Antagonistic effects of NES and NLS motifs determine S. cerevisiae Rna1p subcellular distribution

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1 Journal of Cell Science 112, (1999) Printed in Great Britain The Company of Biologists Limited 1999 JCS Antagonistic effects of NES and NLS motifs determine S. cerevisiae Rna1p subcellular distribution Wenqin Feng, Ann L. Benko, Jia-Hai Lee, David R. Stanford and Anita K. Hopper* Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA *Author for correspondence ( Accepted 21 November 1998; published on WWW 13 January 1999 SUMMARY Nucleus/cytosol exchange requires a GTPase, Ran. In yeast Rna1p is the GTPase activating protein for Ran (RanGAP) and Prp20p is the Ran GDP/GTP exchange factor (GEF). RanGAP is primarily cytosolic and GEF is nuclear. Their subcellular distributions led to the prediction that Ran- GTP hydrolysis takes place solely in the cytosol and GDP/GTP exchange solely in the nucleus. Current models propose that the Ran-GTP/Ran-GDP gradient across the nuclear membrane determines the direction of exchange. We provide three lines of evidence that Rna1p enters and leaves the nuclear interior. (1) Rna1p possesses leucine-rich nuclear export sequences (NES) that are able to relocate a passenger karyophilic protein to the cytosol; alterations of consensus residues re-establish nuclear location. (2) Rna1p possesses other sequences that function as a novel nuclear localization sequence able to deliver a passenger cytosolic protein to the nucleus. (3) Endogenous Rna1p location is dependent upon Xpo1p/Crm1p, the yeast exportin for leucine-rich NES-containing proteins. The data support the hypothesis that Rna1p exists on both sides of the nuclear membrane, perhaps regulating the Ran-GTP/Ran-GDP gradient, participating in a complete RanGTPase nuclear cycle or serving a novel function. Key words: Nucleus/cytosol exchange, RNA nuclear export, Protein nuclear import, RanGAP, Xpo1p/Crm1p INTRODUCTION The nucleus is a site of bi-directional macromolecular traffic. The general traffic flow pattern is import of karyophilic proteins and export of newly synthesized RNAs. However, there are shuttling proteins and RNAs that pass in both directions (for a review see Ohno et al., 1998). Entry and exit proceed through the same nuclear pores (Dworetzky and Feldherr, 1988). Nuclear pores are supramolecular complexes, with approximately distinct proteins, generating channels. The channels allow diffusion of small molecules, but most macromolecules must be transported by an energyrequiring, signal-mediated process (Ohno et al., 1998). Many proteins that enter the nucleus possess recognizable nuclear localization sequences (NLS), which are necessary and sufficient to target proteins to the nuclear surface. Delivery of the NLS-containing proteins to the nuclear surface usually requires interaction with a member of the importin β family and sometimes the aid of importin α (Ohno et al., 1998). Proteins that are exported from the nucleus interior to the cytosol often possess recognizable stretches of amino acids comprising nuclear export sequences (NES). Different types of sequences have been shown to function as NES: a short leucine-rich motif (Wen et al., 1995; Fischer et al., 1995) and a glycine-rich motif (Michael et al., 1995). The leucine-rich NES motifs interact with Xpo1p/Crm1p, a member of the importin β family that acts as an exportin (Ohno et al., 1998). Nucleus/cytosol exchange requires a small GTPase, Ran, and at least four proteins that regulate the Ran-GTP/Ran-GDP states: a GTPase activating activity (GAP), a GAP co-activator (RanBP1), a GDP/GTP exchange activity (GEF) (Melchior et al., 1993a; Moore and Blobel, 1993; for a review see Dasso, 1993) and a small protein, p10 (Nehrbass and Blobel, 1996). In yeast, Ran is encoded by GSP1 and GSP2 (Belhumeur et al., 1993), RanGAP by RNA1 (Bischoff et al., 1995; Corbett et al., 1995; Becker et al., 1995), RanBP1 by YRB1 (Ouspenski et al., 1995), GEF by PRP20 (Aebi et al., 1990; Bischoff and Ponstingl, 1991) and p10 by NTF2 (Corbett and Silver, 1996). Ran of S. cerevisiae and other eukaryotes is located in both the nucleus and the cytosol. RanGAP in S. cerevisiae (Hopper et al., 1990), S. pombe (Melchior et al., 1993b) and vertebrate cells (Matunis et al., 1996) is located primarily in the cytosol. A fraction of the RanGAP pool associates with the outer surface of the nuclear membrane and, in vertebrate cells, but apparently not in yeast, nucleus-association requires a ubiquitin-like modification (Traglia et al., 1996; Koepp et al., 1996; see references in Matunis et al., 1998). RanGEF is located in nuclei of yeast and vertebrates (Aebi et al., 1990; Ohtsubo et al., 1991). The cellular locations of the proteins regulating the Ran GTP/GDP state have raised questions regarding the roles of GTP hydrolysis and exchange in nucleus/cytosol translocation.

