mutations in the yrb1 gene encoding yeast ran-binding ... · copyright 2001 by the genetics society...

Copyright 2001 by the Genetics Society of America

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

Markus Kunzler,*,† 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, California94720-3202 and †Ruprecht-Karls-Universitat Heidelberg, Biochemie-Zentrum Heidelberg, D-69120 Heidelberg, Germany

Manuscript received July 13, 2000Accepted for publication November 21, 2000

ABSTRACTWe 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. Bothmutations 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 importand one also reduces poly(A)1 RNA export, suggesting a primary defect in nucleocytoplasmic trafficking.Consistent with this conclusion, both yrb1ts mutations display deleterious genetic interactions with mutationsin many other genes involved in nucleocytoplasmic transport, including SRP1 (a-importin) and severalb-importin family members. These yrb1ts alleles were isolated by their ability to suppress two different typesof mating-defective mutants (respectively, fus1D and ste5ts), indicating that reduction in nucleocytoplasmictransport enhances mating proficiency. Indeed, in both yrb1ts mutants, Ste5 (scaffold protein for thepheromone 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 thereceptor for pheromone-stimulated nuclear export of Ste5. Our results suggest that reimport of Ste5 intothe nucleus is important in downregulating mating response.

MATING in the yeast Saccharomyces cerevisiae is the nucleus in response to pheromone, as observed for theregulated import and export of other nuclear proteinsculmination of a complex series of events re-

quired for cellular and nuclear fusion of two haploid (Kaffman and O’Shea 1999).Proteins and protein-RNA complexes cross the nu-cells of opposite mating type (Sprague and Thorner

1992). Mating pheromones (secreted peptides) bind clear envelope through nuclear pores comprised of z50different proteins, termed nucleoporins (Ryan andto G-protein-coupled receptors, stimulating a mitogen-

activated protein kinase (MAPK) cascade (Bardwell et Wente 2000). Nucleocytoplasmic transport also re-quires soluble factors. Transport receptors for both im-al. 1994) that evokes dramatic changes in gene transcrip-

tion, cell cycle arrest, and pronounced alterations of port and export (b-importin and its relatives) bind theircargo and shuttle between the cytosol and the nucleo-cell morphology and nuclear reorganization (Leberer

et al. 1997; Stone et al. 2000). Pheromone-activated and plasm (Gorlich and Kutay 1999). The small Ras-likeGTPase Ran and its associated factors confer directional-pheromone-induced gene products required for cell

fusion are deposited at a localized site on the plasma ity to transport (Macara et al. 2000). In S. cerevisiae,GSP1 and GSP2 encode Ran isoforms (Belhumeur etmembrane at the leading edge of a mating projection

(“shmoo tip”; Madden and Snyder 1998). Proteins re- al. 1993; Kadowaki et al. 1993). Ran exists predomi-nantly in its GTP-bound form in the nucleus; in thequired for nuclear fusion are recruited to the nucleus

(Rose 1996). How the signal initiated at the plasma cytosol, Ran is mainly GDP-bound. This asymmetry isimposed by the subcellular distribution of Ran regula-membrane is transmitted into the nucleus to activate

gene expression is still unclear. Two components of the tors: the Ran-specific guanine-nucleotide exchange fac-tor (RanGEF1), the PRP20/SRM1/MTR1 gene productpathway, Ste5 (Pryciak and Huntress 1998; Mahanty

et al. 1999) and Far1 (Blondel et al. 1999), shuttle in S. cerevisiae, is confined to the nucleus, whereas theRan-specific GTPase-activating protein (RanGAP1), thebetween the nucleus and the cytosol, are predominantly

nuclear in naıve cells, but are rapidly ejected from the RNA1 gene product in S. cerevisiae, is located in thecytoplasm. GSP1, PRP20, and RNA1 are all essentialgenes, and recessive mutations in all three block nuclear

Corresponding author: Markus Kunzler, Ruprecht-Karls-Universitat protein import and poly(A)1 RNA export (CorbettHeidelberg, Biochemie-Zentrum Heidelberg (BZH), Im Neuen- and Silver 1997; Oki et al. 1998).heimer Feld 328, 4. OG, D-69120 Heidelberg, Germany.

Transport receptors bind specifically to the GTP-E-mail: [email protected] 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. Kunzler et al.

termini (Gorlich and Kutay 1999). RanGTP-binding isolated (Katz et al. 1987) as an extragenic suppressorof missense mutations in STE5, which encodes a scaffoldto an export receptor enhances its affinity for an export

substrate; conversely, binding of RanGTP to an import protein for the pheromone-activated MAPK cascade(Elion 1998). As described here, both yrb1 mutationsreceptor prevents the binding of an import substrate.

Hence, high RanGTP in the nuclear compartment po- cause clear defects in nucleocytoplasmic trafficking ofvarious proteins, including Ste5, and are able to sup-tentiates association of export cargo with export recep-

tors and triggers release of import cargo from import press the mating defect of a msn5D mutant. These datasuggest that preventing efficient reimport of Ste5 afterreceptors. Ran-binding-protein-1 (RanBP1) is another

protein that binds specifically to RanGTP (Kunzler et its pheromone-induced release from the nucleus sus-tains the mating-competent state.al. 2000; Plafker and Macara 2000). RanBP1 contains

a conserved Ran-binding domain (RBD) of z140 resi-dues (Beddow et al. 1995; Vetter et al. 1999), which

MATERIALS AND METHODSis necessary and sufficient for high-affinity binding ofRanGTP and for nuclear export of RanBP1, at least in Strains and growth conditions: Yeast strains used in thisyeast (Kunzler et al. 2000). Homologous RBDs are study are listed in Table 1. Strains JTY2483 and JTY2484 werefound in other nuclear proteins, like vertebrate obtained by backcrossing strain 381G-42E-P1 three times

against either YPH499 or YPH500. msn5D::TRP1 strain HMK30RanBP2/NUP358 (Yokoyama et al. 1995) and RanBP3was derived from strain LH90 (Blondel et al. 1999) by three(Mueller et al. 1998), and S. cerevisiae nuclear proteins,consecutive backcrosses against the W303-1A derivatives, CRY1S. cerevisiae Nup2 (Booth et al. 1999) and Yrb2 (Taura 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 methodRan-mediated GTP hydrolysis by RanGAP1; in contrast, (Gietz et al. 1992). The fus1D mutation deletes 90% of the

coding sequence (from the FUS1 promoter to codon 460) andRanBP1 acts as a coactivator of RanGAP1-stimulatedwas constructed by a two-step gene disruption method (BoekeGTP hydrolysis by Ran and, moreover, is required foret al. 1987). Heterozygous diploid strain JTY2501 was derivednucleotide hydrolysis when RanGTP is bound to a trans- from CRY3 by transplacing the YRB1 locus on one homolog

port receptor (Gorlich 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 yrb1D::HIS3 construct excised from plas-

mid pMK112n (Table 2). To construct strain HMK21, JTY2501dant, shuttles between the nucleus and the cytoplasm,was transformed with plasmid pMK103, sporulated, and abut is found almost exclusively in the cytosol at steadyMATa His1 Ura1 5-fluoro-orotic acid (5-FOA)-sensitive sporestate (Kunzler et al. 2000; Plafker and Macara 2000), 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::HIS3both in recycling of transport receptors and in release construct excised from plasmid pJON115 (Loeb et al. 1993).

