Mutations in the YRB1 Gene Encoding Yeast Ran-Binding-Protein-1 That Impair Nucleocytoplasmic Transport and Suppress Yeast Mating Defects

Markus Ku¨nzler,*,† Joshua Trueheart,*,1 Claudio Sette,* Eduard Hurt† and Jeremy Thorner* *Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, California 94720-3202 and †Ruprecht-Karls-Universita¨t Heidelberg, Biochemie-Zentrum Heidelberg, D-69120 Heidelberg, Germany Manuscript received July 13, 2000 Accepted for publication November 21, 2000

ABSTRACT We identified two temperature-sensitive (ts) mutations in the essential gene, YRB1, which encodes the yeast homolog of Ran-binding-protein-1 (RanBP1), a known coregulator of the Ran GTPase cycle. Both mutations result in single amino acid substitutions of evolutionarily conserved residues (A91D and R127K, respectively) in the Ran-binding domain of Yrb1. The altered proteins have reduced affinity for Ran (Gsp1) in vivo. After shift to restrictive temperature, both mutants display impaired nuclear protein import and one also reduces poly(A)ϩ RNA export, suggesting a primary defect in nucleocytoplasmic trafficking. Consistent with this conclusion, both yrb1ts mutations display deleterious genetic interactions with mutations in many other genes involved in nucleocytoplasmic transport, including SRP1 (␣-importin) and several ␤-importin family members. These yrb1ts alleles were isolated by their ability to suppress two different types of mating-defective mutants (respectively, fus1⌬ and ste5ts), indicating that reduction in nucleocytoplasmic transport enhances mating proficiency. Indeed, in both yrb1ts mutants, Ste5 (scaffold protein for the pheromone response MAPK cascade) is mislocalized to the cytosol, even in the absence of pheromone. Also, both yrb1ts mutations suppress the mating defect of a null mutation in MSN5, which encodes the receptor for pheromone-stimulated nuclear export of Ste5. Our results suggest that reimport of Ste5 into the nucleus is important in downregulating mating response.

ATING in the yeast Saccharomyces cerevisiae is the nucleus in response to pheromone, as observed for the M culmination of a complex series of events re- regulated import and export of other nuclear proteins quired for cellular and nuclear fusion of two haploid (Kaffman and O’Shea 1999). cells of opposite mating type (Sprague and Thorner Proteins and protein-RNA complexes cross the nu- 50ف Mating pheromones (secreted peptides) bind clear envelope through nuclear pores comprised of .(1992 to G-protein-coupled receptors, stimulating a mitogen- different proteins, termed nucleoporins (Ryan and activated protein kinase (MAPK) cascade (Bardwell et Wente 2000). Nucleocytoplasmic transport also re- al. 1994) that evokes dramatic changes in gene transcrip- quires soluble factors. Transport receptors for both im- tion, cell cycle arrest, and pronounced alterations of port and export (␤-importin and its relatives) bind their cell morphology and nuclear reorganization (Leberer cargo and shuttle between the cytosol and the nucleo- et al. 1997; Stone et al. 2000). Pheromone-activated and plasm (Go¨rlich and Kutay 1999). The small Ras-like pheromone-induced gene products required for cell GTPase Ran and its associated factors confer directional- fusion are deposited at a localized site on the plasma ity to transport (Macara et al. 2000). In S. cerevisiae, membrane at the leading edge of a mating projection GSP1 and GSP2 encode Ran isoforms (Belhumeur et (“shmoo tip”; Madden and Snyder 1998). Proteins re- al. 1993; Kadowaki et al. 1993). Ran exists predomi- quired for nuclear fusion are recruited to the nucleus nantly in its GTP-bound form in the nucleus; in the (Rose 1996). How the signal initiated at the plasma cytosol, Ran is mainly GDP-bound. This asymmetry is membrane is transmitted into the nucleus to activate imposed by the subcellular distribution of Ran regula- gene expression is still unclear. Two components of the tors: the Ran-specific guanine-nucleotide exchange fac- pathway, Ste5 (Pryciak and Huntress 1998; Mahanty tor (RanGEF1), the PRP20/SRM1/MTR1 gene product et al. 1999) and Far1 (Blondel et al. 1999), shuttle in S. cerevisiae, is confined to the nucleus, whereas the between the nucleus and the cytosol, are predominantly Ran-specific GTPase-activating protein (RanGAP1), the nuclear in naı¨ve cells, but are rapidly ejected from the RNA1 gene product in S. cerevisiae, is located in the cytoplasm. GSP1, PRP20, and RNA1 are all essential genes, and recessive mutations in all three block nuclear Corresponding author: Markus Ku¨nzler, Ruprecht-Karls-Universita¨t protein import and poly(A)ϩ RNA export (Corbett Heidelberg, Biochemie-Zentrum Heidelberg (BZH), Im Neuen- heimer Feld 328, 4. OG, D-69120 Heidelberg, Germany. and Silver 1997; Oki et al. 1998). E-mail: [email protected] Transport receptors bind specifically to the GTP- 1 Present address: Microbia, Inc., Cambridge, MA 02139. bound form of Ran via a conserved domain at their N

Genetics 157: 1089–1105 (March 2001) 1090 M. Ku¨nzler et al. termini (Go¨rlich and Kutay 1999). RanGTP-binding isolated (Katz et al. 1987) as an extragenic suppressor to an export receptor enhances its affinity for an export of missense mutations in STE5, which encodes a scaffold substrate; conversely, binding of RanGTP to an import protein for the pheromone-activated MAPK cascade receptor prevents the binding of an import substrate. (Elion 1998). As described here, both yrb1 mutations Hence, high RanGTP in the nuclear compartment po- cause clear defects in nucleocytoplasmic trafficking of tentiates association of export cargo with export recep- various proteins, including Ste5, and are able to sup- tors and triggers release of import cargo from import press the mating defect of a msn5⌬ mutant. These data receptors. Ran-binding-protein-1 (RanBP1) is another suggest that preventing efficient reimport of Ste5 after protein that binds specifically to RanGTP (Ku¨nzler et its pheromone-induced release from the nucleus sus- al. 2000; Plafker and Macara 2000). RanBP1 contains tains the mating-competent state. -resi 140ف a conserved Ran-binding domain (RBD) of dues (Beddow et al. 1995; Vetter et al. 1999), which is necessary and sufficient for high-affinity binding of MATERIALS AND METHODS RanGTP and for nuclear export of RanBP1, at least in Strains and growth conditions: Yeast strains used in this yeast (Ku¨nzler et al. 2000). Homologous RBDs are study are listed in Table 1. Strains JTY2483 and JTY2484 were found in other nuclear proteins, like vertebrate obtained by backcrossing strain 381G-42E-P1 three times RanBP2/NUP358 (Yokoyama et al. 1995) and RanBP3 against either YPH499 or YPH500. msn5⌬::TRP1 strain HMK30 (Mueller et al. 1998), and S. cerevisiae nuclear proteins, was derived from strain LH90 (Blondel et al. 1999) by three S. cerevisiae Nup2 (Booth et al. 1999) and Yrb2 (Taura consecutive backcrosses against the W303-1A derivatives, CRY1 or CRY2. DNA-mediated transformation of yeast cells was per- et al. 1998). Transport receptors block stimulation of formed using a modified version of the lithium acetate method Ran-mediated GTP hydrolysis by RanGAP1; in contrast, (Gietz et al. 1992). The fus1⌬ mutation deletes 90% of the RanBP1 acts as a coactivator of RanGAP1-stimulated coding sequence (from the FUS1 promoter to codon 460) and GTP hydrolysis by Ran and, moreover, is required for was constructed by a two-step gene disruption method (Boeke nucleotide hydrolysis when RanGTP is bound to a trans- et al. 1987). Heterozygous diploid strain JTY2501 was derived from CRY3 by transplacing the YRB1 locus on one homolog port receptor (Go¨rlich and Kutay 1999). These bio- of chromosome IV via transformation with an EcoRI-XbaI frag- chemical activities, and the fact that RanBP1 is abun- ment containing the yrb1⌬::HIS3 construct excised from plas- dant, shuttles between the nucleus and the cytoplasm, mid pMK112n (Table 2). To construct strain HMK21, JTY2501 was transformed with plasmid pMK103, sporulated, and a but is found almost exclusively in the cytosol at steady ϩ ϩ state (Kunzler et al. 2000; Plafker and Macara 2000), MATa His Ura 5-fluoro-orotic acid (5-FOA)-sensitive spore ¨ was chosen. Strain JTY2486 was obtained by transformation of suggest that RanBP1 has a major role in the cytoplasm CRY1 with an EcoRI-SpeI fragment containing the nup2::HIS3 both in recycling of transport receptors and in release construct excised from plasmid pJON115 (Loeb et al. 1993). of export cargo (Peterson et al. 2000). Consistent with Strain HMK29 was constructed analogously using a BamHI- this view, S. cerevisiae YRB1, encoding yeast RanBP1, is HindIII fragment containing a gsp2⌬::LEU2 construct (Kado- essential for cell viability and is required for both nu- waki et al. 1993). Correct transplacements were verified by ϩ Southern hybridization analysis. clear protein import and poly(A) RNA export Unless indicated otherwise, yeast cells were propagated at (Schlenstedt et al. 1995). 30Њ. Rich medium (YP), synthetic complete medium (SC), and A link between yeast mating and the Ran GTPase synthetic minimal medium (SM) were prepared as described cycle was the identification of the srm1-1 mutation, now (Kaiser et al. 1994). Glucose (Glc) or raffinose (Raf) were known to reside in RanGEF1, which suppressed the added as carbon source at a final concentration of 20 g/liter after autoclaving; induction with galactose (Gal) was per- mating defect of cells lacking pheromone receptors and formed by adding Gal (final concentration 2%) to Raf-grown increased the basal expression of a pheromone-respon- cells. Drop-out media (SC lacking the appropriate nutrients) sive reporter gene (Clark and Sprague 1989). Another were used to maintain selection for plasmids. Agar plates con- connection between mating and nucleocytoplasmic taining 5-FOA were prepared as described by Boeke et al. ␣ transport was the finding that the ste21 mutation, identi- (1987). Escherichia coli strain DH5 (Hanahan 1983) was used for propagation of plasmid DNAs. Bacteria were cultivated fied in a screen for enhancers of the mating defect of using standard methods (Sambrook et al. 1989). a temperature-sensitive (ts) mutation in STE4 (encoding Quantitative mating assays: Quantitative mating assays were the ␤-subunit of the pheromone receptor-coupled het- performed as previously described (Sprague 1991). Briefly, erotrimeric G-protein; Akada et al. 1996), resides in MATa strains to be tested and MAT␣ tester strains were pre- Њ MSN5, encoding the nuclear receptor for pheromone- grown at 26 to midlogarithmic phase in selective and rich medium, respectively. Cells were washed with water and 106 stimulated export of Ste5 (Mahanty et al. 1999) and cells of the MATa strains to be tested were mixed with 107 Far1 (Blondel et al. 1999). As described here, we iso- cells of the MAT␣ tester strain. In the case of the experiment lated a yrb1ts mutation as a suppressor of a mutant shown in Table 3, the mixture was spread directly onto pre- (fus1⌬) defective in the cell fusion step of mating. Fus1 cooled (14Њ) SMGlc plates, the plates were incubated for 3 is a pheromone-induced, O-glycosylated, integral mem- days at 14Њ, and then for 3 days at room temperature. The resultant diploid colonies were counted and normalized to brane protein that acts at a late stage in mating the titer of input MATa cells (determined by plating the same (Trueheart and Fink 1989). While mapping this yrb1 dilutions on plates selective for the MATa strain to be tested mutation, we found that it was allelic to a mutation and incubating for 3 days at room temperature). In the case Ran-Binding-Protein-1 and Yeast Mating 1091