2 340 W. Feng and others By the current model, the predicted cellular distributions Ran- GDP in the cytosol and Ran-GTP in the nucleus dictate the directionality of movement (Izaurralde et al., 1997; Richards et al., 1997; Ohno et al., 1998). By previous models GTP hydrolysis provides the energy for the translocation step (Moore and Blobel, 1993; Ohno et al., 1998). Models for the role of the RanGTPase cycle in nucleus/cytosol exchange are dependent upon the cellular locations of Ran and its regulators and also upon the levels of RanGTP and RanGDP in each compartment. Previously, using indirect immunofluorescence, we detected a small nuclear pool of yeast Rna1p when it was expressed in HeLa cells (Traglia et al., 1996). Here we show that yeast Rna1p contains both NES and NLS motifs and that its location is sensitive to Crm1p. Our data provide support for RanGAP function on both sides of the nuclear membrane. MATERIALS AND METHODS Yeast and bacterial strains and methods Yeast strain W303 (MATa ade2-1 ura3-1 his3-1,115 trp1-1 leu2-3,112 can1-100) was used for most experiments. For studying the effects of Xpo1p/Crm1p upon Rna1p subcellular location we used yeast strain W303 xpo1-1 (relevant genotype: xpo1::leu2 ura3-1 and containing plasmid pkw457 encoding xpo1-1; Stade et al., 1997). The affect of Xpo1p/Crm1p upon β-galactosidase passenger location utilized yeast strain ALB11 (MATa ade2-1 ura3-1 his3-1,115 trp1-1 leu2-3,112 can1-100 xpo1/crm1::kan r and containing plasmid pkw457). ALB11 was constructed by disruption of the XPO1/CRM1 gene in W303 with the kanamycin resistance gene (kan r ). Using plasmid pug6 (Güldener et al., 1996) containing the kan r gene as template and oligonucleotides BENK23 (5 -AGAATTTCGCAGATTTCCCTGGCTTTCCTTATTC- TTCCTCAGGTCGACAACCCTTAAT-3 ) and BENK24 (5 -ATA- ATCTGTTGGATCACCTCCGACTTCTTTGATTTGCACGTGGAT- CTGATATCACCTA-3 ) as primers for the polymerase chain reaction (PCR) using Taq DNA polymerase (Fisher Co.), a kan r DNA flanked by sequences from the XPO1 gene was generated. The resulting DNA served as template for a second amplification using the same primers. The second product was used for yeast transformation. Cells in which the kan r marker integrated into the genome were selected by growth in the presence of G418 (Gibco-BRL). Appropriate replacement by xpo1/crm1::kan r at the XPO1/CRM1 locus was confirmed by Southern hybridization (Maniatis et al., 1982). Bacterial strain DH5α was used for all plasmid constructions. Yeast and bacterial growth and transformations followed standard procedures. Plasmids The centromere-containing YCpRNA1 plasmid encoding RNA1 was described previously (Atkinson et al., 1985). Plasmids pfb1-7a and pfb1-67a encoding histone H2B codons 1-14 and histone H2B codons 1-67, respectively, fused to β-galactosidase were also described previously (Moreland et al., 1987). Derivatives of pfb1-7a and pfb1-67a were constructed as follows. The Gle1p NES (codons ) was obtained by PCR using plasmid psw397 (Murphy and Wente, 1996) containing GLE1 as the template and oligonucleotides BENK13 (5 -CTGGATCCA- TAAATGATACTAAAGGC-3 ) and BENK14 (5 -TAGGGATC- CATTACATATTCTAGCC-3 ) as primers. The product was cloned into vector pgem-t (Promega), propagated in bacteria and inserted at the BamHI site of pfb1-67a to generate an in-frame fusion encoding histone H2B(1-67)/Gle1( )/β-galactosidase. pfb1-7arna1p(2-44) and pfb1-67arna1p(2-44) were constructed using plasmid pru35 (Traglia et al., 1989) as the template and primers Rna1pOF (5 -CGCGGATCCGCTACCTTGCACTTC-3 ) and Rna1pOR (5 -CCGGGATCCACAGGTTTTCAAAGC-3 ). The amplified DNA was inserted at the BamHI sites of pfb1-7a and pfb1-67a. pfb1-7arna1p( ) and pfb1-67arna1p( ) were constructed similarly using primers Rna1p5F (5 -CCGGGAT- CCTGGAAGGATAGTTTA-3 ) and Rna1p5R (5 -CCGGGATCCC- ATAGCCGGTAAGAA-3 ). pfb1-7arna1p( ) and pfb1-67arna1p( ) were constructed using primers Rna1p2F (5 -CGCGGATCCGAAA- AGGGAAATTTA-3 ) and Rna1p2R (5 -CCGGGATCCCTCTT- CAAAATCGTC-3 ). Plasmid pgem-t Rna1p( AA), in which the Rna1p L 326 and I 328 were changed to A residues, was generated using primers WQF 27 (5 -GCAAATGGTAACAG- ATTAGATGAAGAT-3 ) and WQF 36 (5 -TTCAGCCTTTTCCAA- TTCAGGTAAATTTCCCTTTTC-3 ) and pgem-t Rna1p( ) as template. PCR amplification followed the protocol described by Hemsley et al. (1989). Plasmids pfb1-67arna1p( LI) and pfb1-67arna1p( AA) were constructed using primers Rna1p2F and Rna1p2F54 (5 -CCGGGATCCTAATCTGTTACCATT- 3 ) and pgem-t Rna1p( ) and pgem-t Rna1p( AA), respectively, as templates. Construction of plasmid pfb1-67arna1p( ) was using primers WQF2F87 (5 -CGCGG- ATCCAATGGTAACAGATTA-3 ) and Rna1p2R and pgem-t Rna1p( ) as template. Oligonucleotides were generated by the Hershey Medical Center Macromolecular Core Facility. Sequences of PCR-amplified fragments were confirmed by DNA sequencing by the same