Strain HMK29 was constructed analogously using a BamHI-of export cargo (Peterson et al. 2000). Consistent withHindIII fragment containing a gsp2D::LEU2 construct (Kado-this view, S. cerevisiae YRB1, encoding yeast RanBP1, iswaki et al. 1993). Correct transplacements were verified byessential for cell viability and is required for both nu- Southern hybridization analysis.

clear protein import and poly(A)1 RNA export Unless indicated otherwise, yeast cells were propagated at(Schlenstedt et al. 1995). 308. Rich medium (YP), synthetic complete medium (SC), and

synthetic minimal medium (SM) were prepared as describedA link between yeast mating and the Ran GTPase(Kaiser et al. 1994). Glucose (Glc) or raffinose (Raf) werecycle was the identification of the srm1-1 mutation, nowadded as carbon source at a final concentration of 20 g/literknown to reside in RanGEF1, which suppressed the 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-grownincreased the basal expression of a pheromone-respon- cells. Drop-out media (SC lacking the appropriate nutrients)

were used to maintain selection for plasmids. Agar plates con-sive reporter gene (Clark and Sprague 1989). Anothertaining 5-FOA were prepared as described by Boeke et al.connection between mating and nucleocytoplasmic(1987). Escherichia coli strain DH5a (Hanahan 1983) was usedtransport was the finding that the ste21 mutation, identi- 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

performed as previously described (Sprague 1991). Briefly,the b-subunit of the pheromone receptor-coupled het-MATa strains to be tested and MATa tester strains were pre-erotrimeric G-protein; Akada et al. 1996), resides ingrown at 268 to midlogarithmic phase in selective and richMSN5, encoding the nuclear receptor for pheromone- 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 MATa tester strain. In the case of the experimentshown in Table 3, the mixture was spread directly onto pre-lated a yrb1ts mutation as a suppressor of a mutantcooled (148) SMGlc plates, the plates were incubated for 3( fus1D) defective in the cell fusion step of mating. Fus1days at 148, and then for 3 days at room temperature. Theis a pheromone-induced, O-glycosylated, integral mem-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

and incubating for 3 days at room temperature). In the casemutation, we found that it was allelic to a mutation

1091Ran-Binding-Protein-1 and Yeast Mating

TABLE 1

Yeast strains

Strain Characteristics Source

YPH499 MATa ura3-52 trp1-D63 his3-D200 leu2-D1 lys2-801am ade2-101oc Sikorski and Hieter (1989)YPH500 MATa ura3-52 trp1-D63 his3-D200 leu2-D1 lys2-801am ade2-101oc Sikorski and Hieter (1989)YPH501 MATa/MATa (YPH499 3 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. FullerCRY2 (W303-1B) MATa ura3-1 trp1-1 his3-11,15 leu2-3,112 ade2-1 can1-100 GAL R. S. FullerCRY3 (W303D) MATa/MATa (CRY1 3 CRY2) R. S. Fuller9399-7B MATa ura3-52 his4-D29 GAL Trueheart and Fink (1989)JY416 MATa ura3-52 leu2-3,112 fus1D This studyJTY2023 MATa ura3-52 trp1-D63 his4D29 ade2-101oc GAL This studyJTY2024 JTY2023 fus1D This studyJTY2025 JTY2024 yrb1-51 (sfo1-1) This studyJTY2488 MATa ura3-52 trp1-D63 his4-D29 fus1D yrb1-51 This studyJTY2501 CRY3 yrb1D::HIS3/YRB1 This studyHMK21 CRY1 yrb1D::HIS3 (pMK103) This studyJTY2026 MATa ura3-52 trp1-D63 his3-D200 leu2-D1 ade2-101oc yrb1-51 This studyJTY2027 MATa ura3-52 trp1-D63 his3-D200 leu2-D1 lys2-801am yrb1-51 This studyJTY2482 MATa/MATa (JTY2026 3 JTY2027) This study381G-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-D63 his3-D200 leu2-D1 lys2-801am ade2-101oc yrb1-52 This studyJTY2484 MATa ura3-52 trp1-D63 his3-D200 leu2-D1 lys2-801am ade2-101oc yrb1-52 This studyJTY2485 MATa/MATa (JTY2483 3 JTY2484) This studyJTY2500 MATa ura3-52 his3-D200 leu2-3,112 trp1-901 canR gal4-542 gal80-538 Inouye et al. (1997a)

ADE2::PGAL-URA3 LYS2::lexop-lacZJTY2486 MATa ura3-1 trp1-1 his3-11,15 leu2-3,112 ade2-1 can1-100 GAL nup2-5::HIS3 This studyHMK29 CRY1 gsp2D::LEU2 This studyHMK30 CRY1 msn5D::TRP1 This studyDC17 MATa 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(see below) revealed that it is identical to the YRB1 gene, andcollected on a 0.45-mm pore filter and incubated for 6 hr at

308 on YPGlc. After the incubation, cells were resuspended because sequence analysis (see below) demonstrated that boththe sfo1-1 and stp52 mutations reside in the YRB1 locus, thesein SMGlc medium and plated in appropriate dilutions onto

SMGlc plates with appropriate nutrients to select for diploids. alleles were renamed yrb1-51 and yrb1-52, respectively.Recovery and analysis of yrb1ts alleles: The base sequenceAs a control for the number of viable MATa cells used in the

mating mixture, 106 cells of the MATa cells were collected on alterations corresponding to the yrb1-51 and yrb1-52 mutationswere determined by cloning and sequencing of DNA isolateda separate filter, incubated as above, resuspended in YPGlc,

and plated on YPGlc plates at appropriate dilutions. Mating from the mutants. The polymerase chain reaction (PCR) wasused to amplify 636-bp products comprising the entire YRB1efficiency was expressed as percentage of the input MATa

haploids that formed diploid colonies. open reading frame (ORF) using genomic DNA from JTY2026(yrb1-51) and 381G-42E-P1 (yrb1-52) as the template and oligo-Isolation of yrb1-51: JTY2024 (MATa fus1D) was mutagen-

ized with ethyl methanesulfonate (108 cells/ml; 3% EMS; 1 hr) nucleotide primers, 59-GGG GAT CCG AAT GTC TAG CGAAGA TAA G-39 (OSFO1) and 59-GGT CTA GAC GCA AGTto 25% survival, and spread on YPGlc plates. After 3 days at

288, z72,000 colonies were replica plated onto precooled AAC AAG C-39 (OSFO5), which corresponded, respectively,to positions 22 to 118 and 1635 to 1616 of the 201-codon(148) SMGlc plates containing uracil (20 mg/liter), on which

2 A600 nm units of JY416 (MATa fus1D) cells had been spread. YRB1 sequence (where 11 is the first base of the initiatorcodon of the ORF) and included restriction sites at theirThese plates were incubated for 4 days at 148. Candidate clones

that gave a positive mating response (35 colonies total) were 59-ends to facilitate cloning of the PCR products. Reactionproducts were isolated, digested with BamHI and XbaI, andrestreaked from the master plate, retested for suppression,

and examined for their ability to grow at various temperatures. inserted into E. coli vector pUC19 for sequencing. Nucleotidesequence of multiple inserts was determined on both strandsA single isolate (JTY2025) displayed a ts phenotype that coseg-

regated with the ability to suppress the mating defect of the using the M13/pUC universal and M13/pUC reverse sequenc-ing primers (New England Biolabs, Beverly, MA) and, whenfus1D cells at 148 (data not shown). The mutation conferring

these phenotypes was initially named sfo1-1 (suppressor of fus necessary, sequence-specific primers. The single-base-pair mu-tations recovered were tested for their ability to confer a tsone). In the course of these crosses, it was shown, first, that

sfo1 was tightly linked to trp1 (no recombinants in 31 tetrads; phenotype by first substituting the mutant YRB1 ORFs (excisedas SalI fragments from the pUC19 derivatives) for the corre-distance #1.6 cM) and, second, by complementation tests,

that the sfo1-1 mutation was allelic to stp52, another suppressor sponding segment in pMK103 and then introducing the entireyrb1-51 and yrb1-52 genes as EcoRI-XbaI fragments (excisedof mating defects that was mapped to the same region (Katz

et al. 1987). Because cloning of the corresponding wild-type from the pMK103 derivatives) into pRS314, yielding the TRP1-


TABLE 2

Plasmids

Plasmid Characteristics Source

YEp24 2-mm URA3 Botstein et al. (1979)pRS314 CEN ARS TRP1 Sikorski and Hieter (1989)pRS424 2-mm TRP1 Sikorski and Hieter (1989)pRS425 2-mm LEU2 Sikorski and Hieter (1989)pUN100 CEN ARS LEU2 Elledge and Davis (1988)YEp352 2-mm URA3 Hill et al. (1986)pGAD424 2-mm LEU2 ADH1p-GAL4TAD-MCS-ADH1t Bartel and Fields (1995)pBTM116 2-mm 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-mm URA3 ADH1p-SV40NLS-GFP-lacZ Lee et al. (1996)pPS817 2-mm URA3 GAL1p-SV40NLS-GFP-lacZ Lee et al. (1996)pGADGFP 2-mm LEU2 ADH1p-SV40NLS-GAL4TAD-GFP Shulga et al. (1996)pNOPGFPAU-NPL3 2-mm ADE2 URA3 NOP1p-GFP-NPL3 Senger et al. (1998)YEplac195AU-L25NLS-GFP 2-mm ADE2 URA3 RPL25NLS-GFP-MEX67t O. Gadal and E. Hurt

