The NTF2 gene encodes an essential, highly conserved protein that functions in nuclear transport in vivo.

The small protein p10/Ntf2p has been implicated in protein import in vitro (Moore, M. S., and Blobel, G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10212-10216; Paschal, B. M., and Gerace, L. (1995) J. Cell Biol. 129, 925-937). Here we present the first evidence that demonstrates an essential in vivo role for the NTF2 gene product in nuclear transport. The NTF2 locus was identified in a screen for temperature-sensitive Saccharomyces cerevisiae mutants defective in the localization of nuclear proteins. Genetic analysis demonstrates that the NTF2 gene is essential for viability in budding yeast. Two temperature-sensitive mutants, ntf2-1 and ntf2-2, that each contain single point mutations in highly conserved amino acid residues show defects in the localization of nuclear proteins but not in the export of poly(A)+ RNA following a shift to the nonpermissive temperature. An epitope-tagged version of Ntf2p was used to show that the protein is concentrated at the nuclear envelope. Finally, the human gene under the control of the yeast promoter fully substitutes for the deleted yeast gene. Taken together, these results demonstrate the exquisite functional conservation of this protein throughout evolution and indicate a critical in vivo role in nuclear transport.

The small protein p10/Ntf2p has been implicated in protein import in vitro ( Here we present the first evidence that demonstrates an essential in vivo role for the NTF2 gene product in nuclear transport. The NTF2 locus was identified in a screen for temperature-sensitive Saccharomyces cerevisiae mutants defective in the localization of nuclear proteins. Genetic analysis demonstrates that the NTF2 gene is essential for viability in budding yeast. Two temperature-sensitive mutants, ntf2-1 and ntf2-2, that each contain single point mutations in highly conserved amino acid residues show defects in the localization of nuclear proteins but not in the export of poly(A) ؉ RNA following a shift to the nonpermissive temperature. An epitope-tagged version of Ntf2p was used to show that the protein is concentrated at the nuclear envelope. Finally, the human gene under the control of the yeast promoter fully substitutes for the deleted yeast gene. Taken together, these results demonstrate the exquisite functional conservation of this protein throughout evolution and indicate a critical in vivo role in nuclear transport.
Bi-directional transport of macromolecules across the nuclear envelope occurs through nuclear pores that are proteinaceous channels in the membrane bilayer (1,2). In addition to the nuclear pores, a number of soluble factors are required for the movement of both proteins and RNA across the nuclear envelope. Recent work has identified a number of these factors and presented models for their roles in the transport reaction (3)(4)(5). GTP hydrolysis mediated by the small GTP-binding protein Ran is required both for the import and export of proteins and for the export of poly(A) ϩ RNA (6 -9). In addition, a number of factors that regulate the GTP hydrolysis activity of Ran play critical roles in transport. These proteins include the cytoplasmic GTPase activating protein RanGAP1/Rna1p (10 -13), the cytoplasmic Ran binding protein 1 (RanBP1)/Yrb1p which stimulates the GAP 1 activity of Rna1p (14 -17), and the nuclear nucleotide exchange factor, RCC1/Prp20p (18). All of these proteins influence both the import of protein and the export of poly(A) ϩ RNA (9,12,17,19,20).
Our goal was to identify novel components of the transport system by using a screen to identify conditional yeast mutants defective in the localization of a nuclear protein. This screen led to the isolation of the NTF2 gene that encodes a protein of approximately 14 kDa with homology to a human protein, PP15 (33). Recently the Ntf2 protein was implicated in transport to the nucleus in vitro by two different studies (34,35). In Xenopus the protein is referred to as p10 and was identified as the previously described (7) cytoplasmic B-2 component required for transport of proteins into the nucleus in vitro (34). p10 was found in a complex with Ran containing a single Ran molecule and a dimer of p10 (34). This finding is consistent with previous experiments that suggested the human PP15 existed as a homodimer in vivo (33). In a second study, Ntf2p was identified as the necessary transport component that was depleted from cytosols following incubation with the HeLa cell O-linked glycoprotein p62 (35), a component of the nuclear pore (36). These results suggested that not only is Ntf2p important for transport to the nucleus in vitro but that it also interacts with the nuclear pore. While these experiments led to the proposal that the NTF2 gene product was important for the import of proteins to the nucleus, they were unable to address the in vivo role of the protein. The data reported here is the first demonstration of an essential in vivo function for Ntf2p in the nuclear transport process.
Mutant Screen-Strains were grown in YEPD at 25°C to a concentration of 1 ϫ 10 7 cells/ml and then shifted to the nonpermissive temperature, 36°C, for 2 h. Cells were fixed and prepared for indirect immunofluorescence as described previously (44). Samples were stained with polyclonal antibodies directed against the nuclear protein Npl3p (45) at a dilution of 1:500 followed by incubation with FITClabeled anti-rabbit antibodies at a 1:1000 dilution and DAPI.