facility. Indirect immunofluorescence and microscopic imaging Indirect immunofluorescence experiments were carried out as described by Pringle et al. (1991) with the modifications previously reported (Hopper et al., 1990). β-galactosidase antigens were detected using a 1:300 or 1:750 dilution of an affinity-purified rabbit anti-βgalactosidase (Hopper et al., 1990) and a 1:400 or 1:750 dilution of FITC-conjugated goat-anti-rabbit secondary antibody (Jackson Immunoresearch Labs). To detect Rna1p, a 1:100 dilution of anti- Rna1p 6142 rabbit sera, preadsorbed versus yeast strain EE1b lacking the Rna1p epitope used to raise the antibody, was prepared and used as previously described (Hopper et al., 1990). Secondary antibody was FITC-conjugated goat-anti-rabbit at a 1:200 dilution. Fluorescence images were obtained using a Nikon Microphot-FX microscope equipped with a 60 objective and a SenSys CCD camera (Photometrics Ltd, Tucson, AZ). Image processing was done using QED software (Pittsburgh, PA). β-galactosidase assay The assay for β-galactosidase activity was performed according to a modified version of the method of Reynolds et al. (1997). RESULTS Rna1p contains NES-like motifs possessing NES activity The vast majority of cellular Rna1p is located in the cytosol. Previously, however, while studying the location of yeast Rna1p in HeLa cells, we noted a small pool of this protein in the nucleus (Traglia et al., 1996). If there is also a small nuclear pool of Rna1p in yeast, then there must be a mechanism to regulate its steady state distribution such that most of the protein is cytosolic. One way to regulate this distribution is for Rna1p to possess a sequence(s) that causes the putative nuclear pool of the protein to be redistributed to the cytosol. Rna1p possesses several leucine-rich regions resembling the NES motifs that are sufficient for nuclear export of particular

3 Fig. 1. Rna1p alignments. GenBank accession numbers: S. cerevisiae Rna1p (Scer Rna1p), (Traglia et al., 1989); S. pombe Rna1p (Spom Rna1p), (Melchior et al., 1993b); human RGP1 (Hum RGP1), (Bischoff et al., 1995); mouse FUG1 (Mus FUG1), (DeGregori et al., 1994); Xenopus laevis (Xl RanGAP1), (Saitoh et al., 1997); sea urchin (StpurRanGAP1), ; C. elegans composed of two complete repeats of the entire Rna1p (Cel repeat 1 and repeat 2), Shading of the alignments is based on the BLOSUM62 scoring matrix (Henikoff and Henikoff, 1992). (A) NES-like motifs of RanGAP homologues. Alignment of S. cerevisiae amino acids and with the same region of several other RanGAPs and A. Functional NES and NLS motifs in RanGAP PKI ** ELALKLAGLDIN ** ELALK LAGLDIN PKI 1 DLPLKLEALAVK DLPLK LEALAVK HIV-1 Rev QLP PLERLTLD QLP P LERLTLD Scer Gle1p ALP LGKLTLY ALP LGKLTLY Scer Rna1p EKGNLP ELEKLEINGN RLDED SDALDLLQSKFD DLEVDDFEE Spom Rna1p IDEKMP DLLFLELNGN RFSEE DDVVDEIREVFS TRGRGELDE Hum RGP1 AMADKA ELEKLDLNGN TLGEEGCEQLQEVLEGFNMAKVLASLSD Mus FUG1 AVADKA ELEKLDLNGN ALGEEGCEQLQEVMDSFNMAKVLASLSD Xl RanGAP1 SVEDKS DLEKLDLNGN CLGEEGCEQVQEILESINMANILGSLSD StpurRanGAP1 SMDTKP HLTLLDLNGN NIG... Cel repeat1 SPKMCH VKVDISVNMF GKDFDSAKARHGKGNIDFGRRGDDELLS Cel repeat2 KFDGLT PKPVLHIHTN SFGDEFSDVAGMAPENVNVGDEDDDLGS B. Mus FUG1 LMV LNHVVRQDYFPKALAP LLLAFVTKPNGALETCSFARHNL LQTLYNI Scer RNA1 WKDSLFELNLNDCLLKTAGSDEVFKVFTEVKFPNLHVLKFEYNEMAQETIEVSFLPAM Spom RNA1 WPN LRELGLNDCLLSARGAAAVVDAFSKLENIGLQTLRLQYNEIELDAVRT LKTV Hum RGP1 LRQ VEVINFGDCLVRSKGAVAIADAIRGGLPK LKELNLSFCEIKRDAA LAVAEAM Mus FUG1 LRQ VEVINFGDCLVRSKGAVAIADAVRGGLPK LKELNLSFCEIKRDAA LVVAEAV Xl RanGAP1 LRQ VEVINFGDCLVRSKGAQAIASALKEGLHK LKDLNLSYCEIKADAA VSLAESV StpurRanGAP1 LSK LEVINFGDCLVRSEGADAIANSLREGVPS LKELNLAFGEIKKEAA VRVAESM Cel repeat1 LQF IEVLDLGDCVCDDPGVLAIIAELDKINRDCLKKVVLSGNNITSDVIDEIGACFN Cel repeat2 WPK LEVLNLSDCLIRDAGCNYIIDHLNPQHHRHLKNVYLCGNELTPPVAKLLIQKWS with four known NES motifs (Wen et al., 1995; Fischer et al., 1995; Murphy and Wente, 1996). The underlined leucines can be individually mutated to alanine without loss of NES activity while mutation of the other two highly conserved amino acids (asterisks) to alanine destroys NES activity. Shading: white on black, identical or similar to hydrophobic residues of known NES motifs; black on light gray, other residues similar or identical between the NES motifs and RanGAPs. (B) Alignments of putative NLS regions. Alignment of two regions of RanGAPs with NLS activity; mouse FUG1 residues and S. cerevisiae Rna1p residues The regions analogous to S. cerevisiae in other RanGAPs are conserved. Shading: white on black, >65% identical or similar; black on light gray, 45-65% identical or similar. 