(unpublished results)pKW430 2-mm URA3 ADH1p-SV40NLS-PKINES-(GFP)2 Stade et al. (1997)pLDB419 2-mm LEU2 YAP1-GFP Yan et al. (1998)

pNOPGFP2L-STE5 This studypSB415 YEp24-NTH1-YRB1 This studypMK102 pUC19-YRB1 This studypMK103 YEp352-YRB1 This studyPMK104 pRSETA-YRB1 This studypMK112n pRS316-yrb1D::HIS3 This studypNOPPATA-GSP1G21V pUN100-NOP1p-ProtA-TEV-GSP1(G21V)-ADH1t Hellmuth et al. (1998)pMK275 pRS314-YRB1 This studypMK277 pRS314-yrb1-51 This studypMK278 pRS314-yrb1-52 This studypMK284n pRS314-YRB1-GFP(S65T) Hellmuth et al. (1998)pMK294-51 pRS314-yrb1-51-GFP(S65T) This studypMK294-52 pRS314-yrb1-52-GFP(S65T) This studypMK291-wt pRS424-YRB1-GFP(S65T) This studypMK291-51 pRS424-yrb1-51-GFP(S65T) This studypMK291-52 pRS424-yrb1-52-GFP(S65T) This studypMK199-wt pGAD424-YRB1 This studypMK199-51 pGAD424-yrb1-51 This studypMK199-52 pGAD424-yrb1-52 This studypMK195-GV pBTM116-GSP1(G21V) This study

marked plasmids pMK277 and pMK278, respectively. Finally, complement the ts phenotype of the yrb1-51 mutant. Thesmallest original isolate (pSB415) contained the YRB1 geneHMK21 (yrb1D [pMK103]) was transformed with either

pMK277, pMK278, or a control plasmid (pMK275) carrying as well as the neighboring gene NTH1 encoding neutral treha-lase. Subsequent subcloning localized the complementing ac-the normal YRB1 gene, plated on 5-FOA plates at 238 to select

against the resident URA3-marked YRB1-containing plasmid tivity to a 1.3-kb chromosomal EcoRI-XbaI fragment containingonly YRB1, which was used to construct pMK102, pMK103,(pMK103), and the resulting isolates were analyzed for their

ability to grow at elevated temperature. and pMK275.To construct a plasmid (pMK112n) carrying the yrb1D::HIS3Construction of plasmids: Standard techniques were used

for the manipulation of recombinant DNA (Sambrook et al. deletion construct, an internal BglII fragment was excisedfrom the YRB1-containing insert in pMK101 and replaced by1989). Plasmid DNA from E. coli was isolated according to

Del Sal et al. (1988). Unless specified otherwise, PCR amplifi- a BamHI fragment containing the HIS3 gene, which was in-serted in the same transcriptional orientation as YRB1. Con-cations were performed using Vent DNA polymerase (New

England Biolabs). Correct sequence of PCR-generated construction of plasmid pNOPPATA-GSP1G21V, which expressesa GTPase-defective mutant form of Gsp1, Gsp1(G21V), fusedstructs was verified by nucleotide sequence analysis. Plasmids

used in this study are listed in Table 2. at its N terminus to a cleavage site (ENLYEQG) for tobaccoetch virus (TEV) protease and to two immunoglobulin G (IgG)The YRB1 gene was isolated from a yeast genomic library

(Carlson and Botstein 1982) carried in a high-copy-number binding domains of Protein A (ProtA), under control of theNOP1 promoter and the ADH1 terminator, has been describedyeast/E. coli shuttle vector (YEp24) by virtue of its ability to


erated STE5 NcoI-BamHI fragment comprising the entireTABLE 3ORF (using primers OSTE5-1, 59-GGGGGGCCATGGGTAT

The yrb1-51 (sfo1-1) mutation suppresses the cold-sensitive GGAAACTCCTACAGAC-39, and OSTE5-2, 59-GGGGGGATmating defect of a fus1D mutant CCCTATATATAATCCATATGG-39) into pNOPPATA (Hell-

muth et al. 1998) and subsequent recloning of the insert asPstI fragment into pNOPGFP2L. This vector is based onPlasmid: mating efficiency (31025)a

pRS425 and contains a 1.4-kb BamHI-PstI NOP1p-GFP cassetteMATa strainb YEp24 YEp24-YRB1 (pSB415) (K. Hellmuth and E. Hurt, unpublished results).

Preparation of rabbit polyclonal anti-Yrb1 antiserum: ToFUS1 YRB1 250 165 generate a (His)6-Yrb1 fusion protein containing all but thefus1D YRB1 [1.0] 0.6 first 10 residues of Yrb1, the corresponding YRB1 coding se-fus1D yrb1-51 (sfo1-1) 39 1.8 quence was excised as a SalI fragment from pMK102 and

ligated into the XhoI site of pRSETA (Invitrogen), yieldinga Mating efficiency is defined as the number of diploidspMK104. For expression in E. coli, strain BL21(DE3)/pLysSformed per number of input haploids of the strain tested.(Studier 1991) was transformed with pMK104 and the fusionThe values given represent the average of three independentprotein was induced by addition of isopropyl-b-d-thio-galacto-trials, each performed in triplicate, and have been normalizedpyranoside (IPTG) to a final concentration of 0.4 mm followedto the mating efficiency of the fus1D mutant.by incubation at 378 for 2 hr. Selection for pMK104 had tob The indicated strains [JTY2023, MATa FUS1 YRB1;be maintained by adding 50 mg/liter carbenicillin (Sigma, St.JTY2024, MATa fus1D YRB1; JTY2025, MATa fus1D yrb1-51Louis), a more stable derivative of ampicillin, to the medium(sfo1-1)] were transformed with either YEp24 (a URA3-markedbecause the fusion protein was relatively toxic to the cells.2-mm DNA vector) or pSB415 (YEp24-YRB1) and mated withThe fusion protein was purified from E. coli using Ni21-chelateJY416 (MATa fus1D), as described in materials and methods.affinity chromatography (Ni-NTA resin; QIAGEN, Chats-worth, CA), according to the manufacturer’s recommenda-tions. The purified protein was used to raise polyclonal anti-previously (Hellmuth et al. 1998). Plasmids pMK294-51 andsera in two adult female New Zealand White rabbits (nos. 1390pMK294-52, and pMK291-wt, pMK291-51, and pMK291-52, ex-and 1391), following standard protocols (Harlow and Lanepressing Yrb1-green fluorescent protein (GFP) fusions under1988), using 600 mg of protein in 50% Titermax (CytRx, Nor-control of the authentic YRB1 promoter, were constructedcross, GA) for the first immunization and 400 mg of proteinby replacing an internal BglII fragment in the YRB1 codingin 50% incomplete Freund’s adjuvant (Sigma) for each of twosequence in pMK284n, which expresses a functional Yrb1-subsequent immunizations administered after 3 and 5 weeks,GFP chimera (Hellmuth et al. 1998), with the correspondingrespectively. Bleeds were taken after 4 weeks (2 ml), 6 weeksfragments from yrb1-51 and yrb1-52, followed by subsequent(50 ml), 7 weeks (2 ml), and 8 weeks (terminal) and storedrecloning of the respective YRB1-GFP gene fusions as EcoRI-in 0.02% sodium azide at 2708. For detection of Yrb1 byNotI fragments into pRS424.immunoblotting the resulting antisera (nos. 1390 and 1391)Fusions of full-length Yrb1 to the Gal4 transcriptional activa-were used as primary antibodies at a dilution of 1:5000.tion domain (TAD) and full-length Gsp1(G21V) to the E. coli