Deletion of NTF2 Coding Region-The entire NTF2 coding region was deleted in the diploid strain PSY845 using a PCR strategy (46). PCR was used to generate a HIS3 (underlined regions shown below) fragment flanked by 45 bp directly 5Ј and 3Ј of the NTF2 coding region using the following oligonucleotides: 5Ј-GTG TAG AAT ACC CCC TAA  GGA ACC CAG GTT TTA ATA CTA TTA TCG GCC TCG TCT AGT  ACA CTG-3Ј and 5Ј-CGA AAA TGA AAA TAC ATG TTT CTG TGG TGA  CTT AAA AAA TCC GCG CGC CTC GTT CAG AAT G-3Ј. Following PCR amplification the resulting HIS3 fragment was used to transform the diploid yeast strain (PSY845) and His ϩ transformants were selected. Integration was verified by PCR and Southern blotting.
Temperature-sensitive ntf2 Mutants-Temperature-sensitive mutants were generated by using PCR mutagenesis and plasmid shuffling as described (41). Briefly, a 1.8-kb genomic fragment encoding Ntf2p was amplified with TaqI polymerase under manufacturer's recommended conditions using the following primers: oligo 1/5Ј-CGA AGT TTG AGA GGA ATT TG-3Ј and oligo 2/5Ј-CAA TAC GAA CAT AAT CCG-3Ј. Gap-repair ligation (40) was achieved by co-transforming the strain PSY852 with the PCR product and plasmid pPS882 linearized with XbaI and BglII. Colonies were selected on synthetic media lacking leucine and containing 5-FOA (fluoroorotic acid) and 7.5 mg/liter of the vital dye Erythrocin B. Colonies that were dead at 36°C but alive at 25°C were identified by uptake of the Erythrocin B dye. Plasmids were rescued from putative temperature-sensitive strains and retransformed into strain PSY852 to demonstrate plasmid linkage of the temperaturesensitive phenotype. Five plasmids were chosen for further analysis, and the open reading frame of NTF2 was entirely sequenced using a U. S. Biochemicals Corp. sequencing kit. Four of these plasmids were pPS919 and the remaining one was pPS920.
Protein Localization-Indirect immunofluorescence microscopy was employed to examine the localization of either an endogenous nuclear protein, Npl3p (44,45), or an artificial reporter protein, SV40-NLS invertase. For experiments that examined the cellular localization of Npl3p, cells were grown in YEPD at room temperature to a density of 1 ϫ 10 7 cells/ml. For the inducible reporter protein, the cells were grown to a density of 5 ϫ 10 6 cells/ml in media lacking leucine with 2% raffinose. Cultures were then induced by the addition of 2% galactose, and growth was continued at room temperature for 2-4 h. Following this induction, cultures were split, and half was maintained at room temperature while half was shifted to 36°C for 2 h. Cells were then prepared for indirect immunofluorescence microscopy as described previously (44). Briefly cells were fixed in 3.7% formaldehyde for 1 h, washed in 0.1 M potassium phosphate buffer, pH 6.5, and resuspended in P solution (0.1 M potassium phosphate buffer, pH 6.5, 1.2 M sorbitol). Cell walls were then digested with 0.5 mg/ml zymolyase. Following digestion, cells were adhered to slides pretreated with 0.3% polylysine and fixed in ice-cold methanol for 6 min followed by 30 s in ice-cold acetone. Incubation with antibodies against Npl3p (1:500 dilution) (45) or invertase (1:10,000 dilution) (42) was followed by incubation with FITC-labeled anti-rabbit antibodies (Jackson ImmunoResearch) at a 1:1000 dilution and with DAPI (1 g/ml).
Poly(A) ϩ RNA Localization-This assay was carried out as described by Amberg et al. (37) with a number of modifications. Cells were grown, fixed, and digested as described above for indirect immunofluorescence microscopy. After cells were adhered to slides they were permeabilized with 0.1% Triton X-100 in P solution for 10 min. Cells were washed once in P solution and equilibrated in 0.1 M triethanolamine (TEA), pH 8.0, for 2 min followed by incubation in 0.1 M TEA, 0.25% acetic anhydride for 10 min. Pre-hybridization was carried out at 37°C for 1 h in hybridization buffer (50% deionized formamide, 4 ϫ SSC, 1 ϫ Denhardt's solution, 125 g/ml tRNA, 10% dextran sulfate, 500 g/ml denatured salmon sperm DNA). The digoxigenin-labeled oligo(dT) probe (50-mer) was labeled with a Genius 6 kit (Boehringer Mannheim). Hybridization was carried out overnight at 37°C. Slides were washed in 2 ϫ SSC (twice), 1 ϫ SSC, and finally in 0.5 ϫ SSC and then blocked in Antibody blocking buffer (0.1 M Tris, pH 9.0, 0.15 M NaCl, 5% heatinactivated fetal calf serum, 0.3% Triton X-100). Samples were incubated in antibody (FITC-conjugated anti-digoxigenin) diluted 1:200 in Antibody blocking buffer for 2 h. Slides were washed twice (10 min and then 30 min) in Antibody wash 1 (0.1 M Tris, pH 9.0, 0.15 M NaCl) and then twice (10 min then 30 min) in Antibody wash 2 (0.1 M Tris, pH 9.5, 0.1 M NaCl, 50 mM MgCl 2 ). As with indirect immunofluorescence microscopy, DAPI was used to visualize chromatin. Epitope-tagged Ntf2p-In order to generate an epitope-tagged version of Ntf2p, a unique SalI site was introduced into the coding region of NTF2 at the N terminus, which is the region of the protein that is least conserved. The altered protein (pPS950) was functional as it was able to rescue the NTF2 null strain. The epitope-tagged version of Ntf2p was made by inserting oligonucleotides encoding the 11-amino acid myc epitope (EQKLISEEDLN) engineered with in-frame SalI sites. This generated a functional epitope-tagged version of Ntf2p (pPS951). The proper insertion of the myc epitope tag was verified by sequence analysis and immunoblotting (see Fig. 7A).