TABLE 1 Yeast strains

Strain Characteristics Source YPH499 MATa ura3-52 trp1-⌬63 his3-⌬200 leu2-⌬1 lys2-801am ade2-101oc Sikorski and Hieter (1989) YPH500 MAT␣ ura3-52 trp1-⌬63 his3-⌬200 leu2-⌬1 lys2-801am ade2-101oc Sikorski and Hieter (1989) YPH501 MATa/MAT␣ (YPH499 ϫ YPH500) Sikorski and Hieter (1989) CRY1 (W303-1A) MATa ura3-1 trp1-1 his3-11,15 leu2-3,112 ade2-1 can1-100 GAL R. S. Fuller CRY2 (W303-1B) MAT␣ ura3-1 trp1-1 his3-11,15 leu2-3,112 ade2-1 can1-100 GAL R. S. Fuller CRY3 (W303D) MATa/MAT␣ (CRY1 ϫ CRY2) R. S. Fuller 9399-7B MATa ura3-52 his4-⌬29 GAL Trueheart and Fink (1989) JY416 MAT␣ ura3-52 leu2-3,112 fus1⌬ This study JTY2023 MATa ura3-52 trp1-⌬63 his4⌬29 ade2-101oc GAL This study JTY2024 JTY2023 fus1⌬ This study JTY2025 JTY2024 yrb1-51 (sfo1-1) This study JTY2488 MATa ura3-52 trp1-⌬63 his4-⌬29 fus1⌬ yrb1-51 This study JTY2501 CRY3 yrb1⌬::HIS3/YRB1 This study HMK21 CRY1 yrb1⌬::HIS3 (pMK103) This study JTY2026 MATa ura3-52 trp1-⌬63 his3-⌬200 leu2-⌬1 ade2-101oc yrb1-51 This study JTY2027 MAT␣ ura3-52 trp1-⌬63 his3-⌬200 leu2-⌬1 lys2-801am yrb1-51 This study JTY2482 MATa/MAT␣ (JTY2026 ϫ JTY2027) This study 381G-42E-P1 MATa ade2-1 lys2 oc tyr1oc his4-580 am trp am ste5-3 yrb1-52 (stp52) CRY1 SUP4-3 ts Katz et al. (1987) JTY2483 MATa ura3-52 trp1-⌬63 his3-⌬200 leu2-⌬1 lys2-801am ade2-101oc yrb1-52 This study JTY2484 MAT␣ ura3-52 trp1-⌬63 his3-⌬200 leu2-⌬1 lys2-801am ade2-101oc yrb1-52 This study JTY2485 MATa/MAT␣ (JTY2483 ϫ JTY2484) This study JTY2500 MATa ura3-52 his3-⌬200 leu2-3,112 trp1-901 canR gal4-542 gal80-538 Inouye et al. (1997a)

ADE2::PGAL-URA3 LYS2::lexop-lacZ JTY2486 MATa ura3-1 trp1-1 his3-11,15 leu2-3,112 ade2-1 can1-100 GAL nup2-5::HIS3 This study HMK29 CRY1 gsp2⌬::LEU2 This study HMK30 CRY1 msn5⌬::TRP1 This study DC17 MAT␣ his1 J. B. Hicks

of the experiment shown in Table 5, the mating mixture was DNA by complementation of the ts phenotype of sfo1-1 cells collected on a 0.45-␮m pore filter and incubated for 6 hr at (see below) revealed that it is identical to the YRB1 gene, and 30Њ on YPGlc. After the incubation, cells were resuspended because sequence analysis (see below) demonstrated that both in SMGlc medium and plated in appropriate dilutions onto the sfo1-1 and stp52 mutations reside in the YRB1 locus, these SMGlc plates with appropriate nutrients to select for diploids. alleles were renamed yrb1-51 and yrb1-52, respectively. As a control for the number of viable MATa cells used in the Recovery and analysis of yrb1ts alleles: The base sequence mating mixture, 106 cells of the MATa cells were collected on alterations corresponding to the yrb1-51 and yrb1-52 mutations a separate filter, incubated as above, resuspended in YPGlc, were determined by cloning and sequencing of DNA isolated and plated on YPGlc plates at appropriate dilutions. Mating from the mutants. The polymerase chain reaction (PCR) was efficiency was expressed as percentage of the input MATa used to amplify 636-bp products comprising the entire YRB1 haploids that formed diploid colonies. open reading frame (ORF) using genomic DNA from JTY2026 Isolation of yrb1-51: JTY2024 (MATa fus1⌬) was mutagen- (yrb1-51) and 381G-42E-P1 (yrb1-52) as the template and oligo- ized with ethyl methanesulfonate (108 cells/ml; 3% EMS; 1 hr) nucleotide primers, 5Ј-GGG GAT CCG AAT GTC TAG CGA to 25% survival, and spread on YPGlc plates. After 3 days at AGA TAA G-3Ј (OSFO1) and 5Ј-GGT CTA GAC GCA AGT ,colonies were replica plated onto precooled AAC AAG C-3Ј (OSFO5), which corresponded, respectively 72,000ف ,28Њ (14Њ) SMGlc plates containing uracil (20 mg/liter), on which to positions Ϫ2toϩ18 and ϩ635 to ϩ616 of the 201-codon 2 A600 nm units of JY416 (MAT␣ fus1⌬) cells had been spread. YRB1 sequence (where ϩ1 is the first base of the initiator These plates were incubated for 4 days at 14Њ. Candidate clones codon of the ORF) and included restriction sites at their that gave a positive mating response (35 colonies total) were 5Ј-ends to facilitate cloning of the PCR products. Reaction restreaked from the master plate, retested for suppression, products were isolated, digested with BamHI and XbaI, and and examined for their ability to grow at various temperatures. inserted into E. coli vector pUC19 for sequencing. Nucleotide A single isolate (JTY2025) displayed a ts phenotype that coseg- sequence of multiple inserts was determined on both strands regated with the ability to suppress the mating defect of the using the M13/pUC universal and M13/pUC reverse sequenc- fus1⌬ cells at 14Њ (data not shown). The mutation conferring ing primers (New England Biolabs, Beverly, MA) and, when these phenotypes was initially named sfo1-1 (suppressor of fus necessary, sequence-specific primers. The single-base-pair mu- one). In the course of these crosses, it was shown, first, that tations recovered were tested for their ability to confer a ts sfo1 was tightly linked to trp1 (no recombinants in 31 tetrads; phenotype by first substituting the mutant YRB1 ORFs (excised distance Յ1.6 cM) and, second, by complementation tests, as SalI fragments from the pUC19 derivatives) for the corre- that the sfo1-1 mutation was allelic to stp52, another suppressor sponding segment in pMK103 and then introducing the entire of mating defects that was mapped to the same region (Katz yrb1-51 and yrb1-52 genes as EcoRI-XbaI fragments (excised et al. 1987). Because cloning of the corresponding wild-type from the pMK103 derivatives) into pRS314, yielding the TRP1- 1092 M. Ku¨nzler et al.

