immunoblotting, but it does not remove Ran/TC4 from cytosol (Ref. 10Paschal B.M. Gerace L. J. Cell Biol. 1995; 129: 925-937Crossref PubMed Scopus (344) Google Scholar and data not shown). We measured the nuclear protein import supported by this cytosol as a function of Ran/TC4 concentration both in the absence and presence of NTF2 (Fig. 5 A). The addition of purified recombinant NTF2 to NTF2-depleted cytosol stimulated transport 2.3-fold, an effect that was altered only slightly by adding low concentrations (<2 μg/ml) of Ran/TC4 protein. However, the addition of Ran/TC4 (25 (μg/ml) to the NTF2-depleted cytosol (Fig. 5 A, lower curve) restored transport to levels that are comparable to those obtained in the presence of both NTF2 and Ran/TC4 (Fig. 5 A, upper curve). These data show that the requirement for NTF2 protein in the nuclear protein import assay can be relieved by supplementing the transport reaction with recombinant Ran/TC4 protein. We note that low levels of NTF2 contributed by the depleted cytosol and the permeabilized cells together with the Ran/TC4 in the depleted cytosol probably accounts for the transport obtained without supplementing the reaction with recombinant NTF2 (Fig. 5 B). Our initial characterization of scNTF2p in the budding yeastS. cerevisiae has provided valuable insight on the role of this protein in cell function. We have found that this protein is required for cell viability, in agreement with observations from other laboratories (19Nehrbass U. Blobel G. Science. 1996; 272: 120-122Crossref PubMed Scopus (149) Google Scholar, 29Corbett A.H. Silver P.A. J. Biol. Chem. 1996; 271: 18477-18484Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Moreover, we observed that depletion of scNTF2p leads to aberrant nuclear morphologies characterized by gross invaginations of the nuclear envelope and juxtaposition of NPCs in stacked nuclear membranes, similar to the morphological phenotypes obtained with mutants of certain NPC proteins (32Aitchison J.D. Rout M.P. Marelli M. Blobel G. Wozniak R.W. J. Cell Biol. 1995; 131: 1133-1148Crossref PubMed Scopus (164) Google Scholar). Most significantly, we have demonstrated that the scNTF2 null mutant can be rescued by GSP1, the S. cerevisiae homologue of Ran/TC4. This provides the first in vivo evidence that certain functions of NTF2 and Ran/TC4 are linked. These linked functions probably relate to nuclear protein import, since both Ran/TC4 and scNTF2 are involved in nuclear import in yeast (29Corbett A.H. Silver P.A. J. Biol. Chem. 1996; 271: 18477-18484Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 33Schlenstedt G. Saavedra C.J. Loeb D.J. Cole C.N. Silver P.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 225-229Crossref PubMed Scopus (168) Google Scholar) as well as in higher eukaryotes (12Melchior F. Gerace L. Curr. Opin. Cell Biol. 1995; 7: 310-318Crossref PubMed Scopus (227) Google Scholar). Our present genetic analysis does not unequivocally demonstrate a direct interaction between GSP1 and scNTF2, and it is formally possible that GSP1 rescues thescNTF2 null mutant by an indirect mechanism. However, addition of recombinant Ran/TC4 to digitonin-permeabilized HeLa cells depleted of NTF2 results in a rescue of nuclear protein import. Taken together, these observations suggest that the rate of nuclear protein import is closely tied to the cellular concentrations of NTF2 and Ran/TC4. The finding that GSP1p levels were elevated only 1.73-fold inscNTF2 null mutants was surprising given that theGSP1 gene was carried on a high copy number (2 μm) plasmid. This may be explained by the fact that overexpression of GSP1 is slightly toxic in the strains used in this study (Fig. 4 A). Therefore, under the conditions of the genetic selection, growth was inhibited both by the repression of scNTF2p expression and by the overexpression of GSP1p from the library plasmid. In vertebrates, both Ran/TC4 and NTF2 are required for transport event(s) that occur during movement of a substrate-NLS receptor complex through the NPC. There is good evidence that during the transport process the GTP form of Ran/TC4 binds to RanBP2, a putative initial substrate/NLS receptor docking site at the NPC and that RanGAP-stimulated hydrolysis at this site is involved in the forward progress of a transport complex (34Melchior F. Guan T. Yokoyama N. Nishimoto T. Gerace L. J. Cell Biol. 1995; 131: 571-581Crossref PubMed Scopus (127) Google Scholar, 35Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1019) Google Scholar). After the transport complex leaves RanBP2, it is not known whether nucleotide exchange on Ran/TC4 and additional rounds of Ran-GTP hydrolysis are required to drive translocation through the nuclear pore (15Rexach M. Blobel G. Cell. 1995; 83: 683-692Abstract Full Text PDF PubMed Scopus (668) Google Scholar, 35Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1019) Google Scholar). The role of NTF2 in nuclear protein import is even less well defined. NTF2 binds directly to several FXFG-type repeat proteins of the NPC as well as to the GDP form of Ran, suggesting it could play a role in modulating the interaction of transport complexes with several discrete NPC sites (14Paschal B.M. Delphin C. Gerace L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7679-7683Crossref PubMed Scopus (79) Google Scholar, 91, 21Hu T.H. Guan T.L. Gerace L. J. Cell Biol. 1996; 134: 589-601Crossref PubMed Scopus (152) Google Scholar). Our observations that a high level of Ran/TC4 can relieve the requirement for NTF2 in cell viability (in yeast) and nuclear protein import (in mammalian cells) suggests that NTF2 may regulate a protein-protein interaction(s) that involves Ran/TC4. For example, dissociation of a substrate-NLS receptor complex from RanBP2 during its transfer to another NPC protein could be promoted by the binding of either NTF2 or Ran-GTP to RanBP2, and in the absence of NTF2 this could occur more rapidly with an increased concentration of Ran/TC4. Alternatively, NTF2 may contribute to binding or dissociation reactions promoted by RanGDP, if the latter itself acts as a soluble factor to promote a step(s) of nuclear import (36Görlich D. Panté N. Kutay U. Aebi U. Bischoff F.R. EMBO J. 1996; 15: 5584-5594Crossref PubMed Scopus (542) Google Scholar). Our observation that NTF2 is dispensable under certain conditions argues against the hypothesis that NTF2 promotes GDP/GTP exchange on Ran/TC4 during nuclear import (19Nehrbass U. Blobel G. Science. 1996; 272: 120-122Crossref PubMed Scopus (149) Google Scholar). In summary, we have demonstrated that the scNTF2 gene encodes an essential protein that is linked to some function(s) ofGSP1, the S. cerevisiae homologue of Ran/TC4. The ability of a high level of Ran/TC4 to relieve the requirement for an otherwise essential gene product in vivo as well as in vitro places constraints on possible functions for NTF2 in nuclear protein import. We favor a role for NTF2 in regulating protein-protein interactions that involves Ran/TC4 at the nuclear pore complex. We thank Drs. Steve Haase, Peter Kaiser, and Steve Reed for gifts of strains and plasmids and for helpful advice. We thank Drs. Janet Leatherwood and Paul Russell for instruction and use of their tetrad dissection apparatus. We thank Dr. David Goldfarb for examining our strains for potential protein import defects and Dr. Pierre Belhumeur for providing the antiserum to GSP1. We also thank Konrad Zeller for technical assistance.