Two-hybrid assay: To assess interactions between LexALexA DNA-binding domain (DBD) were generated via PCR,(DBD)-Gsp1(G21V) and Gal4(TAD)-Yrb1 fusion proteins,using the two-hybrid vectors pGAD424 and pBTM116, respec-strain JTY2500 harboring the E. coli lacZ gene under controltively. Fragments comprising the entire YRB1 ORF were syn-of eight LexA-binding sites was cotransformed with the appro-thesized using 59-CCG AAT TCG GTC CAG GTG GTA GCGpriate pBTM116- and pGAD424-based plasmids. Transfor-AAG ATA AGA AAC CTG TCG-39 (OSFO15) and the M13/mants were grown in SCGlc medium lacking leucine and tryp-pUC reverse sequencing primer (New England Biolabs) astophan to midexponential phase (A546 nm 5 z1) and assayedthe primers and pUC19 carrying the chromosomal YRB1-con-for b-galactosidase acitivity as described previously (Kunzlertaining EcoRI-XbaI fragment (pMK102), or pUC19 carryingand Hurt 1998).the corresponding fragments from the yrb1-51 or yrb1-52 ORFs,

Preparation of yeast cell extracts: Yeast cells were washedas templates. The PCR products were digested with EcoRI andonce with one culture volume of cold phosphate-bufferedPstI and inserted into the corresponding sites in pGAD424,saline (PBS), aliquoted into 1.5-ml microcentrifuge tubesyielding pMK199-wt, pMK199-51, and pMK199-52, respec-(z20 A546 nm units per tube), and stored as pellets at 2708.tively. Similarly, a fragment comprising the entire ORF codingFrozen cell pellets were thawed by adding 0.2 ml cold lysisfor Gsp1(G21V) was produced using 59-GCG AGG CCT TGCbuffer (50 mm Tris-HCl pH 7.5, 150 mm NaCl, 20 mm MgCl2,CCC AGC TGC TAA CGG TGA AG-39 (OGSP7) and RSET10% glycerol, 2 mm DTT, and 1 mm PMSF) and lysed by(59-AAC TGC AGC CAA CTC AGC TTC C-39) as the primers,vigorous vortexing with 0.2 g of acid-washed glass beads (0.45–and E. coli expression vector pRSETB (Invitrogen, Carlsbad,0.6 mm diameter) for six 30-sec periods (separated by 1-minCA) carrying a PCR-mutated genomic PvuII-HindIII fragmentperiods of cooling on ice). The lysate was clarified by centrifu-coding for Gsp1(G21V) as the template. The resulting PCRgation for 5 min at 13,000 3 g at 48 and the protein concentra-product was cleaved with StuI and PstI and inserted into thetion was determined by a dye-binding method (BradfordSmaI and PstI sites of pBTM116, yielding plasmid pMK195-GV.1976) using commercially available reagents (Bio-Rad, Her-Plasmid YEplac195-AU-L25NLS-GFP was derived from YE-cules, CA) and bovine serum albumin (BSA) as the standard.plac195-ADE2-URA3-L25-GFP (Hurt et al. 1998) by removing

Purification of ProtA-TEV-Gsp1(G21V) from yeast: Trans-most of the RPL25 coding sequence, except for the 59-endformants of wild-type strain CRY1, coexpressing ProtA-TEV-that encodes the first 52 residues of L25 (and contains anGsp1(G21V) from pNOPPATA-GSP1G21V and either Yrb1-intron), using a two-step PCR procedure (Giebel and SpritzGFP, Yrb1(A91D)-GFP, or Yrb1(R127K)-GFP from plasmids1990; mutagenic primer, 59-GGG ACA ACT CCA GTG AAApMK284n, pMK294-51, or pMK294-52, respectively, wereAGT CTT CTC TTT GCT CTC GAG TGG AAC AGC CTTgrown in selective medium at 268 to a A546 nm 5 z1.5. Purifica-GGA AGC-39; O. Gadal and E. Hurt, unpublished results).tion of Gsp1(G21V) from these cells was performed essentiallyPlasmid pNOPGFP2L-STE5 expressing a GFP-Ste5 fusion pro-as described (Hellmuth et al. 1998), with the minor modifica-tein from a multicopy-plasmid under control of the consti-

tutive NOP1-promoter was constructed by inserting a PCR-gen- tion that universal buffer (Kunzler and Hurt 1998) was used


throughout the purification, including cell lysis, washing steps, recessive allele of YRB1, which encodes the homolog ofand elution. Elution by cleavage with TEV-protease (GIBCO- mammalian RanBP1 (Ouspenski et al. 1995; Schlen-BRL, Gaithersburg, MD) was performed by incubation for

stedt et al. 1995). Hence, sfo1-1 was redesignated yrb1-51.1 hr at room temperature.A recessive ts mutation, stp52 (sterile pseudorever-Nuclear protein import and RNA export assays: Tempera-

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 MATatransport substrates fused to GFP(S65T), namely SV40NLS-Gal4- ste5-3 3 MATa ste5-3 cross at restrictive temperature(TAD)-GFP (pGADGFP), GFP-Npl3 (pNOPGFPAU-NPL3),

(Katz et al. 1987). The stp52 mutation also suppressedand L25NLS-GFP (YEp195-AU-L25NLS-GFP), were cultivated toother ste5, ste4, and ste7 missense (ts) alleles (Katz etearly exponential phase (A546 nm 5 z0.5) in selective SCGlc

medium at 238, split into two equal portions, and incubated al. 1987). Although the linkage analysis reported byat either 238 or 378 for various periods of time. Strains carrying Katz et al. (1987) assigned the stp52 mutation to theplasmids expressing SV40NLS-GFP-b-galactosidase (pPS817) opposite side of the TRP1 locus from yrb1-51, we foundunder control of the GAL1 promoter were pregrown to early

that a yrb1-51/stp52 diploid strain was still ts, and that theexponential phase (A546 nm 5 z0.5) in selective SCRaf mediumts growth defect of the stp52 mutant could be completelyat 238 before Gal (2%) was added to the cultures and the

cells were incubated at 238 for another hour (to allow mRNA rescued by the cloned YRB1 gene on a plasmid (datasynthesis and export). The induced cultures were split into not shown). These results demonstrated that the stp52two equal portions, and one portion was shifted to 378 for mutation was another recessive allele of YRB1, as was3 hr, while the other portion was maintained at 238 for the

confirmed by sequencing of the mutant DNA (see be-same period. Fluorescence microscopy of living yeast cellslow). Hence, stp52 was redesignated yrb1-52.expressing GFP fusion proteins was done according to Hell-

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 YRB1any 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

first, the physiology of the yrb1 mutants. At 238, yrb1-51constitutively expressed GFP fusion proteins. Poly(A)1 RNAmutant cells grew nearly as well as wild-type cells,was localized by in situ hybridization as described previously

(Segref et al. 1997). whereas the yrb1-52 mutant cells displayed impairedMiscellaneous: SDS-PAGE and immunoblotting were con- growth already under these conditions; both yrb1-51 and

ducted as described previously (Kunzler and Hurt 1998). yrb1-52 cells ceased growth and lost viability within 3–6Multiple sequence alignment was done using the CLUSTALW

hr after shift to 378 (data not shown). Similar results1.7 (Thompson et al. 1994) and BOXSHADE 3.21 [Bioinfor-were observed for the corresponding homozygous dip-matics group of the Swiss Institute for Experimental Cancer

Research (ISREC)] programs. Identities to the Yrb1 sequence loids (data not shown). As judged by immunoblottingwere calculated on the basis of pairwise alignments using the of cell lysates (Figure 1A), after shift to 378 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 stableeven 6 hr after temperature shift. Thus, the yrb1-51 muta-tion appears to destabilize the gene product at higherRESULTStemperature, whereas the yrb1-52 product is stable un-