TABLE 2 Plasmids

Plasmid Characteristics Source YEp24 2-␮m URA3 Botstein et al. (1979) pRS314 CEN ARS TRP1 Sikorski and Hieter (1989) pRS424 2-␮m TRP1 Sikorski and Hieter (1989) pRS425 2-␮m LEU2 Sikorski and Hieter (1989) pUN100 CEN ARS LEU2 Elledge and Davis (1988) YEp352 2-␮m URA3 Hill et al. (1986) pGAD424 2-␮m LEU2 ADH1p-GAL4TAD-MCS-ADH1t Bartel and Fields (1995) pBTM116 2-␮m TRP1 ADH1p-LexADBD-MCS-ADH1t Bartel and Fields (1995) pRSETA AmpR T7-His6-MCS Invitrogen (Carlsbad, CA) pNOPGFP2L pRS425-NOP1p-GFP K. Hellmuth and E. Hurt (unpublished results) pJON115 nup2-5::HIS3 Loeb et al. (1993)

pPS815 2-␮m URA3 ADH1p-SV40NLS-GFP-lacZ Lee et al. (1996) pPS817 2-␮m URA3 GAL1p-SV40NLS-GFP-lacZ Lee et al. (1996) pGADGFP 2-␮m LEU2 ADH1p-SV40NLS-GAL4TAD-GFP Shulga et al. (1996) pNOPGFPAU-NPL3 2-␮m ADE2 URA3 NOP1p-GFP-NPL3 Senger et al. (1998)

YEplac195AU-L25NLS-GFP 2-␮m ADE2 URA3 RPL25NLS-GFP-MEX67t O. Gadal and E. Hurt (unpublished results)

pKW430 2-␮m URA3 ADH1p-SV40NLS-PKINES-(GFP)2 Stade et al. (1997) pLDB419 2-␮m LEU2 YAP1-GFP Yan et al. (1998) pNOPGFP2L-STE5 This study pSB415 YEp24-NTH1-YRB1 This study pMK102 pUC19-YRB1 This study pMK103 YEp352-YRB1 This study PMK104 pRSETA-YRB1 This study pMK112n pRS316-yrb1⌬::HIS3 This study pNOPPATA-GSP1G21V pUN100-NOP1p-ProtA-TEV-GSP1(G21V)-ADH1t Hellmuth et al. (1998) pMK275 pRS314-YRB1 This study pMK277 pRS314-yrb1-51 This study pMK278 pRS314-yrb1-52 This study pMK284n pRS314-YRB1-GFP(S65T) Hellmuth et al. (1998) pMK294-51 pRS314-yrb1-51-GFP(S65T) This study pMK294-52 pRS314-yrb1-52-GFP(S65T) This study pMK291-wt pRS424-YRB1-GFP(S65T) This study pMK291-51 pRS424-yrb1-51-GFP(S65T) This study pMK291-52 pRS424-yrb1-52-GFP(S65T) This study pMK199-wt pGAD424-YRB1 This study pMK199-51 pGAD424-yrb1-51 This study pMK199-52 pGAD424-yrb1-52 This study pMK195-GV pBTM116-GSP1(G21V) This study

marked plasmids pMK277 and pMK278, respectively. Finally, complement the ts phenotype of the yrb1-51 mutant. The HMK21 (yrb1⌬ [pMK103]) was transformed with either smallest original isolate (pSB415) contained the YRB1 gene pMK277, pMK278, or a control plasmid (pMK275) carrying as well as the neighboring gene NTH1 encoding neutral treha- the normal YRB1 gene, plated on 5-FOA plates at 23Њ to select lase. Subsequent subcloning localized the complementing ac- against the resident URA3-marked YRB1-containing plasmid tivity to a 1.3-kb chromosomal EcoRI-XbaI fragment containing (pMK103), and the resulting isolates were analyzed for their only YRB1, which was used to construct pMK102, pMK103, ability to grow at elevated temperature. and pMK275. Construction of plasmids: Standard techniques were used To construct a plasmid (pMK112n) carrying the yrb1⌬::HIS3 for the manipulation of recombinant DNA (Sambrook et al. deletion construct, an internal BglII fragment was excised 1989). Plasmid DNA from E. coli was isolated according to from the YRB1-containing insert in pMK101 and replaced by Del Sal et al. (1988). Unless specified otherwise, PCR amplifi- a BamHI fragment containing the HIS3 gene, which was in- cations were performed using Vent DNA polymerase (New serted in the same transcriptional orientation as YRB1. Con- England Biolabs). Correct sequence of PCR-generated construction of plasmid pNOPPATA-GSP1G21V, which expresses structs was verified by nucleotide sequence analysis. Plasmids a GTPase-defective mutant form of Gsp1, Gsp1(G21V), fused used in this study are listed in Table 2. at its N terminus to a cleavage site (ENLYEQG) for tobacco The YRB1 gene was isolated from a yeast genomic library etch virus (TEV) protease and to two immunoglobulin G (IgG) (Carlson and Botstein 1982) carried in a high-copy-number binding domains of Protein A (ProtA), under control of the yeast/E. coli shuttle vector (YEp24) by virtue of its ability to NOP1 promoter and the ADH1 terminator, has been described Ran-Binding-Protein-1 and Yeast Mating 1093