341 nuclear/cytosolic shuttling proteins (Fischer et al., 1995; Wen et al., 1995; Murphy and Wente, 1996). The similarities led us to test whether these regions have NES function. We initially focused upon a Rna1p leucine-rich motif located between amino acids (aa) 320 and 328, which shows the most similarity to the consensus NES (Fig. 1A), and we designed experiments to test whether this motif functions to redistribute an otherwise karyophilic passenger protein to the cytosol. Plasmid pfb1-67a contains codons for the first 67 amino acids of histone H2B (including its NLS) fused in-frame to E. coli lacz (Moreland et al., 1987). The encoded histone H2B/βgalactosidase fusion protein is located nearly exclusively in the nucleus of yeast cells (Fig. 2A-C; Table 1). To test whether aa have NES activity, RNA1 codons containing the putative NES motif were amplified and inserted into pfb1-67a in-frame at the junction between histone H2B (1-67) and β-galactosidase. For a positive control, a PCR-amplified sequence containing the known NES from Gle1p (Murphy and Wente, 1996) was inserted at the same location in plasmid pfb1-67a. For a negative control, an amplified fragment containing RNA1 codons 2-44, which is leucine-rich but bears little resemblance to NES motifs, was inserted at the same site in the same vector. The plasmids were introduced into wildtype yeast strains and the locations of the fusion proteins were determined by indirect immunofluorescence using a rabbit antibody specific to β-galactosidase (Hopper et al., 1990). Regions including the NES from Gle1p inserted into pfb1-67a caused a portion of β-galactosidase to be located in the cytosol (Table 1). Therefore, as expected, the Gle1p amino acids showed NES activity. In contrast, and also as expected, RNA1 codons 2-44 in the same position in vector pfb1-67a did not result in a change in the nuclear location of β-galactosidase (Fig. 2D-F). Remarkably, the DNA containing RNA1 codons caused a redistribution of the β-galactosidase pool such that the vast majority of the antigen was located in the cytosol (Fig. 2G-I). The data are consistent with the notion that Rna1p aa contain a functional NES motif. If aa within the amplified region act as a canonical NES sequence, then the activity should be dependent upon the penultimate and carboxyl-terminal aliphatic amino acids located at positions 326 and 328 (Wen et al., 1995). To test this, we generated pfb1-67arna1p( AA), in which L at 326 and I at 328 were each changed to A. Despite alteration of the putative NES consensus amino acids, activity was retained as most of the protein was located in the cytosol (Fig. 2J-L). By inspection, Rna1p aa contain another NES-like motif located between aa 333 and 342 (Fig. 1A). Even though this motif is not well conserved phylogenetically and does not align as well with the consensus as does aa , we tested the hypothesis that Rna1p aa contains two NES sequences, one between aa 320 and 328 and the other between aa 333 and 342. To do so we divided the previously amplified region into two parts. Each was inserted at the same site in pfb1-67a and transferred to yeast strain W303. Plasmids pfb1-67arna1p( LI) and pfb1-67arna1p( AA) encode, respectively, the wild-type or mutant motif located between aa 320 and 328 and lack the putative motif at Plasmid pfb1-67arna1p( ) encodes the NES-like motif located between aa 333 and 342 and lacks aa Unlike protein encoded by pfb1-67arna1p( ), which resides

4 342 W. Feng and others Table 1. Location of Rna1p/β-galactosidase fusion proteins in yeast β-galactosidase activity Plasmid Protein location (units/mg protein 10 5 ) pfb1-7a Cytosol 3.0 pfb1-7arna1p(2-44) Cytosol 1.6 pfb1-7arna1p( ) Nucleus and cytosol 1.2 pfb1-7arna1p( ) Cytosol 6.1 pfb1-67a Nucleus 1.7 pfb1-67arna1p(2-44) Nucleus 1.7 pfb1-67arna1p( ) Nucleus and cytosol 1.8 pfb1-67arna1p( ) Cytosol 6.0 pfb1-67arna1p( AA) Cytosol 2.7 pfb1-67arna1p( LI) Cytosol and nucleus 5.5 pfb1-67arna1p( AA) Nucleus 4.7 pfb1-67arna1p( ) Cytosol 6.0 pfb1-67agle1 Nucleus and cytosol 1.3 nearly exclusively in the cytosol (Fig. 3A-C), the fusion protein encoded by pfb1-67arna1p( LI) was located in both the cytosol and the nucleus (Fig. 3D-F). Therefore, aa appear to have less NES activity then does the full-length sequence containing aa In contrast, the fusion protein encoded by pfb1-67arna1p( AA) harboring mutations of the NES consensus sequence was located primarily in the nucleus (Fig. 3G-I). Thus, the NES activity of aa was destroyed by mutation of the conserved aliphatic amino acids. The data support the model that aa function as a conventional NES motif. Fusion protein encoded by pfb1-67arna1p( ) was located nearly exclusively in the cytosol (Fig. 3J-L). Thus, this nonconserved region of Rna1p that has a leucine-rich region that aligns less well with the consensus sequence than does the region encoded by aa , actually possesses stronger NES activity. The data are consistent with the hypothesis that there are two active NES sequences within aa A priori there are two ways that the fusion proteins could have a cytosolic