Immunoblotting-To confirm the presence of the myc epitope in the NTF2 coding region, immunoblotting of strains carrying pPS951 was carried out. Wild type strains were transformed with the plasmid and grown in synthetic media lacking leucine with 2% glucose as the carbon source to a concentration of 3 ϫ 10 7 cells/ml. Samples were prepared as described previously (13) and resolved on 15% polyacrylamide gels (47). Transfer of protein and immunoblotting was carried out by standard methods (48). Immunoblots were probed with the monoclonal anti-myc antibody 9E10 (1:200 dilution). Immunoreactive bands were visualized with the ECL chemiluminescence kit (Amersham Life Sciences, Inc.).
Localization of Ntf2p-Ntf2p was localized either by indirect immunofluorescence microscopy using the epitope-tagged version of the protein or by examining the localization of a functional GFP fusion protein in living cells. For indirect immunofluorescence the wild type strain FY86 was transformed with pPS951 encoding the myc-tagged Ntf2p. Cultures were grown in synthetic media lacking leucine to maintain the plasmid. Cells were digested with 0.5 mg/ml zymolyase, fixed in ice-cold methanol for 30 min (13), and then processed as described above for indirect immunofluorescence. Proteins were visualized by incubation with undiluted anti-myc (9E10) tissue culture supernatant (Ntf2p) or the anti-nucleoporin antibody guinea pig anti-Nup159p (1:200 dilution) (49) followed by incubation with FITC-labeled anti-mouse antibodies (myc) or Texas Red-labeled anti-guinea pig antibodies (Nup159p) at a 1:1000 dilution and with DAPI.
Ntf2p was localized in living cells by using a GFP fusion protein. The fusion protein was generated by fusing GFP in-frame to the N terminus of Ntf2p (pPS886). The naturally fluorescent protein was viewed directly using the FITC channel of a standard fluorescence microscope.
Human NTF2 under the Control of the Yeast NTF2 Promoter-The human NTF2 gene was amplified by PCR from a human Jurkat cell cDNA library with a BglII site engineered at the 5Ј end and a SalI site engineered at the 3Ј end using the following primers: oligo 3/5Ј-GGA AAG ATC TAT GGG AGA CAA GCC AAT TTG GG-3Ј and oligo 4/5Ј-CGC GTC GAC GCA TGA TAT TTG GAG CAT CTG-3Ј. This PCR product was cloned into pBluescript KS (Stratagene) cut with BamHI and SalI. Several hybrid primers were then used to engineer the human open reading frame into the flanking region of the yeast NTF2 gene (50). A brief description of the generation of the 5Ј end of the construct follows. A similar strategy was used to place the 3Ј yeast flanking region directly after the stop codon of the human gene. The human NTF2 gene was reamplified using the following primers: oligo 5/5Ј-CTA TTA TCT TTA TAA TGG GAG ACA AGC CAA TTT G-3Ј and oligo 4. In oligo 5 the region underlined is derived from the region of the yeast genome immediately 5Ј to the yeast NTF2 start codon. In addition the 5Ј-promoter region from 515 bp upstream of the ATG of the yeast gene was amplified with the following primers: oligo 1 and oligo 6/5Ј-CAA ATT GGC TTG TCT CCC ATT ATA AAG ATA ATA G-3Ј. In oligo 6 the underlined region corresponds to the complement of the 5Ј end of the human open reading frame. The two PCR products generated by these amplifications are complementary to one another in the hybrid region that corresponds to the original PCR primers. The resulting PCR products were denatured by heating and then were mixed and allowed to undergo one round of elongation in the absence of any exogenous primers. This extended the noncomplementary regions to create a hybrid PCR product that corresponds to the 515 bp 5Ј of the yeast start codon fused to the human NTF2 open reading frame. This hybrid product was then amplified by readdition of oligos 1 and 4 which correspond to the 5Ј end of the yeast 5Ј-flanking sequence and the 3Ј end of the human NTF2 open reading frame, respectively. The final hybrid construct was digested with XbaI and BglII and cloned into pRS315 (LEU2, CEN (39)) cut with XbaI and BamHI to generate pPS884. Finally, the insert was sequenced to ensure that no mutations had been introduced during the construction of the plasmid.