TABLE 3 erated STE5 NcoI-BamHI fragment comprising the entire ORF (using primers OSTE5-1, 5Ј-GGGGGGCCATGGGTAT The yrb1-51 (sfo1-1) mutation suppresses the cold-sensitive GGAAACTCCTACAGAC-3Ј, and OSTE5-2, 5Ј-GGGGGGAT mating defect of a fus1⌬ mutant CCCTATATATAATCCATATGG-3Ј) into pNOPPATA (Hell- muth et al. 1998) and subsequent recloning of the insert as Plasmid: mating efficiency (ϫ10Ϫ5)a PstI fragment into pNOPGFP2L. This vector is based on pRS425 and contains a 1.4-kb BamHI-PstI NOP1p-GFP cassette MATa strainb YEp24 YEp24-YRB1 (pSB415) (K. Hellmuth and E. Hurt, unpublished results). Preparation of rabbit polyclonal anti-Yrb1 antiserum: To FUS1 YRB1 250 165 generate a (His)6-Yrb1 fusion protein containing all but the fus1⌬ YRB1 [1.0] 0.6 first 10 residues of Yrb1, the corresponding YRB1 coding se- fus1⌬ yrb1-51 (sfo1-1) 39 1.8 quence was excised as a SalI fragment from pMK102 and a Mating efficiency is defined as the number of diploids ligated into the XhoI site of pRSETA (Invitrogen), yielding formed per number of input haploids of the strain tested. pMK104. For expression in E. coli, strain BL21(DE3)/pLysS The values given represent the average of three independent (Studier 1991) was transformed with pMK104 and the fusion ␤ trials, each performed in triplicate, and have been normalized protein was induced by addition of isopropyl- -d-thio-galacto- to the mating efficiency of the fus1⌬ mutant. pyranoside (IPTG) to a final concentration of 0.4 mm followed Њ b The indicated strains [JTY2023, MATa FUS1 YRB1; by incubation at 37 for 2 hr. Selection for pMK104 had to JTY2024, MATa fus1⌬ YRB1; JTY2025, MATa fus1⌬ yrb1-51 be maintained by adding 50 mg/liter carbenicillin (Sigma, St. (sfo1-1)] were transformed with either YEp24 (a URA3-marked Louis), a more stable derivative of ampicillin, to the medium because the fusion protein was relatively toxic to the cells. 2-␮m DNA vector) or pSB415 (YEp24-YRB1) and mated with 2ϩ JY416 (MAT␣ fus1⌬), as described in materials and methods. The fusion protein was purified from E. coli using Ni -chelate affinity chromatography (Ni-NTA resin; QIAGEN, Chats- worth, CA), according to the manufacturer’s recommenda- previously (Hellmuth et al. 1998). Plasmids pMK294-51 and tions. The purified protein was used to raise polyclonal anti- pMK294-52, and pMK291-wt, pMK291-51, and pMK291-52, ex- sera in two adult female New Zealand White rabbits (nos. 1390 pressing Yrb1-green fluorescent protein (GFP) fusions under and 1391), following standard protocols (Harlow and Lane ␮ control of the authentic YRB1 promoter, were constructed 1988), using 600 g of protein in 50% Titermax (CytRx, Nor- by replacing an internal BglII fragment in the YRB1 coding cross, GA) for the first immunization and 400 ␮g of protein sequence in pMK284n, which expresses a functional Yrb1- in 50% incomplete Freund’s adjuvant (Sigma) for each of two GFP chimera (Hellmuth et al. 1998), with the corresponding subsequent immunizations administered after 3 and 5 weeks, fragments from yrb1-51 and yrb1-52, followed by subsequent respectively. Bleeds were taken after 4 weeks (2 ml), 6 weeks recloning of the respective YRB1-GFP gene fusions as EcoRI- (50 ml), 7 weeks (2 ml), and 8 weeks (terminal) and stored NotI fragments into pRS424. in 0.02% sodium azide at Ϫ70Њ. For detection of Yrb1 by Fusions of full-length Yrb1 to the Gal4 transcriptional activa- immunoblotting the resulting antisera (nos. 1390 and 1391) tion domain (TAD) and full-length Gsp1(G21V) to the E. coli were used as primary antibodies at a dilution of 1:5000. LexA DNA-binding domain (DBD) were generated via PCR, Two-hybrid assay: To assess interactions between LexA using the two-hybrid vectors pGAD424 and pBTM116, respec- (DBD)-Gsp1(G21V) and Gal4(TAD)-Yrb1 fusion proteins, tively. Fragments comprising the entire YRB1 ORF were syn- strain JTY2500 harboring the E. coli lacZ gene under control thesized using 5Ј-CCG AAT TCG GTC CAG GTG GTA GCG of eight LexA-binding sites was cotransformed with the appro- AAG ATA AGA AAC CTG TCG-3Ј (OSFO15) and the M13/ priate pBTM116- and pGAD424-based plasmids. Transfor- pUC reverse sequencing primer (New England Biolabs) as mants were grown in SCGlc medium lacking leucine and tryp- and assayed (1ف the primers and pUC19 carrying the chromosomal YRB1-con- tophan to midexponential phase (A546 nm ϭ taining EcoRI-XbaI fragment (pMK102), or pUC19 carrying for ␤-galactosidase acitivity as described previously (Ku¨nzler the corresponding fragments from the yrb1-51 or yrb1-52 ORFs, and Hurt 1998). as templates. The PCR products were digested with EcoRI and Preparation of yeast cell extracts: Yeast cells were washed PstI and inserted into the corresponding sites in pGAD424, once with one culture volume of cold phosphate-buffered yielding pMK199-wt, pMK199-51, and pMK199-52, respec- saline (PBS), aliquoted into 1.5-ml microcentrifuge tubes .A546 nm units per tube), and stored as pellets at Ϫ70Њ 20ف) tively. Similarly, a fragment comprising the entire ORF coding for Gsp1(G21V) was produced using 5Ј-GCG AGG CCT TGC Frozen cell pellets were thawed by adding 0.2 ml cold lysis CCC AGC TGC TAA CGG TGA AG-3Ј (OGSP7) and RSET buffer (50 mm Tris-HCl pH 7.5, 150 mm NaCl, 20 mm MgCl2, (5Ј-AAC TGC AGC CAA CTC AGC TTC C-3Ј) as the primers, 10% glycerol, 2 mm DTT, and 1 mm PMSF) and lysed by and E. coli expression vector pRSETB (Invitrogen, Carlsbad, vigorous vortexing with 0.2 g of acid-washed glass beads (0.45– CA) carrying a PCR-mutated genomic PvuII-HindIII fragment 0.6 mm diameter) for six 30-sec periods (separated by 1-min coding for Gsp1(G21V) as the template. The resulting PCR periods of cooling on ice). The lysate was clarified by centrifu- product was cleaved with StuI and PstI and inserted into the gation for 5 min at 13,000 ϫ g at 4Њ and the protein concentra- SmaI and PstI sites of pBTM116, yielding plasmid pMK195-GV. tion was determined by a dye-binding method (Bradford Plasmid YEplac195-AU-L25NLS-GFP was derived from YE- 1976) using commercially available reagents (Bio-Rad, Her- plac195-ADE2-URA3-L25-GFP (Hurt et al. 1998) by removing cules, CA) and bovine serum albumin (BSA) as the standard. most of the RPL25 coding sequence, except for the 5Ј-end Purification of ProtA-TEV-Gsp1(G21V) from yeast: Trans- that encodes the first 52 residues of L25 (and contains an formants of wild-type strain CRY1, coexpressing ProtA-TEV- intron), using a two-step PCR procedure (Giebel and Spritz Gsp1(G21V) from pNOPPATA-GSP1G21V and either Yrb1- 1990; mutagenic primer, 5Ј-GGG ACA ACT CCA GTG AAA GFP, Yrb1(A91D)-GFP, or Yrb1(R127K)-GFP from plasmids AGT CTT CTC TTT GCT CTC GAG TGG AAC AGC CTT pMK284n, pMK294-51, or pMK294-52, respectively, were -Purifica .1.5ف GGA AGC-3Ј; O. Gadal and E. Hurt, unpublished results). grown in selective medium at 26Њ to a A546 nm ϭ Plasmid pNOPGFP2L-STE5 expressing a GFP-Ste5 fusion pro- tion of Gsp1(G21V) from these cells was performed essentially tein from a multicopy-plasmid under control of the consti- as described (Hellmuth et al. 1998), with the minor modifica- tutive NOP1-promoter was constructed by inserting a PCR-gen- tion that universal buffer (Ku¨nzler and Hurt 1998) was used 1094 M. Ku¨nzler et al. throughout the purification, including cell lysis, washing steps, recessive allele of YRB1, which encodes the homolog of and elution. Elution by cleavage with TEV-protease (GIBCO- mammalian RanBP1 (Ouspenski et al. 1995; Schlen- BRL, Gaithersburg, MD) was performed by incubation for 1 hr at room temperature. stedt et al. 1995). Hence, sfo1-1 was redesignated yrb1-51. Nuclear protein import and RNA export assays: Tempera- A recessive ts mutation, stp52 (sterile pseudorever- ture-sensitive mutants, and their otherwise isogenic wild-type sion), closely linked to TRP1, was isolated as an ex- strains, containing plasmids that express constitutively nuclear tragenic suppressor of the mating defect of a MATa transport substrates fused to GFP(S65T), namely SV40NLS-Gal4- ste5-3 ϫ MAT␣ ste5-3 cross at restrictive temperature (TAD)-GFP (pGADGFP), GFP-Npl3 (pNOPGFPAU-NPL3), (Katz et al. 1987). The stp52 mutation also suppressed and L25NLS-GFP (YEp195-AU-L25NLS-GFP), were cultivated to in selective SCGlc other ste5, ste4, and ste7 missense (ts) alleles (Katz et (0.5ف early exponential phase (A546 nm ϭ medium at 23Њ, split into two equal portions, and incubated al. 1987). Although the linkage analysis reported by at either 23Њ or 37Њ for various periods of time. Strains carrying Katz et al. (1987) assigned the stp52 mutation to the ␤ plasmids expressing SV40NLS-GFP- -galactosidase (pPS817) opposite side of the TRP1 locus from yrb1-51, we found under control of the GAL1 promoter were pregrown to early that a yrb1-51/stp52 diploid strain was still ts, and that the in selective SCRaf medium (0.5ف exponential phase (A546 nm ϭ at 23Њ before Gal (2%) was added to the cultures and the ts growth defect of the stp52 mutant could be completely cells were incubated at 23Њ for another hour (to allow mRNA rescued by the cloned YRB1 gene on a plasmid (data synthesis and export). The induced cultures were split into not shown). These results demonstrated that the stp52 two equal portions, and one portion was shifted to 37Њ for mutation was another recessive allele of YRB1, as was 3 hr, while the other portion was maintained at 23Њ for the same period. Fluorescence microscopy of living yeast cells confirmed by sequencing of the mutant DNA (see be- expressing GFP fusion proteins was done according to Hell- low). Hence, stp52 was redesignated yrb1-52. muth et al. (1998). Cells were concentrated by brief centrifuga- Phenotypic characterization of the yrb1-51 and yrb1- tion and resuspended in the residual growth medium without 52 mutations: To understand how alterations in YRB1 any washing steps. To assay mRNA export, cells were cultivated can suppress mating-defective mutants, we examined, in YPGlc medium as described above for strains harboring constitutively expressed GFP fusion proteins. Poly(A)ϩ RNA first, the physiology of the yrb1 mutants. At 23Њ, yrb1-51 was localized by in situ hybridization as described previously mutant cells grew nearly as well as wild-type cells, (Segref et al. 1997). whereas the yrb1-52 mutant cells displayed impaired Miscellaneous: SDS-PAGE and immunoblotting were con- growth already under these conditions; both yrb1-51 and ducted as described previously (Ku¨nzler and Hurt 1998). yrb1-52 cells ceased growth and lost viability within 3–6 Multiple sequence alignment was done using the CLUSTALW 1.7 (Thompson et al. 1994) and BOXSHADE 3.21 [Bioinfor- hr after shift to 37Њ (data not shown). Similar results matics group of the Swiss Institute for Experimental Cancer were observed for the corresponding homozygous dip- Research (ISREC)] programs. Identities to the Yrb1 sequence loids (data not shown). As judged by immunoblotting were calculated on the basis of pairwise alignments using the of cell lysates (Figure 1A), after shift to 37Њ for 3 hr, ALIGN algorithm from the FASTA package (Pearson and the product of the yrb1-51 allele was hardly detectable, Lipman 1988). whereas the yrb1-52 product remained relatively stable even 6 hr after temperature shift. Thus, the yrb1-51 mutation appears to destabilize the gene product at higher RESULTS temperature, whereas the yrb1-52 product is stable un- Isolation of yrb1ts mutations as suppressors of mating der the same conditions. Upon prolonged incubation defects: The mating deficiency of a fus1⌬ mutant is at 37Њ, an apparent degradation product of Yrb1 accu- much more pronounced at 14Њ than at 30Њ.At14Њ, dip- mulated in yrb1-52 cells, but was also observed in the loid formation in a MATa fus1⌬ϫMAT␣ fus1⌬ cross wild-type control cells. Yrb1 was expressed at similar is Ͻ0.5% that of a MATa FUS1 ϫ MAT␣ fus1⌬ cross levels in MATa, MAT␣, and MATa/MAT␣ cells (data (Table 3). A screen for extragenic suppressors of this not shown) and its level in MATa cells was not elevated in mating defect (see materials and methods) yielded response to treatment with ␣-factor mating pheromone a single mutation, sfo1-1. This suppressor mutation re- (data not shown). producibly enhanced mating competence of a fus1⌬ Examination of the cell morphology revealed that mutant 30–50-fold (Table 3), but did not fully restore haploid yrb1-51 cells arrested mostly as enlarged cells mating proficiency to the level of a FUS1 cell. The sup- with a large bud or as large unbudded cells upon shift pressor segregated 2:2 through two backcrosses against to 37Њ (data not shown; Baümer et al. 2000), which is a fus1⌬ strain and cosegregated with a recessive ts growth reminiscent of cell cycle progression mutants. Similar defect (in Ͼ15 tetrads analyzed per cross). Genetic map- results were previously observed for stp52/yrb1-52 cells ping of the mutation to the right arm of chromosome (Katz et al. 1987; Ouspenski 1998). Another striking IV between CEN4 and the TRP1 gene (data not shown), phenotype of both yrb1ts alleles was the appearance of complementation of the ts growth defect by the wild- chains of elongated nonseparated cells, most evident in type YRB1 gene on a plasmid (data not shown), elimina- homozygous diploids grown on plates at a semipermis- tion of the suppression phenotype by plasmid-borne sive temperature (30Њ; Figure 1B). Such cell elongation YRB1 (Table 3), and nucleotide sequencing of the mu- is diagnostic of mutations that delay G2-M progression tant DNA (see below) all established that sfo1-1 was a (Lew 2000). We observed a similar morphological de- Ran-Binding-Protein-1 and Yeast Mating 1095