location. First, the sequences we mapped could be bona fide NES motifs causing the exit of the protein from the nuclear interior to the cytosol. Second, insertion of Rna1p sequences into the passenger proteins could cause them to misfold, masking the NLS motif and preventing import of the fusion protein into the nucleus. Each of the Rna1pcontaining fusion proteins was enzymatically active (Table 1), which argues in favor of the first explanation. However, to test this in another way we employed a yeast strain with a mutation in the XPO1/CRM1 gene. In yeast the XPO1/CRM1 gene encodes the leucine-rich NES exportin, and mutants harboring the temperature-sensitive xpo1-1/crm1-1 allele are blocked in the export of NES-containing proteins at the nonpermissive temperature (Stade et al., 1997). If the NLS/Rna1p/βgalactosidase could enter the nucleus, but accumulated in the cytosol because the NES motifs caused nuclear export, then preventing export by use of the xpo1-1/crm1-1 strain should result in nuclear pools of the fusion proteins. We compared the location of the histone H2B/Rna1p( )/β-galactosidase fusion protein in wild-type cells to their location in xpo1-1/crm1-1 cells. As expected, the fusion protein was located primarily in the cytosol when wild-type W303 cells were grown at 23 C (data not shown) or when they were shifted to 37 C for 15 minutes (Fig. 4A-C). When xpo1-1/crm1-1 mutant cells were grown at 23 C the location of the fusion protein was indistinguishable from its location in strain W303 (data not shown). In contrast, when the mutant cells were incubated at 37 C for 15 minutes the histone H2B/Rna1p( )/β-galactosidase fusion protein was either uniformly distributed throughout the nucleus and the cytosol or there was biased nuclear location for the fusion protein (Fig. 4D-F,D -F ). Although we do not understand the reason why all the cells do not stain identically, the data show that the location of the fusion protein containing the NES-like motifs is sensitive to Xpo1p/Crm1p. The dependence of location of the fusion protein upon Xpo1p/Crm1p function supports the hypothesis that Rna1p aa contain bona fide NES motifs. A novel motif able to move Rna1p into the nucleus The nuclear pools of Rna1p that we detected using HeLa cells could have resulted from diffusion of this approx. 46 kda protein into the nucleus or from mis-sorting during mitosis. In this case, the NES motifs would serve to re-sort inappropriately located Rna1p back to the cytosol. Alternatively, it is possible that a fraction of Rna1p normally resides in the nucleus and the NES motifs serve to regulate the nucleus/cytosol distribution. According to the later model, Rna1p may possess an NLS. By inspection Rna1p has no sequences resembling known NLS motifs (for reviews see Dingwall and Laskey, 1991; Michael et al., 1995, 1997). Our previous studies provided no evidence for an NLS in the first 187 aa of the Rna1p (Hopper et al., 1990). To determine whether there is(are) an NLS in the remaining portion of Rna1p, we assayed Rna1p regions for NLS activity. Plasmid pfb1-7a encodes β-galactosidase fused to histone H2B codons 1-14 that lack the NLS (Moreland et al., 1987) and therefore cells possessing pfb1-7a have β-galactosidase located nearly exclusively in the cytosol (Fig. 5A-C). We inserted amplified Rna1p regions into the junction between histone H2B codons 1-14 and lacz of pfb1-7a and determined the cellular location of the resulting fusion proteins. Regions encoding Rna1p aa 2-44 or (Table 1) inserted into plasmid pfb1-7a encoded protein that was located exclusively in the cytosol. Thus, there is no evidence for an NLS in Rna1p aa 2-44 or In contrast, pfb1-7arna1p( ) encoded a β-galactosidase fusion protein with distinct nuclear pools (Fig. 5D-F). Therefore, Rna1p aa are sufficient to deliver an otherwise cytosolic passenger protein into the nucleus and therefore act as an NLS motif. Since Rna1p aa did not deliver the entire pool of passenger protein to the nucleus, we tested whether they function like the hnrnp motifs in having both NLS and NES activities (Michael et al., 1995, 1997). pfb1-67arna1p( ) encodes a fusion protein consisting of histone H2B NLS, Rna1p aa and β-galactosidase. When introduced into yeast the resulting fusion protein had a greater cytosolic pool than did the protein encoded by pfb1-67a lacking the Rna1p sequences (compare Figs 5G-I and 2A-C, Table 1). The data are consistent with the model that Rna1p aa contain sequences that function in both nuclear delivery and nuclear export or cytoplasmic retention. Matunis et al. (1998) recently reported a putative NLS for mouse RanGAP (FUG1). This NLS is located between aa 541 and 589 in a C-terminal extension that is lacking in lower eukaryotes. The FUG1 NLS is similar to Rna1p in that both contain numerous hydrophobic amino acids (Fig. 1B).