RESULTS
In order to isolate components of the nuclear transport system, we chose to screen for temperature-sensitive yeast mutants defective in the localization of nuclear proteins. The temperature-sensitive library used was generated by ultraviolet mutagenesis of the wild type strains FY86 and FY23 (37). Each temperature-sensitive mutant was grown in YEPD at the permissive temperature (25°C) and then shifted to the nonpermissive temperature (36°C) for 2 h. Following the shift to the nonpermissive temperature, cells were fixed for indirect immunofluorescence microscopy, and the localization of the nuclear protein, Npl3p, was examined. After screening approximately 1200 mutant strains, six strains that mislocalized the nuclear protein were identified. Each strain was crossed to strains containing mutations in genes that had previously been implicated in nuclear transport including rna1-1 (12), npl3-1 (45), and prp20 -1 (13,19,37) to identify mutants in these known genes. This approach identified an allele of prp20 which confirmed the ability of this screen to identify mutants in the nuclear transport machinery. Therefore, further characterization of the unidentified mutants was undertaken.
Identification of the NTF2 Locus-One mutant was found to have a defect in the localization of both Npl3p and the reporter protein H 2 B-NLS-␤-galactosidase (data not shown). The mutant strain was back-crossed three times to the parent strain (FY23) to yield the working strain PSY854. In all tetrads examined, temperature sensitivity and protein mislocalization co-segregated as a single locus suggesting that a single mutation was responsible for both phenotypes. The genomic DNA corresponding to the mutant locus was cloned by complementation of the temperature-sensitive phenotype by transforming the strain with a yeast genomic library (38). Several clones that rescued the temperature-sensitive phenotype were identified. Restriction digests demonstrated that all clones contained overlapping inserts. The clone that contained the smallest complementing fragment was chosen for further analysis. This clone, pPS902, contained a 13-kb insert that mapped to a previously sequenced region of chromosome V corresponding to approximately 7000 -20,000 bp of cosmid 9537 (Genbank Accession U18778). Subcloning demonstrated that the fragment responsible for restoring both temperature resistance and correct localization of nuclear proteins was a 7.9-kb NsiI-NcoI fragment encoding two complete genes transcribed in opposite directions from one another (Fig. 1, panel A). The first gene corresponds to a 3.5-kb open reading frame encoding the PSL1 gene that is allelic with the previously described sec3 locus (51, The NsiI-NcoI fragment corresponds to pPS917; the NsiI-ScaI fragment corresponds to pPS903; the XbaI-XbaI fragment corresponds to pPS853; and the XbaI-BglII fragment corresponds to pPS916. The ability of these plasmids to complement the temperature-sensitive (TS) and protein localization (Localization) phenotypes are shown on the right. The SpeI sites used to create the probe for Southern blotting are indicated. The deletion found in the PSY845 mutant strain is indicated at the top. B, the amino acid homology between the human PP15 protein and Ntf2p from S. cerevisiae is shown. Identical amino acids are shaded. The site of insertion of the myc epitope is indicated by the underlined amino acid (L). This is also the amino acid altered in the epitope-tagged version of Ntf2p (L to V). The mutations found in ntf2-1 and ntf2-2 are indicated by asterisks. The mutation in ntf2-1 changes amino acid 83 from methionine (M) to threonine (T). The ntf2-2 mutation changes amino acid 91 from aspartic acid (D) to glycine (G). 52). The second gene (YER009w) encodes a small protein identified by homology to the small human protein PP15 (Fig. 1,  panel B) (33) and later shown to encode a homologue of p10/ Ntf2p (34,35,53). In order to determine which gene was mutated in strain PSY854, further subcloning was undertaken to separate the two open reading frames. This analysis revealed that the temperature sensitivity and the nuclear localization defect were separable. A 4-kb NsiI-ScaI fragment encoding only the PSLI/SEC3 gene (pPS903) was able to restore temperature resistance; however, the same clone was unable to rescue the protein localization defect (Fig. 1, panel A). In contrast, either a 3.8-kb XbaI fragment (pPS883) or a 1.9-kb XbaI-BglII fragment (pPS916) was able to restore protein localization to the mutant but was unable to restore temperature resistance (Fig. 1, panel A). Thus, it seemed that the two phenotypes resulted from changes in both genes. In order to determine the nature of the mutation in the mutant strain PSY854, Southern blotting was carried out. A 900-bp SpeI fragment corresponding to the N terminus of PSL1/SEC3 and a portion of the NTF2 open reading frame was used to probe genomic DNA prepared from either the mutant strain, PSY854, or the parent strain, FY86. Results indicate that the mutant strain contains a deletion of approximately 250 bp in this region (see Fig. 1, panel A). Subsequent mapping with smaller probes indicates that the small deletion eliminates the start codon of the PSL1/SEC3 gene and a portion of the NTF2 promoter. Since one phenotype of deletion of the PSL1/SEC3 gene is temperature sensitivity on rich media (52) and no defect in nuclear transport is observed, 2 we conclude that the temperature-sensitive phenotype results from deletion of the functional PSL1/SEC3 gene and that the nuclear transport defect results from a deletion of a portion of the promoter region of the NTF2 gene.