nonical cell division cycle (cdc) mutations, and might indicate a role of Yrb1 at multiple stages of the cell cycle. Consistent with such a notion, the yrb1-51 mutation interferes with both the G1/S transition and the passage through mitosis (Baümer et al. 2000), and displayed synthetic growth defects when combined with two different cdc28 alleles (cdc28-4 and cdc28-1N) that are diagnostic for different cell cycle stages (G1 and G2/M, respectively; Table 4). Yrb1-51 and Yrb1-52 are altered in conserved residues of the Ran-binding domain and defective for Ran-binding in vivo: To determine the nature of the alterations in the mutant proteins, PCR was used to recover the YRB1 coding sequences from the mutant strains (see materials and methods). The DNA sequence of each mutant ORF contained a single point mutation, both of which alter a highly conserved residue in the RBD (Figure 2A). The yrb1-51 mutation is a C-to-A transver- sion on the coding strand at position 272 (where ϩ1is the first base of the initiator ATG), which substitutes Asp for Ala at codon 91 (A91D). The yrb1-52 allele is a G-to-A transition on the coding strand at position 380, which substitutes Lys for Arg at codon 127 (R127K). On the basis of homology modeling of Yrb1 on the crystal structure of the first RBD (RanBD1) in mammalian Nup358 (RanBP2) complexed with Ran bound to Figure 1.—Effects of yrb1-51 and yrb1-52 mutations on in a nonhydrolyzable GTP analog (Vetter et al. 1999), vivo stability of Yrb1 and cell morphology. (A) Stability of A91D replaces a nonpolar residue in the hydrophobic normal and mutant Yrb1 at restrictive temperature. Haploid strain YPH499 (YRB1) and its congenic derivatives, JTY2026 core of Yrb1 with a bulkier, charged residue (Figure (yrb1-51) and JTY2483 (yrb1-52), were grown at 23Њ in SCGlc 2B). This change should destabilize the global fold of medium to midexponential phase, shifted to 37Њ, and samples Yrb1, consistent with the rapid degradation of this mu- were withdrawn at the indicated times. Total protein was ex- tant protein observed at restrictive temperature (Figure tracted from each sample and analyzed by SDS-PAGE and 1A). Two other existent yrb1 alleles, yrb1-1 and yrb1-2 immunoblotting using a rabbit polyclonal anti-Yrb1 antiserum (no. 1390). A band that cross-reacts nonspecifically with the (Schlenstedt et al. 1995), alter residues (F187 and L93, anti-Yrb1 antiserum served as a loading control. The asterisk respectively) that project into the same hydrophobic indicates a major degradation product of Yrb1. (B) Morphol- pocket as A91. In contrast, R127K makes a seemingly ogy of homozygous diploid yrb1ts cells. Strains YPH501 (YRB1/ modest change in a surface-exposed residue that forms YRB1), JTY2482 (yrb1-51/yrb1-51), and JTY2485 (yrb1-52/yrb1- a bridge to residues in the long C-terminal “arm” of 52) were cultivated on YPGlc plates at 16Њ or 30Њ, as indicated, and viewed by Nomarski optics. Ran that embraces the RBD (Figure 2B), an alteration unlikely to disrupt the overall structure, consistent with the observed stability of the mutant protein at restrictive fect in srp1-31ts/srp1-31ts diploids (data not shown). temperature (Figure 1A). We confirmed that each muta- Srp1/␣-importin is the adaptor necessary for recogni- tion was both necessary and sufficient to confer the ts tion and nuclear import of proteins that contain a clas- phenotype of the corresponding allele by inserting each sical nuclear localization signal (NLS) by the Kap95/ mutant DNA into a plasmid and introducing it into a ␤-importin receptor (Enenkel et al. 1995). At 37Њ, srp1- yrb1⌬ background (see materials and methods). 31 cells are impaired in import of NLS-containing re- Two independent approaches demonstrated that the porter proteins and arrest uniformly as large-budded yrb1-51 and yrb1-52 mutations interfere with Yrb1-Ran cells indicative of a defect in mitosis (Loeb et al. 1995). (Gsp1) interaction in vivo. The GTPase-deficient form Correspondingly, degradation of Clb2, whose destruc- of Gsp1, Gsp1(G21V), binds more strongly to Yrb1 than tion is required for exit from mitosis, is impaired in normal Gsp1 (Schlenstedt et al. 1995); hence, we used srp1-31 cells (Loeb et al. 1995); likewise, degradation of Gsp1(G21V) in our analyses. First, we applied the two- Clb2 and of two anaphase inhibitors, Pds1 and Sic1, is hybrid method using full-length wild-type Yrb1, Yrb1 also impaired in yrb1-51 cells (Baümer et al. 2000). De- (A91D), or Yrb1(R127K) fused to the Gal4 transcrip- spite these similarities, the absence of a uniform cell tional activation domain [Gal4(TAD)] and full-length cycle arrest phenotype distinguishes the yrb1-51 and yrb1- Gsp1(G21V) fused to the LexA DNA-binding domain 52 mutations from the srp1-31 mutation and from ca- [LexA(DBD)] in a reporter strain carrying a chromo- 1096 M. Ku¨nzler et al.

TABLE 4 Summary of genetic interactions between yrb1-51 (and yrb1-52) and nucleocytoplasmic transport factors

Mutation Source Genetic interactiona Ran GTPase cycle rna1-1 Atkinson et al. (1985) ϩϩ (sl) prp20-1 Aebi et al. (1990) ϩ (ϩ) srm1-1 Clark and Sprague (1989) ϩ (nd) prp20-10 Fleischmann et al. (1996) Ϫ (nd) gsp1-1, -2 Wong et al. (1997) ϩϩ (nd) gsp2::HIS3 Kadowaki et al. (1993) ϩ (nd) yrb2::HIS3 I. Macara (personal communication) Ϫ (sl) nup2::HIS3 Loeb et al. (1993) ϩ (sl) Nucleoporins nsp1ts Nehrbass et al. (1993) ϩ (nd) nup133::HIS3 Doye et al. (1994) ϩ (ϩ) nup116::URA3 Bailer et al. (1998) ϩ (ϩ) Nuclear import receptors srp1-31 Loeb et al. (1995) sl (sl) srp1-49 Schroeder et al. (1999) sl (sl) rsl1-4 Koepp et al. (1996) sl (sl) mtr10::HIS3 Senger et al. (1998) sl (sl) kap104::HIS3 Aitchison et al. (1996) sl (nd) pse1-1 Seedorf and Silver (1997) ϩ (nd) yrb4::HIS3 Schlenstedt et al. (1997) Ϫ (nd) pse1-1 yrb4::HIS3 Seedorf and Silver (1997) sl (nd) Nuclear export receptors los1::HIS3 Hellmuth et al. (1998) Ϫ (nd) msn5::TRP1 Blondel et al. (1999) sup (sup) cse1-1 Xiao et al. (1993) sl (nd) xpo1-1 Stade et al. (1997) ϩ (sl) crm1-1, -2, -3 Yan et al. (1998); F. Stutz (personal communication) Ϫ (nd) Others cdc28-4 Reed (1980) ϩ (nd) cdc28-1N Piggott et al. (1982) ϩ (nd) rat1-1 Amberg et al. (1992) Ϫ (nd) nsr1::URA3 Kondo and Inouye (1992) Ϫ (nd) plc1::HIS3 Flick and Thorner (1993) ϩ (nd) Abbreviations in parentheses indicate phenotype observed with the yrb1-52 allele. a Ϫ, no synthetic growth phenotype; ϩ, synthetic growth defect (see text for details); sl, synthetic lethality; nd, not determined; sup, no synthetic growth defect but extragenic suppression of mating defect (see Table 5). somally inserted copy of E. coli lacZ under control of muth et al. (1998), eluted by cleavage with TEV-protease eight LexA-operator sites. In this system, Yrb1(A91D) (see materials and methods), and analyzed by SDS- showed a reproducible reduction (Ͼ3-fold) and Yrb1 PAGE, Coomassie blue staining, and immunoblotting (R127K) showed a dramatic reduction (Ͼ50-fold) in using a polyclonal anti-Yrb1 antisera (no. 1391). Exami- interaction with Gsp1(G21V) compared to wild-type nation of the input material (Figure 3B, load), and flow- Yrb1 (Figure 3A). Immunoblotting with anti-Yrb1 antise- through fractions (data not shown), demonstrated that rum (see materials and methods) and anti-Gsp1 anti- expression of normal and mutant Yrb1-GFP fusions was bodies (gift of P. Belhumeur) showed that all constructs comparable, as were their stabilities during purification. were expressed at equivalent levels (data not shown). No detectable Yrb1(R127K)-GFP copurified with Gsp1 These results were confirmed by a biochemical pro- (G21V), whereas significant amounts of both wild-type cedure (Figure 3B). Yrb1-GFP, Yrb1(A91D)-GFP, or Yrb1-GFP and endogenous Yrb1 were retained by the Yrb1(R127K)-GFP, produced from the authentic YRB1 same beads (Figure 3B). A detectable amount of Yrb1 promoter on CEN plasmids, were expressed in wild-type (A91D)-GFP copurified with ProtA-Gsp1(G21V), but its cells (strain CRY1) also producing a ProtA-(TEV site)- level was markedly less than the amount of wild-type Gsp1(G21V) from the constitutive NOP1 promoter on Yrb1-GFP retained under the same conditions (Figure 3B). a CEN plasmid. After growth at 26Њ, protein complexes yrb1-51 and yrb1-52 mutants are defective in nuclear bound to bead-immobilized ProtA-(TEV site)-Gsp1(G21V) protein and RNA transport: To determine if yrb1-51 and were recovered from cell extracts as described in Hell- yrb1-52 cause defects in nuclear protein import and RNA Ran-Binding-Protein-1 and Yeast Mating 1097

Figure 2.—Positions of the altered residues in Yrb1 resulting from the yrb1-51 and yrb1-52 mutations. (A) Alignment of various Ran-binding domains (RBDs). Sequences shown are grouped into three subfamilies (RanBP1, RanBP2, and RanBP3), on the basis of certain shared sequence characteristics, and include the following (with GenBank accession num- bers): S. cerevisiae Yrb1 (L38489), Schizosaccharomyces pombe Sbp1 (D86381), mouse RanBP1 (X56045), human RanBP1 (X83617), Xenopus laevis RanBP1 (U09128), Arabidopsis thali- ana RanBP1 (U62742), mouse RanBP2 nucleoporin (X87337), human NUP358 (D38076), Bos taurus RanBP2 nucleoporin (L41691), Caenorhabditis elegans Ranup96 (Z34801), S. cerevisiae Nup2 (X69964), S. pombe Hba1 (U38783), and human RanBP3 (Y08697). Sequence of S. cerevisiae Yrb2/Nup36 is from the Swiss Protein Database (accession no. P40517). The mouse and bovine RanBP2 are incomplete because they are derived from partial cDNA clones. An insert of 24 residues (possibly an intron) was omitted from the actual C. elegans Ranup96 sequence to optimize its alignment to the other RBDs. Identi- ties shared by 11 (or more) of the RBDs shown are indicated by white-on-black letters; chemically similar residues are shown as black-on-grey letters. The positions mutated in yrb1-51, A91A, and in yrb1-52, R127K, are indicated at the top. (B) Positions of the residues (A91 and R127) altered in the yrb1- 51 and yrb1-52 mutants, respectively, have been modeled on the ﬁrst RBD (RanBD1) in human NUP358 complexed with Ran bound to a nonhydrolyzable GTP analog (Vetter et al. 1999). Blue, Yrb1; purple, Ran; and red, GTP analog.