Isolation of yrb1ts mutations as suppressors of mating der the same conditions. Upon prolonged incubationdefects: The mating deficiency of a fus1D mutant is at 378, an apparent degradation product of Yrb1 accu-much more pronounced at 148 than at 308. At 148, dip- mulated in yrb1-52 cells, but was also observed in theloid formation in a MATa fus1D 3 MATa fus1D cross wild-type control cells. Yrb1 was expressed at similaris ,0.5% that of a MATa FUS1 3 MATa fus1D cross levels in MATa, MATa, and MATa/MATa cells (data(Table 3). A screen for extragenic suppressors of this not shown) and its level in MATa cells was not elevated inmating defect (see materials and methods) yielded response to treatment with a-factor mating pheromonea single mutation, sfo1-1. This suppressor mutation re- (data not shown).producibly enhanced mating competence of a fus1D Examination of the cell morphology revealed thatmutant 30–50-fold (Table 3), but did not fully restore haploid yrb1-51 cells arrested mostly as enlarged cellsmating proficiency to the level of a FUS1 cell. The sup- with a large bud or as large unbudded cells upon shiftpressor segregated 2:2 through two backcrosses against to 378 (data not shown; Baumer et al. 2000), which isa fus1D strain and cosegregated with a recessive ts growth reminiscent of cell cycle progression mutants. Similardefect (in .15 tetrads analyzed per cross). Genetic map- results were previously observed for stp52/yrb1-52 cellsping of the mutation to the right arm of chromosome (Katz et al. 1987; Ouspenski 1998). Another strikingIV between CEN4 and the TRP1 gene (data not shown), phenotype of both yrb1ts alleles was the appearance ofcomplementation of the ts growth defect by the wild- chains of elongated nonseparated cells, most evident intype 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 (308; Figure 1B). Such cell elongationYRB1 (Table 3), and nucleotide sequencing of the mu- is diagnostic of mutations that delay G2-M progression

(Lew 2000). We observed a similar morphological de-tant DNA (see below) all established that sfo1-1 was a


nonical cell division cycle (cdc) mutations, and mightindicate a role of Yrb1 at multiple stages of the cellcycle. Consistent with such a notion, the yrb1-51 muta-tion interferes with both the G1/S transition and thepassage through mitosis (Baumer et al. 2000), and dis-played synthetic growth defects when combined withtwo different cdc28 alleles (cdc28-4 and cdc28-1N) thatare diagnostic for different cell cycle stages (G1 andG2/M, respectively; Table 4).

Yrb1-51 and Yrb1-52 are altered in conserved residuesof the Ran-binding domain and defective for Ran-bind-ing in vivo: To determine the nature of the alterationsin the mutant proteins, PCR was used to recover theYRB1 coding sequences from the mutant strains (seematerials and methods). The DNA sequence of eachmutant ORF contained a single point mutation, bothof 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 11 isthe first base of the initiator ATG), which substitutesAsp for Ala at codon 91 (A91D). The yrb1-52 allele is aG-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 thecrystal structure of the first RBD (RanBD1) in mamma-lian 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 hydrophobicnormal and mutant Yrb1 at restrictive temperature. Haploid core of Yrb1 with a bulkier, charged residue (Figurestrain YPH499 (YRB1) and its congenic derivatives, JTY2026

2B). This change should destabilize the global fold of(yrb1-51) and JTY2483 (yrb1-52), were grown at 238 in SCGlcYrb1, consistent with the rapid degradation of this mu-medium to midexponential phase, shifted to 378, and samples

were withdrawn at the indicated times. Total protein was ex- tant protein observed at restrictive temperature (Figuretracted from each sample and analyzed by SDS-PAGE and 1A). Two other existent yrb1 alleles, yrb1-1 and yrb1-2immunoblotting using a rabbit polyclonal anti-Yrb1 antiserum (Schlenstedt et al. 1995), alter residues (F187 and L93,(no. 1390). A band that cross-reacts nonspecifically with the

respectively) that project into the same hydrophobicanti-Yrb1 antiserum served as a loading control. The asteriskpocket as A91. In contrast, R127K makes a seeminglyindicates a major degradation product of Yrb1. (B) Morphol-

ogy of homozygous diploid yrb1ts cells. Strains YPH501 (YRB1/ modest change in a surface-exposed residue that formsYRB1), JTY2482 (yrb1-51/yrb1-51), and JTY2485 (yrb1-52/yrb1- a bridge to residues in the long C-terminal “arm” of52) were cultivated on YPGlc plates at 168 or 308, as indicated, Ran that embraces the RBD (Figure 2B), an alterationand viewed by Nomarski optics.

unlikely to disrupt the overall structure, consistent withthe observed stability of the mutant protein at restrictivetemperature (Figure 1A). We confirmed that each muta-fect in srp1-31ts/srp1-31ts diploids (data not shown).

Srp1/a-importin is the adaptor necessary for recogni- tion was both necessary and sufficient to confer the tsphenotype of the corresponding allele by inserting eachtion and nuclear import of proteins that contain a clas-

sical nuclear localization signal (NLS) by the Kap95/ mutant DNA into a plasmid and introducing it into ayrb1D background (see materials and methods).b-importin receptor (Enenkel et al. 1995). At 378, srp1-

31 cells are impaired in import of NLS-containing re- Two independent approaches demonstrated that theyrb1-51 and yrb1-52 mutations interfere with Yrb1-Ranporter proteins and arrest uniformly as large-budded

cells indicative of a defect in mitosis (Loeb et al. 1995). (Gsp1) interaction in vivo. The GTPase-deficient formof Gsp1, Gsp1(G21V), binds more strongly to Yrb1 thanCorrespondingly, degradation of Clb2, whose destruc-

tion is required for exit from mitosis, is impaired in normal Gsp1 (Schlenstedt et al. 1995); hence, we usedGsp1(G21V) in our analyses. First, we applied the two-srp1-31 cells (Loeb et al. 1995); likewise, degradation of

Clb2 and of two anaphase inhibitors, Pds1 and Sic1, is hybrid method using full-length wild-type Yrb1, Yrb1(A91D), or Yrb1(R127K) fused to the Gal4 transcrip-also impaired in yrb1-51 cells (Baumer et al. 2000). De-

spite these similarities, the absence of a uniform cell tional activation domain [Gal4(TAD)] and full-lengthGsp1(G21V) fused to the LexA DNA-binding domaincycle arrest phenotype distinguishes the yrb1-51 and yrb1-

52 mutations from the srp1-31 mutation and from ca- [LexA(DBD)] in a reporter strain carrying a chromo-


TABLE 4

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

Mutation Source Genetic interactiona

Ran GTPase cyclerna1-1 Atkinson et al. (1985) 11 (sl)prp20-1 Aebi et al. (1990) 1 (1)srm1-1 Clark and Sprague (1989) 1 (nd)prp20-10 Fleischmann et al. (1996) 2 (nd)gsp1-1, -2 Wong et al. (1997) 11 (nd)gsp2::HIS3 Kadowaki et al. (1993) 1 (nd)yrb2::HIS3 I. Macara (personal communication) 2 (sl)nup2::HIS3 Loeb et al. (1993) 1 (sl)

Nucleoporinsnsp1ts Nehrbass et al. (1993) 1 (nd)nup133::HIS3 Doye et al. (1994) 1 (1)nup116::URA3 Bailer et al. (1998) 1 (1)

Nuclear import receptorssrp1-31 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) 1 (nd)yrb4::HIS3 Schlenstedt et al. (1997) 2 (nd)pse1-1 yrb4::HIS3 Seedorf and Silver (1997) sl (nd)

Nuclear export receptorslos1::HIS3 Hellmuth et al. (1998) 2 (nd)msn5::TRP1 Blondel et al. (1999) sup (sup)cse1-1 Xiao et al. (1993) sl (nd)xpo1-1 Stade et al. (1997) 1 (sl)crm1-1, -2, -3 Yan et al. (1998); F. Stutz (personal communication) 2 (nd)

Otherscdc28-4 Reed (1980) 1 (nd)cdc28-1N Piggott et al. (1982) 1 (nd)rat1-1 Amberg et al. (1992) 2 (nd)nsr1::URA3 Kondo and Inouye (1992) 2 (nd)plc1::HIS3 Flick and Thorner (1993) 1 (nd)