In order to confirm that there was no mutation in the coding region of the NTF2 gene, this portion of the genome was cloned by gap-repair from the mutant strain (40, 54) and completely sequenced. No mutations were identified. Consistent with a deletion in the region between the two genes the NTF2/PSL1 promoter could not be gap-repaired. Finally, to demonstrate that this locus was genetically linked to the observed phenotypes, a strain carrying a complete deletion of PSL1 was crossed to the mutant strain, PSY854. The resulting diploid strain was temperature-sensitive, and all spores generated from the diploid were also temperature-sensitive. These findings confirmed that the temperature sensitivity of the PSY854 strain is linked to the loss of a functional PSL1/SEC3 gene. However, the nuclear import defect associated with the promoter mutation in the NTF2 locus suggested that Ntf2p might be important for nuclear transport and led us to investigate further the in vivo function of Ntf2p.
NTF2 Is Essential for Viability in Saccharomyces cerevisiae-In order to determine whether the NTF2 gene was essential for viability, the entire NTF2 open reading frame starting at the ATG and ending at the stop codon was replaced with the HIS3 gene in the diploid strain PSY845. This strain was sporulated, and tetrads were dissected. In all cases only two viable spores were recovered, and they were never HIS ϩ (data not shown). Furthermore, when the diploid strain was transformed with pPS853 (URA3, CEN) encoding NTF2, four viable spores were occasionally obtained, and the spores that were HIS ϩ were never able to grow on FOA demonstrating that they were unable to live without the wild type plasmid encoding NTF2. These results indicate that the NTF2 gene is essential for viability in S. cerevisiae. An identical finding was recently reported by Nehrbass and Blobel (53).
Novel Temperature-sensitive Alleles of ntf2-To confirm the results obtained with the mutant strain, PSY854, and demonstrate that NTF2 is required for nuclear transport in vivo, we generated new conditional alleles of NTF2 using the PCR mutagenesis and plasmid shuffle technique described under "Experimental Procedures" (41). Five plasmids that allowed growth at 25°C but not at the restrictive temperature of 36°C were chosen for further analysis. The plasmids were retransformed into the deletion strain by a plasmid shuffle, and in all five cases the temperature-sensitive phenotype was found to be plasmid-linked. The inserts encoding NTF2 on all five plasmids were completely sequenced. In all cases, single base changes were identified within the coding region of NTF2 that resulted in a change in a single conserved amino acid residue. In four of the plasmids the same mutation was identified. This mutation changed base 248 from a T to a C and resulted in the change of methionine (M) 83 to threonine (T) (Fig. 1, panel B). This mutant was designated ntf2-1. The second mutation was a change of A to G at base 272 that changed amino acid 91 from aspartic acid (D) to glycine (G) and was designated ntf2-2 ( Fig.  1, panel B).
To characterize the growth of the mutant strains, the mutant plasmids, pPS919 and pPS920, were transformed into the NTF2 deletion strain, PSY852, by plasmid shuffle. As a control the plasmid pPS882 encoding wild type NTF2 was employed. Cultures were grown at the permissive temperature to a concentration of 0.2 ϫ 10 7 cells/ml in media lacking leucine or in YEPD. Cultures were then split, and half was maintained at the permissive temperature, and half was shifted to the nonpermissive temperature of 36°C. Samples were removed from each culture at 1-h intervals, and the cell number was determined by counting. Relative cell counts were the same regardless of the media employed, although all cells grew more slowly in synthetic media. Both ntf2-1 and ntf2-2 strains grow more slowly than the wild type strain at the 25°C (Fig. 2) although the effect is far more pronounced for the ntf2-2 allele. However, both strains cease growth rapidly following a shift to the nonpermissive temperature (Fig. 2). This cessation of growth is not observed for the strain carrying a wild type copy of NTF2.
Nuclear Proteins Are Mislocalized in ntf2 Mutants-To determine whether the temperature-sensitive alleles of NTF2 were defective in nuclear transport, protein localization was examined in these mutants. Initial experiments examined the localization of a galactose-inducible reporter protein composed FIG. 2. Growth curves for ntf2 mutant strains. Wild type and mutant ntf2 strains were grown at the permissive temperature (25°C). Cultures were then split, and half was maintained at the permissive temperature (25°C), and half was shifted to the nonpermissive temperature (36°C) as described under "Experimental Procedures." The relative increase in cell number (Fold Cell Growth) is plotted versus time. The point at which cells were shifted to the nonpermissive temperature is indicated by the arrow. Results are indicated for the wild type (Ⅺ), ntf2-1 (q), and ntf2-2 strains (E).