5 Functional NES and NLS motifs in RanGAP 343 Therefore, it appears that at least part of the FUG1 carboxylterminal extension that is not found in lower eukaryotes may be redundant with internal sequences. If true, the mouse gene may contain two active NLS motifs. Rna1p accumulates in the nucleus in a xpo1-1/crm1-1 strain Our data show that Rna1p possesses leucine-rich sequences that act as canonical NES motifs when appended to an otherwise karyophilic passenger protein and other sequences that function as a novel NLS when appended to an otherwise cytosolic passenger protein. If these sequences impart the same activity to the endogenous Rna1p, Rna1p should be able to enter and leave the nuclear interior and its movement should be dependent upon gene products involved in nucleus/cytosol distribution. As the vast majority of the pool of Rna1p is located in the cytosol, we tested whether inhibition of the nuclear export process would alter the distribution such that a nuclear pool would be detected. The location of Rna1p was determined using an antiserum specific for Rna1p (Hopper et al., 1990). As this antiserum barely detects endogenous levels of Rna1p, we conducted experiments to locate Rna1p in cells expressing slightly amplified levels encoded by plasmid YCpRNA1 (maintained at approx. 1 copy/cell; Atkinson et al., 1985), as well as in cells expressing solely endogenous levels. We studied the distribution of Rna1p in wild-type and mutant yeast harboring the xpo1-1/crm1-1 allele. Yeast cells lacking the epitope used to raise the anti-rna1p sera (Hopper et al., 1990) have very little signal (Fig. 6A-B). As expected, in yeast cells possessing a wild-type chromosomal RNA1 or YCpRNA1 in addition to the chromosomal RNA1 gene in either the wild-type (Fig. 6C-D and O-P, respectively) or the xpo1-1/crm1-1 mutant strain (Fig. 6I-J and U-V), Rna1p is located primarily in the cytosol when the cells are grown at 23 C. Also, as expected (Traglia et al., 1996), when XPO1 cells are incubated at 37 C a small portion of Rna1p locates to the nuclear membrane (Fig. 6E-H,Q-T). In contrast, when xpo1-1/crm1-1 cells containing either endogenous levels of Rna1p (Fig. 6K-N) or the slightly amplified levels (Fig. 6W-Z) are incubated at the nonpermissive temperature for 15 minutes (Fig. 6K-L and W- X) or 30 minutes (Fig. 6M-N and Y-Z), Rna1p has a different subcellular distribution. Within 15 minutes at the nonpermissive temperature, the distribution changes from a predominantly cytosolic location to one in which many, if not most, cells contain substantial nuclear Rna1p pools. The nuclear distribution is quite pronounced 30 minutes post-shift. These results are reminiscent of the studies of Stade et al. (1997), who showed that nucleus/cytosol shuttling proteins have a nuclear location by 15 minutes post shift to the nonpermissive temperature in xpo1-1 cells. Thus, Rna1p location is dependent upon Xpo1p/Crm1p. Moreover, as the rate of Rna1p nuclear accumulation is indistinguishable from other proteins with bona fide NLS and NES motifs, the data are consistent with the hypothesis that Rna1p achieves this nuclear location via an active process. DISCUSSION Four lines of evidence support the hypothesis that Rna1p possesses functional NES motifs: (1) Rna1p contains motifs that align well with canonical leucine-rich NES motifs; (2) the tested sequences behave as functional NES in that they cause an otherwise karyophilic protein to locate in the cytosol; (3) NES activity is dependent upon those amino acids that are necessary for function of authentic NES motifs; (4) the location of proteins containing Rna1p NES-like motifs is dependent upon Xpo1p/Crm1p, the exportin for leucine-rich NEScontaining cargo. Here we show that Rna1p has at least two functional leucine-rich NES motifs. Inspection of the Rna1p sequence leads to the prediction that Rna1p contains several other such motifs. It is striking that Rna1p subcellular location is dependent upon Crm1p, as according to current models the Crm1p/Ran- GTP/cargo complex would be dissociated by Rna1p, the RanGAP activity and in this case RanGAP is the cargo. That the trimeric complex does not dissociate before nuclear export could