Abbreviations in parentheses indicate phenotype observed with the yrb1-52 allele.a 2, no synthetic growth phenotype; 1, 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(see materials and methods), and analyzed by SDS-eight LexA-operator sites. In this system, Yrb1(A91D)

showed a reproducible reduction (.3-fold) and Yrb1 PAGE, Coomassie blue staining, and immunoblottingusing a polyclonal anti-Yrb1 antisera (no. 1391). Exami-(R127K) showed a dramatic reduction (.50-fold) in

interaction with Gsp1(G21V) compared to wild-type nation of the input material (Figure 3B, load), and flow-through fractions (data not shown), demonstrated thatYrb1 (Figure 3A). Immunoblotting with anti-Yrb1 antise-

rum (see materials and methods) and anti-Gsp1 anti- expression of normal and mutant Yrb1-GFP fusions wascomparable, as were their stabilities during purification.bodies (gift of P. Belhumeur) showed that all constructs

were expressed at equivalent levels (data not shown). No detectable Yrb1(R127K)-GFP copurified with Gsp1(G21V), whereas significant amounts of both wild-typeThese results were confirmed by a biochemical pro-

cedure (Figure 3B). Yrb1-GFP, Yrb1(A91D)-GFP, or Yrb1-GFP and endogenous Yrb1 were retained by thesame beads (Figure 3B). A detectable amount of Yrb1Yrb1(R127K)-GFP, produced from the authentic YRB1

promoter on CEN plasmids, were expressed in wild-type (A91D)-GFP copurified with ProtA-Gsp1(G21V), but itslevel was markedly less than the amount of wild-typecells (strain CRY1) also producing a ProtA-(TEV site)-

Gsp1(G21V) from the constitutive NOP1 promoter on Yrb1-GFP retained under the same conditions (Figure 3B).yrb1-51 and yrb1-52 mutants are defective in nucleara CEN plasmid. After growth at 268, protein complexes

bound to bead-immobilized ProtA-(TEV site)-Gsp1(G21V) protein and RNA transport: To determine if yrb1-51 andyrb1-52 cause defects in nuclear protein import and RNAwere recovered from cell extracts as described in Hell-


Figure 2.—Positions of the altered residues in Yrb1 re-sulting from the yrb1-51 and yrb1-52 mutations. (A) Alignmentof various Ran-binding domains (RBDs). Sequences shownare grouped into three subfamilies (RanBP1, RanBP2, andRanBP3), on the basis of certain shared sequence characteris-tics, and include the following (with GenBank accession num-bers): S. cerevisiae Yrb1 (L38489), Schizosaccharomyces pombeSbp1 (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. cerevisiaeNup2 (X69964), S. pombe Hba1 (U38783), and human RanBP3(Y08697). Sequence of S. cerevisiae Yrb2/Nup36 is from theSwiss Protein Database (accession no. P40517). The mouseand bovine RanBP2 are incomplete because they are derivedfrom partial cDNA clones. An insert of 24 residues (possiblyan intron) was omitted from the actual C. elegans Ranup96sequence to optimize its alignment to the other RBDs. Identi-ties shared by 11 (or more) of the RBDs shown are indicatedby white-on-black letters; chemically similar residues are shownas 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 onthe first RBD (RanBD1) in human NUP358 complexed withRan 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, oneof which was maintained at 238 and the other shiftedleles (Schlenstedt et al. 1995), we first examined the

distribution of poly(A)1-RNA by in situ hybridization. to 378. Samples were withdrawn at various times foranalysis. Results were more readily visualized in diploidMutant or wild-type control cells were grown to midex-

ponential phase at permissive temperature (238), and cells because of their larger size; however, similar find-


ings were made with haploid cells (data not shown). Inhomozygous yrb1-51/yrb1-51 diploids shifted to 378 for2 hr, there was a rapid and clear-cut nuclear accumula-tion of poly(A)1-RNA in every cell (Figure 4A); thiseffect was readily apparent in z50% of the cells even1 hr after the shift (data not shown). Poly(A)1 RNAaccumulated in the nucleus in a distinctly punctate pat-tern, a feature seen in mutants that have a strong RNAexport 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 mutantsand nuclear RNA accumulation was not as complete(some cytosolic poly(A)1 RNA signal remains even 2 hrafter shift to 378). In striking contrast, yrb1-52/yrb1-52diploids did not show any accumulation of poly(A)1

RNA (even 5 hr after shift to 378), just like the wild-typecontrol (Figure 4A). Although yrb1-51 cells manifested aclear defect in RNA export, neither yrb1-51 mutants noryrb1-52 mutants had any detectable effect on the nuclearexport of proteins containing a leucine-rich NES, suchas 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 muta-tions on nuclear protein import, four different GFPfusions of nuclear proteins were examined. To assessthe a-importin/Srp1 and b-importin/Kap95/Rsl1-depen-dent pathway, two chimeras containing the SV40 NLSwere used: a galactose-inducible SV40NLS-GFP-b-galactos-idase, which is so large it cannot diffuse out of thenucleus 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 ofthe nucleus unless ongoing import occurs continuouslyFigure 3.—Yrb1(A91D) (yrb1-51) and Yrb1(R127K) (yrb1-(Kunzler and Hurt 1998). The third reporter was a52) bind Gsp1(G21V) with reduced affinity in vivo. (A) Interac-fusion of GFP to Npl3, an mRNA-binding protein, whosetion between wild-type Yrb1, Yrb1(A91D), or Yrb1(R127K) and

Gsp1(G21V) was determined using the two-hybrid method as nuclear entry depends on the importin, Kap111/Mtr10described in materials and methods. In brief, Yrb1 proteins (Senger et al. 1998). GFP-Npl3 accumulates rapidly inwere fused to the Gal4(TAD), and Gsp1(G21V) was fused to

the cytoplasm if import is impaired because Npl3 contin-the LexA(DBD). In the recipient strain (JTY2500), the E. coliuously shuttles between the nucleus and the cytosol.lacZ gene is under control of eight LexA-operator elements.The fourth transport substrate was constitutively ex-b-Galactosidase activity is expressed in arbitrary units. Each

value represents the average of single measurements made on pressed and composed of GFP fused to the NLS ofthree 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)

sonal communication). Transport of L25NLS-GFP intowas assessed by copurification. Cultures of strain CRY1 (YRB1the nucleus utilizes two different import receptors,GSP1) carrying CEN plasmids expressing a ProtA-TEV-Kap121/Pse1 and Kap123/Yrb4 (Schlenstedt et al.Gsp1(G21V) fusion from the NOP1 promoter and either wild-

type Yrb1-GFP, Yrb1(A91D)-GFP, or Yrb1(R127K)-GFP ex- 1997). Like SV40NLS-Gal4TAD-GFP, L25NLS-GFP is smallpressed from the authentic YRB1 promoter were grown in enough to diffuse out of the nucleus unless its importselective SCGlc medium at 268. Extracts were prepared and

occurs continuously.the ProtA-TEV-Gsp1(G21V) was purified on IgG-SepharoseControl strains or yrb1-51 and yrb1-52 mutants carrying(Pharmacia, Uppsala, Sweden) and eluted by digestion with

the reporter plasmids described above were cultivatedrecombinant TEV protease (GIBCO-BRL, Gaithersburg, MD).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

ducible reporter, transformants were grown to midexpo-polyclonal anti-Yrb1 antiserum (no. 1391). Endogenous Yrb1nential phase in Raf-containing medium at permissiveserved as a control to confirm equivalent loading and function-temperature and induced for 1 hr by addition of 2%ality of the immobilized Gsp1(G21V).Gal (to allow for mRNA synthesis and export) beforeshift to 378. For all four reporter proteins, there was a


Figure 4.—Nuclear transport defects in yrb1ts mutants. (A)To examine poly(A)1RNA export, cultures of homozygousdiploid strains YPH501 (YRB1/YRB1), JTY2482 (yrb1-51/yrb1-51), and JTY2485 (yrb1-52/yrb1-52) were grown in YPGlc at238 to early exponential phase, split into two equal portions,and incubated for another 2 hr either at 238 or 378. Afterfixation with formaldehyde, cells were stained with the DNAdye 49,6-diamidino-2-phenylindole, analyzed by in situ hybrid-ization using a CY3-labeled oligo(dT) probe to visualize thesubcellular distribution of poly(A)1RNA, and viewed by fluo-rescence microscopy using appropriate band-pass filters. (Band C) To examine nuclear protein import, the same strainsas in A were transformed with multicopy plasmids expressingeither SV40NLS-Gal4TAD-GFP (B) or L25NLS-GFP (C), respec-tively, cultivated and shifted as in A, and viewed directly byfluorescence microscopy and Nomarski optics.