of the SV40-NLS fused to invertase (42). Both wild type and mutant strains were first grown at the permissive temperature to a concentration of 1 ϫ 10 7 cells/ml in media lacking leucine with raffinose (2%) as the carbon source. Galactose was then added to a final concentration of 2%, and cultures were induced for 2 h at 25°C. Following the induction, cultures were split, and half was maintained at the permissive temperature, and half was shifted to the nonpermissive temperature (36°C) for 2 h. Samples were then processed for indirect immunofluorescence as described under "Experimental Procedures." At the permissive temperature, the reporter protein is restricted to the nucleus of wild type cells. Most of the mutant cells also efficiently localize the reporter to the nucleus; however, several ntf2-1 cells mislocalize the protein even at the permissive temperature (Fig. 3, panel A). In addition, while the ntf2-2 cells localize the reporter primarily to the nucleus, several of the cells also appear to have some protein in the cytoplasm. These findings are not surprising since the growth curves shown in Fig. 2, panel A, demonstrate that growth of the mutant strains is somewhat retarded even at 25°C. Following a shift to the nonpermissive temperature, wild type cells still efficiently localize the reporter protein to the nucleus (Fig. 3, panel B). This is not the case for the two ntf2 mutant alleles. In both mutants the nuclear reporter protein is grossly mislocalized to the cytoplasm. Similar results were obtained when the localization of a second reporter protein containing the H 2 B-NLS fused to ␤-galactosidase (43) was examined (data not shown). These results suggest that Ntf2p plays a role in the localization of nuclear proteins in vivo.
To examine further the role of Ntf2p in protein transport, the localization of an endogenous nuclear protein Npl3p (45) was examined. Wild type cells were able to efficiently localize this protein to the nucleus following a shift to the nonpermissive temperature as evidenced by co-localization with the chromatin binding dye, DAPI (Fig. 4). In contrast, while Npl3p was localized primarily to the nucleus in the ntf2-1 and ntf2-2 alleles at the permissive temperature (data not shown), the protein was mislocalized throughout the cells following a 2-h shift to the nonpermissive temperature (Fig. 4). These results indicate that functional Ntf2p is required for the efficient localization of different NLS-bearing proteins to the nucleus.
RNA Export in ntf2 Mutants-In addition to proteins, RNA must also be transported into and out of the nucleus (55). To investigate the role of Ntf2p in RNA trafficking, we examined poly(A) ϩ RNA localization in the ntf2 mutant alleles using a previously described fluorescence in situ hybridization assay (37). Following a 2-h shift to the nonpermissive temperature, no accumulation of poly(A) ϩ RNA in the nucleus was observed in either ntf2-1 or ntf2-2 cells (Fig. 5). As a control poly(A) ϩ RNA export was examined in a mutant that is known to be defective in this process, prp20 -1 (56,57). prp20 -1 mutant cells accumulate a significant amount of poly(A) ϩ RNA in the nucleus under the same conditions where no nuclear accumulation is observed in the ntf2 mutants. These results suggest that the NTF2 gene product participates more prominently in the import of proteins to the nucleus than the export of poly(A) ϩ RNA from the nucleus.
Localization of Ntf2p-A functional epitope-tagged version of Ntf2p was generated by inserting an 11-amino acid myc epitope at the third codon of the protein as described under "Experimental Procedures" (also see Fig. 1, panel B). The plasmid encodes a functional version of Ntf2p as it is able to rescue the strain deleted for NTF2 (data not shown). Previous attempts to epitope-tag Ntf2p at the C terminus produced nonfunctional proteins that were unable to rescue the NTF2 null strain. These results suggest that the C terminus of the protein is critical for its in vivo function. This hypothesis is also supported by the fact that the temperature-sensitive mutants isolated were all in the C-terminal portion of the protein.
To confirm that the myc-tagged protein was expressed, immunoblotting was performed. Wild type strains were transformed with vector alone, a plasmid encoding untagged Ntf2p (pPS950), a plasmid encoding myc-tagged Ntf2p (pPS951), or a plasmid encoding another myc-tagged protein, Prp20p-myc. Cultures were grown in synthetic media lacking leucine to maintain the plasmids. Lysates were prepared, and 10 g of each lysate was resolved by polyacrylamide gel electrophoresis (47) and immunoblotted by standard procedures (48). Blots were probed with a 1:200 dilution of anti-myc (9E10) tissue  culture supernatant (Fig. 6, panel A). A band of approximately 14-kDa molecular mass is observed only in the strain carrying the epitope-tagged version of Ntf2p. The predicted molecular mass of S. cerevisiae Ntf2p is 14.3 kDa. These results confirm the expression of a functional epitope-tagged Ntf2p.
The epitope-tagged Ntf2p was employed to determine the intracellular localization of the protein using indirect immunofluorescence microscopy. Ntf2p was also localized in living cells using a functional Ntf2p fusion protein consisting of the NTF2 open reading frame with GFP fused to the N terminus. Previous work has shown that GFP fusion proteins are useful for the intracellular localization of proteins in living cells because in the absence of any other protein moieties the GFP protein is localized diffusely throughout the cell (44,58). Similar results were obtained regardless of whether Ntf2p was localized by indirect immunofluorescence or in live cells as a GFP fusion protein (Fig. 6).