be explained if Rna1p does not have catalytic activity when complexed to Crm1p. Alternatively, as it has been reported that dissociation of complexes consisting of the importin β CAS, Ran-GTP and cargo requires both Rna1p and Ran-binding protein Yrb1p (Kutay et al., 1997b), it is possible that the Crm1p/Ran-GTP/Rna1p trimeric complex requires Yrb1p for dissociation and that the predicted low nuclear pool of Yrb1p would result in stability of the complex in the nucleoplasm. Rna1p is not the only protein to interact with Ran that possesses NES motifs. Vertebrate RanBP1 has an essential NES and accumulates in the nucleus when nuclear export is blocked (Richards et al., 1996; Pasquinelli et al., 1997). It has been proposed that the NES functions as a protection mechanism to prevent inappropriate accumulation of RanBP1 in the nucleus, especially in organisms with an open mitosis (Richards et al., 1996). Even though yeast has a closed mitosis the argument could be made that yeast Rna1p NES motifs serve to deliver to the cytosol Rna1p that inappropriately becomes located in the nucleus. We do not favor the model that Rna1p NES motifs function solely to prohibit inadvertent Rna1p nuclear pools because Rna1p apparently actively accumulates in the nuclear interior. Two lines of data support this: (1) Rna1p contains a sequence that functions as a classical NLS motif and (2) inhibition of nuclear export by the crm1-1 mutation causes Rna1p to accumulate in the nucleus with kinetics consistent with active nuclear import. The sequence of Rna1p that functions to deliver an otherwise cytosolic passenger protein to the nucleus bears no resemblance to previously characterized NLS motifs and would not be expected to interact with the classical importin α and β import adapter and receptor. It is possible that it is the founder for a new family of NLS motifs that interacts with different members of the importin β family. The yeast genome encodes a large family of importin β-like proteins (Görlich et al., 1997). Although the substrates for some of these remain unknown, recently it has been reported that particular members of the family are responsible for the nuclear import of proteins with nonclassical NLS motifs (Rout et al., 1997; Schlenstedt et al., 1997). It is also possible that the Rna1p NLS-motif binds to another karyophilic protein and enters the nucleus in a complex with this protein; however, in this later case the karyophilic Rna1p-binding protein would fit the definition of a classical import adapter. Whatever the mechanism, Rna1p

6 344 W. Feng and others Fig. 2. Rna1p amino acids have NES activity. Plasmids pfb1-67a (A-C), pfb1-67arna1p(2-44) (D-F), pfb1-67arna1p( ) (G-I) and pfb1-67arna1p( AA) (J-L) encoding H2B amino acids 1-67 fused to the indicated Rna1p amino acids and to β-galactosidase were transferred to yeast strain W303. The cellular locations of the passenger β-galactosidase were determined by indirect immunofluorescence. (A,D,G,J) FITC location of β-galactosidase; (B,E,H,K) location of nuclear and mitochondrial DNA as determined by DAPI staining; (C,F,I,L) converged images of A-B, D-E, G-H, J-K, respectively. Bar, 10 µm. Fig. 3. Rna1p amino acids contain two functional NES motifs. Plasmids pfb1-67arna1p( ) (A-C), pfb1-67arna1p( ) (D-F), pfb1-67arna1p( AA) (G-I) and pfb1-67arna1p( ) (J-L), encoding H2B amino acids 1-67 fused to the indicated Rna1p amino acids and β-galactosidase, were transferred to yeast strain W303. The cellular locations of the passenger β-galactosidase were determined by indirect immunofluorescence. (A,D,G,J) FITC location of β-galactosidase; (B,E,H,K) DAPI staining of DNA; (C,F,I,L) converged images of A- B, D-E, G-H, J-K respectively. Bar, 10 µm. Fig. 4. The function of the putative Rna1p NES depends upon Xpo1p/Crm1p. The location of the Rna1p/β-galactosidase fusion protein encoded by plasmid pfb1-67arna1p( ) in yeast strains W303 (A-C) and ALB11 (D-F and D -F ) was determined by indirect immunofluorescence. Cells were grown at 23 C and then shifted to 37 C for 15 minutes. (A,D,D ) FITC location of β-galactosidase; (B,E,E ) DAPI staining of DNA; (C,F,F ) converged images of A-B, D-E and D -E, respectively. Bar, 10 µm.