significant cytoplasmic accumulation in yrb1-51 and yrb1- in haploids and for the other two reporters (data notshown). In yrb1-52 cells, the defect was noticeable even52 mutants after shift to 378, compared to wild-type cells,

indicating a general defect in nuclear protein import. at permissive temperature. Our results showing a defectin nuclear protein import in yrb1-52 cells are at oddsResults for SV40NLS-Gal4TAD-GFP (Figure 4B) and L25NLS-

GFP (Figure 4C) reporters in homozygous diploid with a previously published report on the same mutant(Ouspenski 1998).strains are shown; but, similar results were obtained


To exclude the possibility that cytoplasmic localiza-tion of the reporter proteins in yrb-51 and yrb1-52 cellswas due to “leakiness” of the mutant nuclei, accumula-tion of constitutively expressed SV40NLS-GFP-b-galactosi-dase (encoded by pPS815; Lee et al. 1996) was examinedin the same strains before and after temperature shift.In contrast to the short-term assay with the inducibleversion of the same reporter protein (see above), noincreased cytoplasmic GFP signal was observed (datanot 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 nucleocy-toplasmic transport factors: As an independent meansto confirm that yrb1-51 and yrb1-52 compromise nucleo-cytoplasmic trafficking even at permissive temperature,

Figure 5.—Genetic interactions between yrb1ts alleles andgenetic interactions of these alleles with mutations in 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 strainsnucleocytoplasmic transport were examined. Strains carrying a mutation in another gene of interest. The resulting

diploids were subjected to sporulation, and growth of individ-carrying mutations of interest were crossed with strainsual spores from tetratype asci was examined at various temper-carrying the yrb1-51 or yrb1-52 mutation, and the re-atures. Left, genetic interaction of yrb1-51 with nup2::HIS3 issulting diploids were sporulated. Double mutant segre- manifested by the extremely poor growth (“1” in Table 4) of

gants from tetratype asci were compared to each single the yrb1-51 nup2::HIS3 double mutant at 288, a temperaturemutant segregant and to the wild-type segregant for clearly permissive for the congenic yrb1-51 and nup2::HIS3

single mutants (yrb1-51 alone has a restrictive temperature oftheir ability to grow at various temperatures (see Figure318 under these conditions and nup2::HIS3 alone has no obvi-5). Mutations tested included alterations in genes en-ous growth defect even at higher temperatures). Right, a yrb1-coding components of the Ran GTPase cycle, nuclear 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 strain4). The plc1D::HIS3 mutation (Flick and Thorner 1993) carrying a ts mutation, srp1-31, in a-importin. The resulting

diploid was sporulated and individual spores from a tetratypewas tested since there is evidence for a role of PLC1-ascus were streaked on medium containing 5-FOA to count-encoded phosphatidylinositol-specific phospholipase Cerselect against the plasmid. Genetic interaction of yrb1-51in mRNA export (York et al. 1999; J. Flick, personal 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 atterious genetic interactions with many of these other any temperature, whereas the congenic yrb1-51 and srp1-31

single mutants are able to grow at permissive temperaturesclasses of mutants that affect nucleocytoplasmic trans-(here shown at 268). Other double mutant combinationsport (Table 4). Combination of the yrb1-52 mutationtested and their phenotype are listed in Table 4.with at least 12 of these mutations revealed essentially

the same growth defects. For xpo1-1 yrb1-52 andyrb2D::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 theyrb1-51 (Table 4). Equally pronounced growth defects only components of the mating pheromone responsewere 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) andtors (Table 4). In all these cases, the double mutant was yrb1-52 was isolated as an extragenic suppressor of anot viable at any temperature (synthetic lethality); for ste5 missense mutation (Katz et al. 1987). Hence, wethe other genetic interactions observed, double mutants constructed a functional GFP-Ste5 chimera and exam-were viable at the permissive temperature (238) but re- ined its subcellular localization in yrb1-51 and yrb1-52vealed a restrictive temperature that was considerably mutants (and in control cells) in the absence of phero-(.38; 11) or slightly (#38; 1) lower than the one of mone to avoid the complications of signal-inducedany 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 beforeconditions. (Mahanty et al. 1999; Pryciak and Huntress 1998).

Nucleocytoplasmic trafficking of Ste5 is altered in In contrast, no nuclear accumulation was observed inyrb1-51 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

between nucleus and cytoplasm (Mahanty et al. 1999),of the phenotypes of yrb1-51 and yrb1-52 mutants, includ-


TABLE 5

Suppression of the partial mating defect of a msn5D nullmutant by the yrb1-51 and yrb1-52 mutations

Genotypea Mating efficiency (%)b

MSN5 YRB1 92.3 6 10msn5D YRB1 20 6 12MSN5 yrb1-51 96.6 6 4.7msn5D yrb1-51 89 6 12.2MSN5 YRB1 67.3 6 18.7msn5D YRB1 18.3 6 2.8MSN5 yrb1-52 89.3 6 8.9msn5D yrb1-52 70.6 6 24.5

a MATa spores of the indicated genotype from a cross be-tween strains HMK30 (msn5D), and JTY2027 (yrb1-51) andJTY2484 (yrb1-52), respectively, were assayed for quantitativemating with the MATa tester strain DC17 as described inmaterials and methods.

b Mating efficiencies are the ratio of the number of diploidsformed to the number of viable input MATa haploids, are theaverage of three independent trials, and are given as percent-age 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, nuclearmulticopy plasmid were cultivated, shifted, and viewed as de-

envelope formation, maintenance of chromatin struc-scribed in the legend to Figure 4, B and C.ture and nuclear (and nucleolar) organization, chromo-some segregation, DNA replication, RNA metabolism,and cell cycle progression. It is still a matter of somethe observed mislocalization in cells with defective Yrb1

could be due, in principle, either to inhibition of Ste5 debate whether the pleiotropic phenotypes of alter-ations in the Ran GTPase cycle are due solely to theimport into the nucleus or enhancement of Ste5 export

from the nucleus. Since we have demonstrated that yrb1- established role of Ran in nucleocytoplasmic transport(Gorlich and Kutay 1999) or whether Ran has addi-51 and yrb1-52 clearly impede nuclear import of various

reporter proteins, the former possibility seems more tional roles in the cell. Recent studies using in vitrosystems have implicated Ran function directly in micro-likely.

To obtain further evidence that suppression of mating tubule organization (Kahana and Cleveland 1999)and nuclear envelope formation (Hetzer et al. 2000;defects by yrb1-51 and yrb1-52 arises from impairment

of nuclear import of Ste5, we tested whether these yrb1ts Zhang and Clarke 2000). Our evidence indicates thatthe Ran GTPase cycle is linked to the signaling eventsmutations could suppress the mating defect of a msn5D

mutant. Msn5 is thought to be the nuclear receptor required for mating as a consequence of the role ofRan in nucleocytoplasmic transport.required for pheromone-stimulated export of Ste5 from

the nucleus (Mahanty et al. 1999). Each yrb1ts mutant A decade ago, before the function of the essential Ranregulator, RanGEF1, was fully appreciated, a ts mutationwas crossed against a msn5D strain and the wild-type

single mutant, and double mutant spores from the re- (srm1-1) in its yeast homolog (SRM1/PRP20/MTR1) wasisolated as a suppressor of the mating defect of haploidsulting tetratype asci were tested for their relative mating

proficiency using a quantitative mating assay performed cells lacking pheromone receptors (Clark and Sprague1989). Unlike other ts mutations in yeast RanGEF1 iden-at semipermissive temperature (308) for the yrb1ts alleles.