export, as observed before for the yrb1-1 and yrb1-2 al- the cultures were then split into two equal portions, one leles (Schlenstedt et al. 1995), we ﬁrst examined the of which was maintained at 23Њ and the other shifted distribution of poly(A)ϩ-RNA by in situ hybridization. to 37Њ. Samples were withdrawn at various times for Mutant or wild-type control cells were grown to midex- analysis. Results were more readily visualized in diploid ponential phase at permissive temperature (23Њ), and cells because of their larger size; however, similar ﬁnd- 1098 M. Ku¨nzler et al.

ings were made with haploid cells (data not shown). In homozygous yrb1-51/yrb1-51 diploids shifted to 37Њ for 2 hr, there was a rapid and clear-cut nuclear accumulation of poly(A)ϩ-RNA in every cell (Figure 4A); this of the cells even %50ف effect was readily apparent in 1 hr after the shift (data not shown). Poly(A)ϩ RNA accumulated in the nucleus in a distinctly punctate pat- tern, a feature seen in mutants that have a strong RNA export defect, such as mex67-5 (Segref et al. 1997). How- ever, onset of the RNA export defect in the yrb1-51/ yrb1-51 cells was slower than that in mex67-5 mutants and nuclear RNA accumulation was not as complete (some cytosolic poly(A)ϩ RNA signal remains even 2 hr after shift to 37Њ). In striking contrast, yrb1-52/yrb1-52 diploids did not show any accumulation of poly(A)ϩ RNA (even 5 hr after shift to 37Њ), just like the wild-type control (Figure 4A). Although yrb1-51 cells manifested a clear defect in RNA export, neither yrb1-51 mutants nor yrb1-52 mutants had any detectable effect on the nuclear export of proteins containing a leucine-rich NES, such

as SV40NLS-PKINES-GFP (Stade et al. 1997) or yAP1-GFP (Yan et al. 1998; data not shown). To monitor the effect of the yrb1-51 and yrb1-52 mutations on nuclear protein import, four different GFP fusions of nuclear proteins were examined. To assess the ␣-importin/Srp1 and ␤-importin/Kap95/Rsl1-dependent pathway, two chimeras containing the SV40 NLS

were used: a galactose-inducible SV40NLS-GFP-␤-galactosidase, which is so large it cannot diffuse out of the nucleus after it has been delivered there (Lee et al.

1996), and a constitutively expressed SV40NLS-Gal4TAD- GFP, which, due to its small size, can diffuse out of Figure 3.—Yrb1(A91D) (yrb1-51) and Yrb1(R127K) (yrb1- the nucleus unless ongoing import occurs continuously 52) bind Gsp1(G21V) with reduced affinity in vivo. (A) Interac- (Ku¨nzler and Hurt 1998). The third reporter was a tion between wild-type Yrb1, Yrb1(A91D), or Yrb1(R127K) and fusion of GFP to Npl3, an mRNA-binding protein, whose Gsp1(G21V) was determined using the two-hybrid method as nuclear entry depends on the importin, Kap111/Mtr10 described in materials and methods. In brief, Yrb1 proteins (Senger et al. 1998). GFP-Npl3 accumulates rapidly in were fused to the Gal4(TAD), and Gsp1(G21V) was fused to the LexA(DBD). In the recipient strain (JTY2500), the E. coli the cytoplasm if import is impaired because Npl3 contin- lacZ gene is under control of eight LexA-operator elements. uously shuttles between the nucleus and the cytosol. ␤-Galactosidase activity is expressed in arbitrary units. Each The fourth transport substrate was constitutively ex- value represents the average of single measurements made on pressed and composed of GFP fused to the NLS of three independent transformants; error bars indicate standard ribosomal protein L25 (O. Gadal and E. Hurt, per- deviation of the mean. (B) Binding of Yrb1 to Gsp(G12V) was assessed by copurification. Cultures of strain CRY1 (YRB1 sonal communication). Transport of L25NLS-GFP into GSP1) carrying CEN plasmids expressing a ProtA-TEV- the nucleus utilizes two different import receptors, Gsp1(G21V) fusion from the NOP1 promoter and either wild- Kap121/Pse1 and Kap123/Yrb4 (Schlenstedt et al. type Yrb1-GFP, Yrb1(A91D)-GFP, or Yrb1(R127K)-GFP ex- 1997). Like SV40NLS-Gal4TAD-GFP, L25NLS-GFP is small pressed from the authentic YRB1 promoter were grown in enough to diffuse out of the nucleus unless its import selective SCGlc medium at 26Њ. Extracts were prepared and the ProtA-TEV-Gsp1(G21V) was purified on IgG-Sepharose occurs continuously. (Pharmacia, Uppsala, Sweden) and eluted by digestion with Control strains or yrb1-51 and yrb1-52 mutants carrying recombinant TEV protease (GIBCO-BRL, Gaithersburg, MD). the reporter plasmids described above were cultivated Equal fractions of the load and the eluate of each column in selective medium, shifted to restrictive temperature, were resolved by SDS-PAGE and analyzed, as indicated, by and examined by fluorescence microscopy. For the in- Coomassie blue staining and immunoblotting using a rabbit polyclonal anti-Yrb1 antiserum (no. 1391). Endogenous Yrb1 ducible reporter, transformants were grown to midexpo- served as a control to confirm equivalent loading and function- nential phase in Raf-containing medium at permissive ality of the immobilized Gsp1(G21V). temperature and induced for 1 hr by addition of 2% Gal (to allow for mRNA synthesis and export) before shift to 37Њ. For all four reporter proteins, there was a Ran-Binding-Protein-1 and Yeast Mating 1099

Figure 4.—Nuclear transport defects in yrb1ts mutants. (A) To examine poly(A)ϩRNA export, cultures of homozygous diploid strains YPH501 (YRB1/YRB1), JTY2482 (yrb1-51/yrb1- 51), and JTY2485 (yrb1-52/yrb1-52) were grown in YPGlc at 23Њ to early exponential phase, split into two equal portions, and incubated for another 2 hr either at 23Њ or 37Њ. After fixation with formaldehyde, cells were stained with the DNA dye 4Ј,6-diamidino-2-phenylindole, analyzed by in situ hybridization using a CY3-labeled oligo(dT) probe to visualize the subcellular distribution of poly(A)ϩRNA, and viewed by fluorescence microscopy using appropriate band-pass filters. (B and C) To examine nuclear protein import, the same strains as in A were transformed with multicopy plasmids expressing either SV40NLS-Gal4TAD-GFP (B) or L25NLS-GFP (C), respectively, cultivated and shifted as in A, and viewed directly by fluorescence microscopy and Nomarski optics.

signiﬁcant cytoplasmic accumulation in yrb1-51 and yrb1- in haploids and for the other two reporters (data not 52 mutants after shift to 37Њ, compared to wild-type cells, shown). In yrb1-52 cells, the defect was noticeable even indicating a general defect in nuclear protein import. at permissive temperature. Our results showing a defect

Results for SV40NLS-Gal4TAD-GFP (Figure 4B) and L25NLS- in nuclear protein import in yrb1-52 cells are at odds GFP (Figure 4C) reporters in homozygous diploid with a previously published report on the same mutant strains are shown; but, similar results were obtained (Ouspenski 1998). 1100 M. Ku¨nzler et al.

To exclude the possibility that cytoplasmic localization of the reporter proteins in yrb-51 and yrb1-52 cells was due to “leakiness” of the mutant nuclei, accumulation of constitutively expressed SV40NLS-GFP-␤-galactosidase (encoded by pPS815; Lee et al. 1996) was examined in the same strains before and after temperature shift. In contrast to the short-term assay with the inducible version of the same reporter protein (see above), no increased cytoplasmic GFP signal was observed (data not shown), demonstrating that the nuclei in the yrb1- 51 and yrb1-52 mutants were not more “leaky” than wild- type nuclei. Genetic interactions of yrb1 mutations with nucleocytoplasmic transport factors: As an independent means to confirm that yrb1-51 and yrb1-52 compromise nucleocytoplasmic trafficking even at permissive temperature, ts genetic interactions of these alleles with mutations in Figure 5.—Genetic interactions between yrb1 alleles and components of the nucleocytoplasmic transport machinery. genes encoding a variety of other factors involved in yrb1-51 (or yrb1-52) mutant strains were crossed with strains nucleocytoplasmic transport were examined. Strains carrying a mutation in another gene of interest. The resulting carrying mutations of interest were crossed with strains diploids were subjected to sporulation, and growth of individ- carrying the yrb1-51 or yrb1-52 mutation, and the re- ual spores from tetratype asci was examined at various temper- sulting diploids were sporulated. Double mutant segre- atures. Left, genetic interaction of yrb1-51 with nup2::HIS3 is manifested by the extremely poor growth (“ϩ” in Table 4) of gants from tetratype asci were compared to each single the yrb1-51 nup2::HIS3 double mutant at 28Њ, a temperature mutant segregant and to the wild-type segregant for clearly permissive for the congenic yrb1-51 and nup2::HIS3 their ability to grow at various temperatures (see Figure single mutants (yrb1-51 alone has a restrictive temperature of 5). Mutations tested included alterations in genes en- 31Њ under these conditions and nup2::HIS3 alone has no obvi- coding components of the Ran GTPase cycle, nuclear ous growth defect even at higher temperatures). Right, a yrb1- 51 mutant carrying a URA3-marked multicopy plasmid ex- import and export receptors, and nucleoporins (see Table pressing wild-type YRB1 (pMK103) was crossed with a strain 4). The plc1⌬::HIS3 mutation (Flick and Thorner 1993) carrying a ts mutation, srp1-31,in␣-importin. The resulting was tested since there is evidence for a role of PLC1- diploid was sporulated and individual spores from a tetratype encoded phosphatidylinositol-specific phospholipase C ascus were streaked on medium containing 5-FOA to count- in mRNA export (York et al. 1999; J. Flick, personal erselect against the plasmid. Genetic interaction of yrb1-51 with srp1-31 is manifested by the inviability or “synthetic lethal- communication). The yrb1-51 mutation displayed dele- ity” (“sl” in Table 4) of the yrb1-51 srp1-31 double mutant at terious genetic interactions with many of these other any temperature, whereas the congenic yrb1-51 and srp1-31 classes of mutants that affect nucleocytoplasmic trans- single mutants are able to grow at permissive temperatures port (Table 4). Combination of the yrb1-52 mutation (here shown at 26Њ). Other double mutant combinations with at least 12 of these mutations revealed essentially tested and their phenotype are listed in Table 4. the same growth defects. For xpo1-1 yrb1-52 and yrb2⌬::HIS3 yrb1-52, the growth defect was even more ing, presumably, the ability of these mutations to sup- severe than for the corresponding double mutant with press mating defects. Far1 and Ste5 are currently the yrb1-51 (Table 4). Equally pronounced growth defects only components of the mating pheromone response were observed when yrb1-51 was combined with muta- pathway known to shuttle between nucleus and cyto- tions in genes encoding certain nuclear transport recep- plasm (Blondel et al. 1999; Mahanty et al. 1999) and tors (Table 4). In all these cases, the double mutant was yrb1-52 was isolated as an extragenic suppressor of a not viable at any temperature (synthetic lethality); for ste5 missense mutation (Katz et al. 1987). Hence, we the other genetic interactions observed, double mutants constructed a functional GFP-Ste5 chimera and exam- were viable at the permissive temperature (23Њ) but re- ined its subcellular localization in yrb1-51 and yrb1-52 vealed a restrictive temperature that was considerably mutants (and in control cells) in the absence of phero- (Ͼ3Њ; ϩϩ) or slightly (Յ3Њ; ϩ) lower than the one of mone to avoid the complications of signal-induced any of the two single mutants (synthetic growth defect). changes. In wild-type cells at steady state, GFP-Ste5 accu- Thus, the functions of Yrb1(A91D) and Yrb1(R127K) mulated in the nucleus, even though cytoplasmic stain- are at least partially defective even under permissive ing was also evident (Figure 6), as observed before conditions. (Mahanty et al. 1999; Pryciak and Huntress 1998). Nucleocytoplasmic trafficking of Ste5 is altered in In contrast, no nuclear accumulation was observed in yrb1-51 and yrb1-52 cells: Our results indicate that im- either of the two yrb1ts mutants, even at permissive tem- paired nucleocytoplasmic transport is the primary cause perature (Figure 6). Since Ste5 shuttles continuously of the phenotypes of yrb1-51 and yrb1-52 mutants, includ- between nucleus and cytoplasm (Mahanty et al. 1999), Ran-Binding-Protein-1 and Yeast Mating 1101