In living cells expressing the Ntf2p-GFP fusion protein a punctate fluorescent pattern is observed (Fig. 6, panel B). This pattern is strikingly reminiscent of staining observed with numerous anti-nucleoporin antibodies (1). To determine whether Ntf2p co-localizes with nucleoporins, wild type cells were co-stained with anti-myc and anti-Nup159p antibodies (49). Results indicate that Ntf2p is localized in a pattern similar to that observed for Nup159p (Fig. 6, compare panels C and D). Since Nup159p has previously been shown to be a component of the nuclear pore (49,59), the anti-Nup159p staining defines the nuclear rim. Thus, Ntf2p is concentrated at nuclear pores, the site at which bi-directional transport into and out of the nucleus occurs. In control experiments, no anti-myc staining was observed in cells transformed with vector alone or with a plasmid encoding untagged Ntf2p.
The NTF2 Gene Is Highly Conserved-Yeast Ntf2p and the human PP15 protein are 45% identical to one another at the amino acid level. In order to determine whether the two proteins are functionally conserved, we tested whether the human gene could replace the yeast gene. The human PP15 (NTF2) gene was amplified from a Jurkat cell cDNA library as described under "Experimental Procedures." A CEN plasmid containing the human PP15 open reading frame in the context of the yeast NTF2 flanking sequence was constructed. This plasmid was transformed into the NTF2 deletion strain, PSY852, by a plasmid shuffle technique. As controls, the yeast NTF2 gene and the vector alone were also transformed into this strain. The resulting strains containing wild type NTF2 on a URA3 plasmid and the test plasmids with LEU2 markers were then streaked to medium containing FOA to eliminate the URA3 plasmid. As seen in Fig. 7 (panel A), the human gene was able to fully restore growth to the strain carrying a complete deletion of the yeast NTF2 gene. In addition, no defects in the localization of nuclear proteins were detected when the strain containing the human gene was examined as described above (Fig. 7, panel B). DISCUSSION This study provides the first evidence that the product of the NTF2 gene plays an important role in nuclear transport in vivo. We have shown that the NTF2 gene product is essential for viability in S. cerevisiae and that temperature-sensitive yeast strains bearing conditional alleles of NTF2 are unable to efficiently localize proteins to the nucleus but are still able to export poly(A) ϩ RNA. Furthermore, two different approaches demonstrate that Ntf2p is concentrated at the nuclear envelope in vivo. Finally, Ntf2p function is exceedingly well-conserved as the human NTF2 gene can functionally substitute for the deleted yeast NTF2 gene.  6. Ntf2p is concentrated at nuclear pores. A, an epitopetagged version of Ntf2p was generated as described under "Experimental Procedures." To confirm that the myc-tagged protein was expressed, the plasmid encoding it, pPS951, was transformed into wild type cells. As controls, the wild type strain was either transformed with a vector, with an untagged version of Ntf2p (pPS950), or with a plasmid encoding another myc-tagged protein, Prp20p-myc. Lysates from each strain were made, and 10 g of protein from each was resolved by polyacrylamide gel electrophoresis through 15% gels. An immunoblot is shown. The lanes are as follows: vector alone (1), untagged-Ntf2p (2) Previous work has shown that Ntf2p is important for the transport of proteins into the nucleus in vitro (34,35). However, neither of these studies was able to address the in vivo role of Ntf2p. It is only through the use of a genetic system such as S. cerevisiae and the generation of conditional alleles of the gene that an in vivo role can be inferred. Thus, genetic studies provide the essential complement to the biochemical studies that elucidate in vitro protein function.
In the past several years a combination of biochemical and genetic approaches has been used to demonstrate that bi-directional transport through the nuclear pores involves a cycle of highly regulated GTP hydrolysis (3,4,60). Several proteins that regulate the GTP hydrolysis activity of the small GTPbinding protein Ran have been identified including the exchange factor RCC1/Prp20p (18), the GTPase activating protein RanGAP1/Rna1p (11,61), and the GAP activating protein RanBP1/Yrb1p (14 -17). It is likely that the NTF2 gene also functions in the regulation of the Ran-GTP cycle. This hypothesis is consistent with the fact that the Xenopus Ntf2 protein (p10) was isolated in a complex with Ran (34).
The evidence collected thus far suggests that the regulation of Ran function mediated by Ntf2p occurs at the stage of translocation of macromolecules through the nuclear pore. In fact, Ntf2p is not required for targeting of NLS-containing substrates to the nuclear rim in vitro and has previously been shown to interact with the nucleoporin p62 in HeLa cell extracts (35). Our observation that Ntf2p is localized at the nuclear rim in vivo provides further evidence that the function of Ntf2p is carried out at the nuclear pore. Given this information, several models for Ntf2p function can be proposed. For instance, Ntf2p may be involved in recycling the Ran⅐GTP complex away from the nuclear pore complex after NLS proteins have been appropriately docked or it may function in the dissociation of the Ran⅐NLS-receptor complex at the pore (32). Further in vivo and in vitro studies will be needed to test these models for Ntf2p function.