Both the yrb1-51 and yrb1-52 mutations restored the mat- tified subsequently (Amberg et al. 1993), srm1-1 doesnot cause dramatic nuclear accumulation of poly(A)1ing efficiency of the msn5D mutant to essentially the

wild-type level (Table 5). This suppression is consistent RNA at restrictive temperature (Kadowaki et al. 1993;and its effect on nuclear protein import was not exam-with the idea that a higher cytoplasmic pool of Ste5 (due

to its inefficient nuclear import in the yrb1ts mutants) ined), which left open the possibility that the RanGTPase cycle had some role in mating distinct fromcompensates for its inefficient pheromone-stimulated

export from the nucleus (due to the msn5D mutation). its function in nucleocytoplasmic transport. We have


shown here that two independently isolated suppressors be required for cytoskeletal reorganization, which isnecessary for polarized cell growth toward the matingof early and late defects in the mating pathway are

alterations in another essential Ran regulator, RanBP1, partner (Blondel et al. 1999; Shimada et al. 2000). Therole of recruitment of Ste5 to the shmoo tip may bethat clearly impair nuclear protein import, even at per-

missive temperature. We found further that the Ste5 similar, given the interaction of Ste5 with Bem1, a pro-tein involved in polarized cell growth (Leberer et al.scaffold protein, which shuttles between nucleus and

cytoplasm and is required for pheromone response, is 1997). Since relocalization of Far1 (Blondel et al. 1999;Shimada et al. 2000) and Ste5 (Pryciak and Huntressmislocalized to the cytosol in these mutants. Consistent

with this finding, both yrb1ts alleles suppressed the partial 1998; Mahanty et al. 1999) is transient, a nuclear importdefect that kept these proteins out of the nucleus longermating defect of a null mutation in MSN5, which en-

codes the nuclear receptor required for pheromone- might increase mating efficiency in cells in which thecell fusion process would otherwise be nonoptimal.stimulated export of Ste5.

In retrospect, it may seem obvious that perturbation In agreement with the above model, both the yrb1-51and the yrb1-52 mutations increased the cytoplasmicof nucleocytoplasmic transport could affect yeast mating

since response to pheromone requires: (a) that some concentration of Ste5 and were able to suppress themating defect of a msn5 null mutation. The MSN5 genesignal carrier enter the nucleus to induce the transcrip-

tion of genes, (b) that RNPs containing newly synthe- encodes a nuclear export receptor of the b-importinfamily (Kaffman et al. 1998; DeVit and Johnston 1999)sized mRNAs for mating-specific components exit the

nucleus, and (c) that the translation products of some and was also identified as STE21 because a null mutationat this locus confers a partial mating defect (Akada et al.of those transcripts be recruited back into the nucleus

in support of late mating events, like karyogamy (Rose 1996; Alepuz et al. 1999; Blondel et al. 1999). Impairednuclear protein import in the yrb1ts mutants keeps Ste51996). Accordingly, it is now known that many compo-

nents of the pheromone response machinery, both pre- (and perhaps other proteins required for mating) inthe cytosol longer, and thus there is less need for theirexisting and pheromone induced, are localized to the

nucleus. On the other hand, it has been shown that continuous Msn5-dependent reexport from the nu-cleus. Also consistent with the above model, MATa yrb1-two components, Far1 (Blondel et al. 1999) and Ste5

(Pryciak and Huntress 1998; Mahanty et al. 1999), 51 and MATa yrb1-52 mutants are able to respond nor-mally to a-factor under conditions (by inducing therapidly relocalize from the nucleus to the site of cell

fusion (shmoo tip) upon pheromone treatment. Our pheromone-responsive reporter FUS1-lacZ) and by un-dergoing G1 arrest (as judged by the standard haloresults suggest the following mechanism to explain the

suppression of mating defects by mutations in nucleocy- bioassay; data not shown); hence, these mutants are notdefective in early signaling events. If, however, proteinstoplasmic transport factors.

Because both yrb1ts alleles described here are defective required for late events in mating are maintained longerin the cytosol of yrb1 mutants due to defective nuclearin nuclear protein import, but only one seems impaired

in poly(A)1 RNA export, yet both act as suppressors of protein import, the efficiency of mating would be en-hanced, as observed. Finally, such a model would alsomating defects, it is presumably the import defect that

leads to suppression. Also, it should be recalled that explain the spectrum of mating defects that are sup-pressed by yrb1-51, yrb1-52, and srm1-1. All of the muta-yrb1-51 was isolated on the basis of rescue of the mating

debility of a fus1D mutant, which has an intact phero- tions suppressed are in gene products that are targeted,directly or indirectly, to the shmoo tip after pheromonemone response pathway and is only partially mating

defective. Likewise, yrb1-52 was isolated on the basis of treatment and most are involved in the pheromone-induced remodeling of the cortical cytoskeleton (Leb-its ability to rescue ste5 missense mutations (and can do

so for ste4 and ste7 missense mutations), but is unable erer et al. 1997). Pheromone receptors (Ste2 and Ste3)are localized at the shmoo tip and participate in cellto rescue null alleles in these same genes (Katz et al.

1987). These considerations indicate that residual sig- polarity determination and mating partner discrimina-tion (Jackson et al. 1991). Ste4 (Gb), when releasednaling in the pheromone response pathway must be

present for suppression to occur. How could impair- from the receptors as the Gbg complex in response topheromone binding, is responsible for direct recruit-ment of nuclear protein import improve the mating

efficiency of such partially mating-defective mutants? ment of major regulators of cytoskeletal structure andcell polarity, including Ste20 (Leeuw et al. 1998), Far1One reasonable scenario is that nuclear import of cer-

tain signaling components results in downregulation of (Shimada et al. 2000), and Ste5 (Inouye et al. 1997b;Pryciak and Huntress 1998). These components in-the mating response. If so, mutations that reduce the

rate of nuclear entry of such a factor(s) should enhance teract with other molecules, like Bem1, that contributeto reorganization of the cytoskeleton (Leberer et al.the efficiency of mutants that are only partially defective

in mating. Both Far1 and Ste5 are reasonable candidates 1997). Even Ste7, which encodes the MAPK kinase(MAPKK) of the pheromone-responsive MAPK cascade,for such factors. For Far1, its pheromone-induced relo-

calization from the nucleus to the shmoo tip seems to and which is suppressed by yrb1-52, albeit rather weakly


by a European Molecular Biology Organization Long-Term Fellowship(Katz et al. 1987), is bound to Ste5, which becomesand funds provided by the Swiss National Science Foundation (to M.tethered at the shmoo tip via its interaction with Gbg.Kunzler), by National Science Foundation Postdoctoral Fellowship

Finally, Fus1 is an integral membrane protein localized DMB-8807575 and National Cancer Institute Postdoctoral Traineeshipto the tip of the mating projection and is involved in CA09041 (to J. Trueheart), by a Postdoctoral Fellowship from the

Italian-American Cancer Foundation (to C. Sette), by National Insti-the cell fusion step of mating (Trueheart and Finktutes of Health Research Grant GM21841 (to J. Thorner), and by1989). Indeed, mutational analysis of Fus1 indicates thatfacilities provided by the Berkeley campus Cancer Research La-its large O-glycosylated exocellular domain is dispens-boratory.

able for its function and that an SH3 domain and twopotential actin-binding motifs in its relatively short, cyto-solic, C-terminal tail are essential for its function, sug-

LITERATURE CITEDgesting that the primary role of Fus1 in the matingAebi, M., M. W. Clark, U. Vijayraghavan and J. Abelson, 1990process is its contribution to modifying the structure

A yeast mutant, PRP20, altered in mRNA metabolism and mainte-of the cytoskeleton (J. Trueheart and J. Thorner,nance of the nuclear structure, is defective in a gene homologous

unpublished results). to the human gene RCC1, which is involved in the control ofchromosome condensation. Mol. Gen. Genet. 224: 72–80.A primary defect of the analyzed yrb1ts mutants in

Aitchison, J. D., G. Blobel and M. P. Rout, 1996 Kap104p: anucleocytoplasmic transport would also explain the ob- karyopherin involved in the nuclear transport of messenger RNAserved mitotic phenotypes, since similar mitotic distur- binding proteins. Science 274: 624–627.

Akada, R., L. Kallal, D. I. Johnson and J. Kurjan, 1996 Geneticbances have been reported for mutants deficient inrelationships between the G protein bg complex, Ste5p, Ste20pother factors involved in nucleocytoplasmic transport, and Cdc42p: investigation of effector roles in the yeast phero-

for example, Srp1 (a-importin; Loeb et al. 1995), and mone response pathway. Genetics 143: 103–117.Alepuz, P. M., D. Matheos, K. W. Cunningham and F. Estruch,Cse1 (Xiao et al. 1993), which is required for reexport

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