TABLE 5 Suppression of the partial mating defect of a msn5⌬ null mutant by the yrb1-51 and yrb1-52 mutations

Genotypea Mating efﬁciency (%)b MSN5 YRB1 92.3 Ϯ 10 msn5⌬ YRB1 20 Ϯ 12 MSN5 yrb1-51 96.6 Ϯ 4.7 msn5⌬ yrb1-51 89 Ϯ 12.2 MSN5 YRB1 67.3 Ϯ 18.7 msn5⌬ YRB1 18.3 Ϯ 2.8 MSN5 yrb1-52 89.3 Ϯ 8.9 msn5⌬ yrb1-52 70.6 Ϯ 24.5 a MATa spores of the indicated genotype from a cross between strains HMK30 (msn5⌬), and JTY2027 (yrb1-51) and JTY2484 (yrb1-52), respectively, were assayed for quantitative mating with the MAT␣ tester strain DC17 as described in materials and methods. b Mating efﬁciencies are the ratio of the number of diploids formed to the number of viable input MATa haploids, are the average of three independent trials, and are given as percentage together with the standard deviations of the mean.

DISCUSSION Ran GTPase is one of the most highly conserved pro- Figure 6.—Localization of GFP-Ste5 in yrb1ts mutants. Hap- teins in nucleated cells (Macara et al. 2000; Sazer and loid strains YPH499 (YRB1), JTY2026 (yrb1-51), and JTY2483 Dasso 2000). Ran action has been implicated in nucleo- (yrb1-52) expressing a GFP-Ste5 fusion protein from a cytoplasmic transport, microtubule assembly, nuclear multicopy plasmid were cultivated, shifted, and viewed as described in the legend to Figure 4, B and C. envelope formation, maintenance of chromatin structure and nuclear (and nucleolar) organization, chromosome segregation, DNA replication, RNA metabolism, the observed mislocalization in cells with defective Yrb1 and cell cycle progression. It is still a matter of some could be due, in principle, either to inhibition of Ste5 debate whether the pleiotropic phenotypes of alter- import into the nucleus or enhancement of Ste5 export ations in the Ran GTPase cycle are due solely to the from the nucleus. Since we have demonstrated that yrb1- established role of Ran in nucleocytoplasmic transport 51 and yrb1-52 clearly impede nuclear import of various (Go¨rlich and Kutay 1999) or whether Ran has addi- reporter proteins, the former possibility seems more tional roles in the cell. Recent studies using in vitro likely. systems have implicated Ran function directly in micro- To obtain further evidence that suppression of mating tubule organization (Kahana and Cleveland 1999) defects by yrb1-51 and yrb1-52 arises from impairment and nuclear envelope formation (Hetzer et al. 2000; of nuclear import of Ste5, we tested whether these yrb1ts Zhang and Clarke 2000). Our evidence indicates that mutations could suppress the mating defect of a msn5⌬ the Ran GTPase cycle is linked to the signaling events mutant. Msn5 is thought to be the nuclear receptor required for mating as a consequence of the role of required for pheromone-stimulated export of Ste5 from Ran in nucleocytoplasmic transport. the nucleus (Mahanty et al. 1999). Each yrb1ts mutant A decade ago, before the function of the essential Ran was crossed against a msn5⌬ strain and the wild-type regulator, RanGEF1, was fully appreciated, a ts mutation single mutant, and double mutant spores from the re- (srm1-1) in its yeast homolog (SRM1/PRP20/MTR1) was sulting tetratype asci were tested for their relative mating isolated as a suppressor of the mating defect of haploid proficiency using a quantitative mating assay performed cells lacking pheromone receptors (Clark and Sprague at semipermissive temperature (30Њ) for the yrb1ts alleles. 1989). Unlike other ts mutations in yeast RanGEF1 iden- Both the yrb1-51 and yrb1-52 mutations restored the mat- tified subsequently (Amberg et al. 1993), srm1-1 does ing efficiency of the msn5⌬ mutant to essentially the not cause dramatic nuclear accumulation of poly(A)ϩ wild-type level (Table 5). This suppression is consistent RNA at restrictive temperature (Kadowaki et al. 1993; with the idea that a higher cytoplasmic pool of Ste5 (due and its effect on nuclear protein import was not exam- to its inefficient nuclear import in the yrb1ts mutants) ined), which left open the possibility that the Ran compensates for its inefficient pheromone-stimulated GTPase cycle had some role in mating distinct from export from the nucleus (due to the msn5⌬ mutation). its function in nucleocytoplasmic transport. We have 1102 M. Ku¨nzler et al. shown here that two independently isolated suppressors be required for cytoskeletal reorganization, which is of early and late defects in the mating pathway are necessary for polarized cell growth toward the mating alterations in another essential Ran regulator, RanBP1, partner (Blondel et al. 1999; Shimada et al. 2000). The that clearly impair nuclear protein import, even at per- role of recruitment of Ste5 to the shmoo tip may be missive temperature. We found further that the Ste5 similar, given the interaction of Ste5 with Bem1, a pro- scaffold protein, which shuttles between nucleus and tein involved in polarized cell growth (Leberer et al. cytoplasm and is required for pheromone response, is 1997). Since relocalization of Far1 (Blondel et al. 1999; mislocalized to the cytosol in these mutants. Consistent Shimada et al. 2000) and Ste5 (Pryciak and Huntress with this finding, both yrb1ts alleles suppressed the partial 1998; Mahanty et al. 1999) is transient, a nuclear import mating defect of a null mutation in MSN5, which en- defect that kept these proteins out of the nucleus longer codes the nuclear receptor required for pheromone- might increase mating efficiency in cells in which the stimulated export of Ste5. cell fusion process would otherwise be nonoptimal. In retrospect, it may seem obvious that perturbation In agreement with the above model, both the yrb1-51 of nucleocytoplasmic transport could affect yeast mating and the yrb1-52 mutations increased the cytoplasmic since response to pheromone requires: (a) that some concentration of Ste5 and were able to suppress the signal carrier enter the nucleus to induce the transcrip- mating defect of a msn5 null mutation. The MSN5 gene tion of genes, (b) that RNPs containing newly synthe- encodes a nuclear export receptor of the ␤-importin sized mRNAs for mating-specific components exit the family (Kaffman et al. 1998; DeVit and Johnston 1999) nucleus, and (c) that the translation products of some and was also identified as STE21 because a null mutation of those transcripts be recruited back into the nucleus at this locus confers a partial mating defect (Akada et al. in support of late mating events, like karyogamy (Rose 1996; Alepuz et al. 1999; Blondel et al. 1999). Impaired 1996). Accordingly, it is now known that many compo- nuclear protein import in the yrb1ts mutants keeps Ste5 nents of the pheromone response machinery, both pre- (and perhaps other proteins required for mating) in existing and pheromone induced, are localized to the the cytosol longer, and thus there is less need for their nucleus. On the other hand, it has been shown that continuous Msn5-dependent reexport from the nu- two components, Far1 (Blondel et al. 1999) and Ste5 cleus. Also consistent with the above model, MATa yrb1- (Pryciak and Huntress 1998; Mahanty et al. 1999), 51 and MATa yrb1-52 mutants are able to respond nor- rapidly relocalize from the nucleus to the site of cell mally to ␣-factor under conditions (by inducing the fusion (shmoo tip) upon pheromone treatment. Our pheromone-responsive reporter FUS1-lacZ) and by un- results suggest the following mechanism to explain the dergoing G1 arrest (as judged by the standard halo suppression of mating defects by mutations in nucleocy- bioassay; data not shown); hence, these mutants are not toplasmic transport factors. defective in early signaling events. If, however, proteins Because both yrb1ts alleles described here are defective required for late events in mating are maintained longer in nuclear protein import, but only one seems impaired in the cytosol of yrb1 mutants due to defective nuclear in poly(A)ϩ RNA export, yet both act as suppressors of protein import, the efficiency of mating would be en- mating defects, it is presumably the import defect that hanced, as observed. Finally, such a model would also leads to suppression. Also, it should be recalled that explain the spectrum of mating defects that are sup- yrb1-51 was isolated on the basis of rescue of the mating pressed by yrb1-51, yrb1-52, and srm1-1. All of the muta- debility of a fus1⌬ mutant, which has an intact phero- tions suppressed are in gene products that are targeted, mone response pathway and is only partially mating directly or indirectly, to the shmoo tip after pheromone defective. Likewise, yrb1-52 was isolated on the basis of treatment and most are involved in the pheromone- its ability to rescue ste5 missense mutations (and can do induced remodeling of the cortical cytoskeleton (Leb- so for ste4 and ste7 missense mutations), but is unable erer et al. 1997). Pheromone receptors (Ste2 and Ste3) to rescue null alleles in these same genes (Katz et al. are localized at the shmoo tip and participate in cell 1987). These considerations indicate that residual sig- polarity determination and mating partner discrimina- naling in the pheromone response pathway must be tion (Jackson et al. 1991). Ste4 (G␤), when released present for suppression to occur. How could impair- from the receptors as the G␤␥ complex in response to ment of nuclear protein import improve the mating pheromone binding, is responsible for direct recruit- efficiency of such partially mating-defective mutants? ment of major regulators of cytoskeletal structure and One reasonable scenario is that nuclear import of cer- cell polarity, including Ste20 (Leeuw et al. 1998), Far1 tain signaling components results in downregulation of (Shimada et al. 2000), and Ste5 (Inouye et al. 1997b; the mating response. If so, mutations that reduce the Pryciak and Huntress 1998). These components in- rate of nuclear entry of such a factor(s) should enhance teract with other molecules, like Bem1, that contribute the efficiency of mutants that are only partially defective to reorganization of the cytoskeleton (Leberer et al. in mating. Both Far1 and Ste5 are reasonable candidates 1997). Even Ste7, which encodes the MAPK kinase for such factors. For Far1, its pheromone-induced relo- (MAPKK) of the pheromone-responsive MAPK cascade, calization from the nucleus to the shmoo tip seems to and which is suppressed by yrb1-52, albeit rather weakly Ran-Binding-Protein-1 and Yeast Mating 1103