7 Functional NES and NLS motifs in RanGAP 345 Fig. 5. Rna1p possesses a functional NLS between aa 258 and 315. Plasmids pfb1-7a (A-C), pfb1-7arna1p( ) (D-F) and pfb1-67arna1p( ) (G-I) were transferred to yeast strain W303 and the location of the Rna1p/β-galactosidase fusion protein was determined by indirect immunofluorescence. (A,D,G) FITC location of the fusion protein; (B,E,H) DAPI staining of DNA. (C,F,I) Converged images of A-B, D-E and G-H, respectively. Bar, 10 µm. possesses sequences that function to move Rna1p to the nuclear interior and other sequences that move Rna1p from the nuclear interior to the cytosol, and its subcellular distribution is dependent upon at least some of the machinery that regulates nuclear import and export. The data are consistent with the notion that Rna1p could function on both sides of the nuclear membrane. It has been proposed that the ratio of RanGDP in the cytosol to RanGTP in the nucleus determines the directionality of nucleus/cytosol exchange. This view is based, in part, upon binding studies of complexes of Ran, receptors and targeted cargo. The data support a model in which RanGDP/GTPexchange functions to release imported cargo from import receptors and, conversely, Ran-catalyzed GTP hydrolysis functions to release exported cargo from export receptors (Rexach and Blobel, 1995; Kutay et al., 1997a). This view has also been supported by studies in Xenopus oocytes and mammalian cells, in which nucleus/cytosol exchange is Fig. 6. Location of Rna1p in wild-type and xpo1-1/crm1-1 yeast strains. W303 xpo1-1/crm1-1 (I-N and U-Z) and the parental W303 strains (C- H and O-T) either containing (O-Z) or lacking (C-N) YCpRNA1 were grown at 23 C (C-D,I-J,O-P,U-V) and then incubated at 37 C for 15 minutes (E-F,K-L,Q-R,W-X) or 30 minutes (G-H,M-N,S-T,Y-Z) prior to indirect immunofluorescence analysis. Rna1p was located using rabbit antiserum 6142 (Hopper et al., 1990). The antibody does not detect protein in yeast strain EE1b lacking the epitope (A-B). (A,C,E,G,I,K,M,O,Q,S,U,W,Y) FITC location of Rna1p; (B,D,F,H,J,L,N,P,R,T,V,X,Z) DAPI staining of DNA. Arrowheads highlight cells where the distribution of Rna1p is primarily cytosolic; arrows indicate cells where there is a nuclear pool of Rna1p. Bar, 10 µm.

8 346 W. Feng and others monitored upon introduction of Ran dominant-negative mutants blocked in GTP- or GDP-bound forms, introduction of RanGAP into nuclei, or depletion of RanGEF in cells with a temperature-sensitive RCC1 gene (Richards et al., 1997; Kutay et al., 1997a; Izurralde et al., 1997). According to this model, nuclear pools of RanGAP, we predict, would serve to regulate the nuclear levels of RanGTP and thereby could regulate nucleus/cytosol exchange. Previously (Tung et al., 1992, 1995) we reported that the RanGTPase cycle appears to be sensitive to the carbon source in the medium, camp and to Reg1p, encoding a regulatory subunit of protein phosphatase type 1 (Tu and Carlson, 1995; Huang et al., 1995). If the subcellular location of Rna1p responds to environmental conditions and a signal transduction cascade, this could regulate nucleus/cytosol exchange in response to environmental stimuli. While many lines of data support the model that RanGTP hydrolysis per se is not necessary for nucleus/cytosol exchange, some observations are not readily explained by this model. For example, yeast mutations that block Ran in the GTP-bound state prevent export of poly(a)-rna (Schlenstedt et al., 1995) and even in mammalian cells, export of particular RNAs appears to require GTP hydrolysis (Izurralde et al., 1997). In yeast, the temperature-sensitive rna1-1 and prp20 mutations each rapidly affect both nuclear import and nuclear export (Amberg et al., 1992, 1993; Forrester et al., 1992; Kadowaki et al., 1993). If the role of Rna1p is to release RNA from transporters on the cytosolic side of the nucleus, one might expect to view accumulation near the nuclear membrane at early times after the block; however, the mutants accumulate RNA rather uniformly throughout the nucleus. The phenotypes of the mutants and our current studies indicating possible nuclear pools of Rna1p could be interpreted to support an alternative view of the role of the Ran cycle (Traglia et al., 1996). According to this view, there may be a complete Ran cycle on both sides of the yeast nuclear membrane, and GTP to GDP hydrolysis could play a role in the import and export processes. If correct, then there must be a mechanism to regulate the Ran cycle so that it does not cycle futilely. Moreover, one would need to provide an explanation for the fact that most Rna1p is cytosolic while Prp20p is located in the nucleus. A difficulty here is in determining whether part or all of the pools in each location are active and/or in contact with Ran. Other types of models are also compatible with our data. Since components of the Ran cycle affect processes in addition to nucleus/cytosol exchange (for a review see Sazer, 1996), it is possible that the nuclear pools of Rna1p participate in a process(es) distinct from exchange. This would be consistent with the observations that some mrnas that accumulate at the nonpermissive temperature in rna1-1 cells possess 5 and 3 extensions (Forrester et al., 1992), as if transcription initiation and termination site selection are aberrant. Rna1p could also regulate the GTP/GDP levels of a GTPase other than Ran. The putative small GTPase encoded by GTR1 that has genetic interactions with RNA1 and PRP20 is a candidate protein (Nakashima et al., 1996). Future studies of rna1 variants that encode active Rna1p proteins located solely in the nucleus or solely in the cytosol should resolve whether nuclear Rna1p pools regulate the Ran-GTP/Ran-GDP gradient, affect export directly or function in a novel path. This work was supported by a grant from NIH to A. 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