Our results suggest that NTF2 plays an important role in the translocation of proteins into the nucleus. Recent work demonstrates that the critical components of the nuclear transport apparatus have been extensively conserved throughout evolution (4). It had been known for some time that the ultrastructure of the nuclear pore complex is conserved (36,62), and it is now becoming clear that this is also the case for individual components of the pore complex (63). Conservation of structure and function is also the rule for the soluble factors that mediate the transport reaction. For example, human RAN is 82% identical to yeast RAN (Gsp1p) at the amino acid level. Functional conservation is apparent for both the GAP, Rna1p, and the exchange factor, RCC1/Prp20p. Rna1p from the yeast Schizosaccharomyces pombe complements the temperature-sensitive rna1-1 mutant in S. cerevisiae (64) and the human RCC1 protein is able to complement the temperature-sensitive S. cerevisiae mutant prp20 -1 (65). Our findings demonstrate that Ntf2p is yet another conserved component of nuclear import machinery.
The data presented in this article suggest that the primary role of Ntf2p is in the import of proteins to the nucleus rather than in the export of poly(A) ϩ RNA. Under conditions where protein transport into the nucleus is clearly compromised, no nuclear accumulation of poly(A) ϩ RNA is observed. The same phenotype has been associated with mutants in other components of the nuclear transport system. Thus far mutations in either subunit of the NLS receptor have only been reported to cause defects in the localization of nuclear proteins but not in the export of poly(A) ϩ RNA (13,66). It is possible that yet unidentified alleles of these two genes or even of NTF2 may have primary defects in RNA export; however, the current interpretation is that these components contribute to the movement of proteins into the nucleus, whereas other factors function in the export of RNA and/or proteins.
Experiments carried out with Npl3p do suggest that export of protein from the nucleus (at least Npl3p) continues in ntf2 mutants. Npl3p is a poly(A) ϩ RNA binding protein that shuttles rapidly between the cytoplasm and the nucleus (44,45,67). The current model for Npl3p function suggests that Npl3p exits the nucleus in conjunction with poly(A) ϩ RNA and then is re-imported into the nucleus (41). Therefore the localization of Npl3p is dynamic in nature. The nuclear localization reported for the protein (44,45) simply reflects the fact that this highly abundant protein is recycled rapidly from the cytoplasm to the nucleus. Npl3p accumulates in the cytoplasm in ntf2 mutants (see Fig. 4). This finding is consistent with a model in which Npl3p continues to exit the nucleus at the nonpermissive temperature (otherwise no cytoplasmic pool would be observed) but re-import is slowed. Although recycling of Npl3p to the nucleus FIG. 7. Human Ntf2p can fully substitute for S. cerevisiae Ntf2p. A, the yeast NTF2 gene, the human NTF2 (PP15) gene, and the vector were transformed into the NTF2 deletion strain, PSY854, as described under "Experimental Procedures." The transformed strains were streaked to FOA plates to eliminate the wild type NTF2 gene present on a URA3 plasmid. The plate is shown. The strain containing vector alone is unable to grow because the NTF2 gene is essential for viability. However, both the control strain containing the wild type yeast NTF2 gene (pPS882) and the strain containing the human NTF2 gene (pPS884) grow normally indicating that human Ntf2p can functionally replace yeast Ntf2p. B, in order to demonstrate that the ability of human Ntf2p to substitute for yeast Ntf2p is complete, the localization of a nuclear protein was examined in strains carrying each of these plasmids. Under all conditions examined no defect in the localization of nuclear proteins was observed in strains carrying the human gene. The localization of the nuclear protein, Npl3p, in null strains covered by the endogenous Yeast gene (A-C) or the Human gene (D-F) is shown. Npl3p was localized by indirect immunofluorescence using polyclonal antibodies against Npl3p (A, D). The corresponding DAPI (B, E) and Nomarski (C, F) images are shown. is slowed, there is a sufficient nuclear pool to maintain wild type levels of poly(A) ϩ RNA export at least until the cells begin to lose viability. Similar results have been obtained with other mutants defective in nuclear transport including the npl3-1 mutant (41,45). When this mutant strain is grown at the semi-permissive temperature of 30°C, Npl3p accumulates in the cytoplasm, but no defect in the export of poly(A) ϩ RNA from the nucleus is observed (41).
Our results demonstrate that the NTF2 gene product is essential for the appropriate localization of nuclear proteins in vivo, that the protein is concentrated at the site of translocation across the nuclear envelope, and that the function of the protein has been extremely well-conserved throughout evolution. These data complement the previous in vitro results and provide the first evidence for an in vivo role for Ntf2p in the import of proteins to the nucleus.