(Katz et al. 1987), is bound to Ste5, which becomes by a European Molecular Biology Organization Long-Term Fellowship tethered at the shmoo tip via its interaction with G␤␥. and funds provided by the Swiss National Science Foundation (to M. Ku¨nzler), by National Science Foundation Postdoctoral Fellowship Finally, Fus1 is an integral membrane protein localized DMB-8807575 and National Cancer Institute Postdoctoral Traineeship to the tip of the mating projection and is involved in CA09041 (to J. Trueheart), by a Postdoctoral Fellowship from the the cell fusion step of mating (Trueheart and Fink Italian-American Cancer Foundation (to C. Sette), by National Insti- 1989). Indeed, mutational analysis of Fus1 indicates that tutes of Health Research Grant GM21841 (to J. Thorner), and by its large O-glycosylated exocellular domain is dispens- facilities provided by the Berkeley campus Cancer Research La- boratory. able for its function and that an SH3 domain and two potential actin-binding motifs in its relatively short, cytosolic, C-terminal tail are essential for its function, suggesting that the primary role of Fus1 in the mating LITERATURE CITED process is its contribution to modifying the structure Aebi, M., M. W. Clark, U. Vijayraghavan and J. Abelson, 1990 of the cytoskeleton (J. Trueheart and J. Thorner, A yeast mutant, PRP20, altered in mRNA metabolism and maintenance of the nuclear structure, is defective in a gene homologous unpublished results). to the human gene RCC1, which is involved in the control of A primary defect of the analyzed yrb1ts mutants in chromosome condensation. Mol. Gen. Genet. 224: 72–80. Aitchison, J. D., G. Blobel and M. P. Rout, 1996 Kap104p: a nucleocytoplasmic transport would also explain the ob- karyopherin involved in the nuclear transport of messenger RNA served mitotic phenotypes, since similar mitotic distur- binding proteins. Science 274: 624–627. bances have been reported for mutants deficient in Akada, R., L. Kallal, D. I. Johnson and J. Kurjan, 1996 Genetic relationships between the G protein ␤␥ complex, Ste5p, Ste20p other factors involved in nucleocytoplasmic transport, and Cdc42p: investigation of effector roles in the yeast phero- for example, Srp1 (␣-importin; Loeb et al. 1995), and mone response pathway. Genetics 143: 103–117. Cse1 (Xiao et al. 1993), which is required for reexport Alepuz, P. M., D. Matheos, K. W. Cunningham and F. Estruch, 1999 The Saccharomyces cerevisiae RanGTP-binding protein of Srp1 from the nucleus (Ku¨nzler and Hurt 1998). Msn5p is involved in different signal transduction pathways. Ge- Both yrb1-51 and yrb1-52 caused cytoplasmic accumula- netics 153: 1219–1231. tion of two different reporter proteins with the SV40 Amberg, D. C., A. L. Goldstein and C. N. Cole, 1992 Isolation and characterization of RAT1: an essential gene of Saccharomyces NLS and displayed synthetic lethality with two different cerevisiae required for the efficient nucleocytoplasmic trafficking srp1ts mutations, with a rsl1ts mutation (␤-importin), and of mRNA. Genes Dev. 6: 1173–1189. with a cold-sensitive cse1 allele, suggesting that impair- Amberg, D. C., M. Fleischmann, I. Stagljar, C. N. Cole and M. Aebi, 1993 Nuclear PRP20 protein is required for mRNA ex- ment of the import of nuclear proteins with a classical port. EMBO J. 12: 233–241. NLS may explain the mitotic defects observed. Atkinson, N. S., R. W. Dunst and A. K. Hopper, 1985 Characteriza- Although our study cannot rigorously rule out the tion of an essential Saccharomyces cerevisiae gene related to RNA processing: cloning of RNA1 and generation of a new allele with possibility that Ran or RanBP1 may play some role in a novel phenotype. Mol. Cell. Biol. 5: 907–915. the mating pathway independent of their functions in Bailer, S. M., S. Siniossoglou, A. V. Podtelejnikov, A. Hellwig, M. nucleocytoplasmic transport, based on the findings pre- Mann et al., 1998 Nup116p and Nup100p are interchangeable through a conserved motif which constitutes a docking site for sented here, the observed suppression of mating defects the mRNA transport factor Gle2p. EMBO J. 17: 1107–1119. and the defects in mitosis caused by the yrb1-51 and Bardwell, L., J. G. Cook, C. Inouye and J. Thorner, 1994 Signal yrb1-52 mutations are most likely direct consequences propagation and regulation in the mating pheromone pathway of the yeast Saccharomyces cerevisiae. Dev. Biol. 166: 363–379. of impaired import of nuclear proteins. Because of its Bartel, P. L., and S. Fields, 1995 Analyzing protein-protein interac- relatively slow onset, the apparent mRNA export defect tions using the two-hybrid system. Methods Enzymol. 254: 241– 263. manifested by the yrb1-51 allele may reflect an indirect Baümer, M., M. Ku¨nzler, P. Steigemann, G. H. Braus and S. consequence of a primary defect in import. Irniger, 2000 Yeast Ran-binding protein Yrb1p is required for efficient proteolysis of the cell cycle regulatory proteins Pds1p We thank Markus Aebi (Federal Institute of Technology, Zu¨rich, and Sic1p. J. Biol. Chem. 275: 38929–38937. Switzerland), Pierre Belhumeur (McGill University, Montreal), Laura Beddow, A. L., S. A. Richards, N. R. Orem and I. G. Macara, Davis (Brandeis University, Waltham, MA), Gerald Fink (Massachus- 1995 The Ran/TC4 GTPase-binding domain: identification by setts Insitute of Technology, Boston), Molly Fitzgerald-Hayes (Univer- expression cloning and characterization of a conserved sequence sity of Massachussetts, Amherst, MA), Anita Hopper (Pennsylvania motif. Proc. Natl. Acad. Sci. USA 92: 3328–3332. State University, Hershey, PA), Masayasu Nomura (University of Cali- Belhumeur, P., A. Lee, R. Tam, T. DiPaolo, N. Fortin et al., 1993 fornia, Irvine, CA), Pamela Silver (Dana Farber Cancer Center, Bos- GSP1 and GSP2, genetic suppressors of the prp20-1 mutant in Saccharomyces cerevisiae: GTP-binding proteins involved in the ton), Françoise Stutz (Centre Hospitalier Universitaire Vaudois, Lau- maintenance of nuclear organization. Mol. Cell. Biol. 13: 2152– sanne, Switzerland), Alan Tartakoff (Case Western Reserve University, 2161. Cleveland), Linda S. Huang (University of California, San Francisco), Blondel, M., P. M. Alepuz, L. S. Huang, S. Shaham, G. Ammerer and Karsten Weis (University of California, Berkeley, CA) for the et al., 1999 Nuclear export of Far1p in response to pheromones generous gifts of reagents; Stefan Irniger (Georg-August-Universita¨t, requires the export receptor Msn5/Ste21p. Genes Dev. 13: 2284– Go¨ttingen, Germany) and Jeff Flick (Vanderbilt University, Nashville, 2300. TN) for sharing unpublished results; and Stephanie Richards and Ian Boeke, J. D., J. Trueheart, G. Natsoulis and G. R. Fink, 1987 5-Flu- oroorotic acid as a selective agent in yeast molecular genetics. Macara (University of Virginia, Charlottesville, VA) for advice and Methods Enzymol. 154: 164–175. material assistance at the early stages of this work. We are grateful to Booth, J. W., K. D. Belanger, M. I. Sannella and L. I. 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