MG G

Mol Gen Genet (1992) 236:145-154

© Springer-Verlag 1992

STESO, a novel gene required for activation of conjugation at an early step in mating in Saccharomyces cerevisiae Massoud Ramezani Rad, Gang Xn and Cornelis P. Hollenberg Institut f/Jr Mikrobiologie, Heinrich-Heine-Universit/it, Universit/itsstral3e 1, W-4000 D/isseldorf 1, F R G Received April 18, 1992 / Accepted July 4, 1992

Summary. A new gene, STE50, which plays an essential role in cell differentiation in Saccharomyees cerevisiae was detected and analysed. S T E 5 0 expression is not cell type-specific and its expression in M A Ta and M A Ta cells is unaffected by pheromones. When present on a high copy number plasmid, S T E 5 0 causes supersensitivity to a-pheromone, and increases the level of a-pheromoneinduced transcription of FUS1 in haploid a cells. Mutants bearing either of the two gene disruptions, ste50-1 or ste50-2, are sterile and have a modulated sensitivity to a-pheromone. The overexpression of S T E 4 (G~) in wildtype cells elicits a constitutive growth arrest signal, however this phenotype is suppressed by a C-terminal truncation mutation in S T E 5 0 (ste50-2). In contrast, the constitutive activation of the pheromone response pathway caused by disruption of GPA1 (G~) is not suppressed in ste50-2 mutants. The ste50-2 mutation partially suppresses the desensitisation defect of the sst2-1 mutation, and the resulting ste50-2 sst2-1 mutants restore fertility. Our result sindicate that the ste50-2 mutant may have a defect in adaptation (hyperadaptation), and suggest a possible interaction of STE50-2 with the G~ subunit of the G protein. Key words: Cell differentiation - G protein - Adaptation - S T E 5 0 - Yeast

Introduction A unifying view of the cell cycle encompasses transitions from one regulatory state to another (Hartwell and Weinert 1989). The crucial features are the existence of two transition control points, at the G2/M boundary and during G1 phase. G1 is the time interval at which cells must either decide to proceed into S phase or exit the cell cycle either by undergoing mating or arresting in response to nutrient limitation, or by entering meiosis if Correspondence to: Dr. M. Ramezani Rad

diploid. Hartwell et al. (1974) have postulated a gating event, referred to as START, which cells must complete in order to enter a new division cycle. Yeast haploid cells exhibit a choice of either proliferation or differentiation depending on the presence of, and their sensitivity to, mating pheromones. Mating pheromones block the Saccharomyces cerevisiae cell cycle in G1 at START and thus allow cells to enter the mating pathway. Gl-arrested cells become large and pearshaped ("shmoos') and are able to aggregate, fuse, and undergo karyogamy (for recent reviews see Cross et al. 1988; Herskowitz 1990). It is known that mating pheromones act through a cell surface receptor and a G protein-linked signalling system. The S T E 2 and S T E 3 genes, respectively, encode the a and a pheromone receptors. The primary structures of these receptors predict a seven transmembrane domain protein and thus they are similar in overall organisation to mammalian G protein-coupled receptors (Burkholder and Hartwell 1985; Nakayama et al. 1985; Hagen et al. 1986). GPA1 (SCG1), STE4, and STE18 encode the a, [3, and 3' subunits, respectively, of the heterotrimeric G protein (Nakafuku et al. 1987; Dietzel and Kurjan 1987; Whiteway et al. 1989) which is functionally coupled to the S T E 2 and S T E 3 receptors. The [3 and 3' subunits of the yeast G protein appear to be essential for receptor coupling (Blumer and Thorner, 1990). Disruption of GPA 1 causes constitutive activation of the pheromone response pathway (Dietzel and Kurjan 1987; Miyajima et al. 1987; Jahng et al. 1988; Nakayama et al. 1988; Blinder et al. 1989), but only if functional S T E 4 or STE18 products are present (Nakayama et al. 1988; Whiteway et al. 1989). A constitutive signal is also elicited when the wild-type S T E 4 product is overexpressed (Cole et al. 1990; Nomoto et al. 1990; Whiteway et al. 1990). Overexpression of the S T E 4 product in stel8 mutants does not elicit a constitutive signal (Cole et al. 1990; Nomoto et al. 1990; Whiteway et al. 1990), which supports the view that the S T E 4 and STE18 products act as a [33' complex. Additional elements have been identified that regulate the activity of the G protein-linked response pathway. If

146 pheromone-treated cells do not mate, they are able to recover from the effects of pheromone and resume the mitotic cell cycle. Recovery involves several independent processes that adapt or desensitise cells in the continued presence of pheromone. Cells deficient in the S S T 2 product are supersensitive to pheromone and fail to recover from pheromone-induced arrest (Chan and Otto 1982). Indeed, overexpression of the G~ subunit (Dietzel and Kurjan 1987; Miyajima et al. 1987) partially suppresses the pheromone hypersensitivity o f s s t 2 mutants (Kurjan and Dietzel 1987). The overexpression o f G~ suppresses the constitutive signal that is generated by overexpression o f the S T E 4 product (Cole et al. 1990; N o m o t o et al. 1990; Whiteway et al. 1990). These and other findings suggest that the G~ subunit plays a positive role in the transduction of signals that stimulate desensitisation to pheromone and recovery from pheromoneinduced cell cycle arrest. Irie et al. (1991) recently identified a gene, S G V 1 , which encodes a C D C 2 8 / c d c 2 related protein kinase, that may play a role in this G~stimulated response. Here, we report the isolation and

characterisation of a gene, S T E 5 0 , which is involved in the activation (sensitisation) of the pheromone response pathway. In this study, we examine the requirement for S T E 5 0 for cell differentiation in S. cerevisiae. In addition we have characterised a C-terminal truncation mutation, s t e 5 0 - 2 , which suppresses the constitutive growth arrest phenotype associated with overproduction o f G~ (Ste4) and promotes cellular recovery in S. eerevisiae. Part of this work was presented at the 15th International Conference on Yeast Genetics and Molecular Biology in 1990 (Ramezani Rad et al. 1990).

Materials and methods M e d i a a n d strains. Yeast media were prepared as described in Sherman et al. (1986). Y E P D or SD (synthetic medium) were titrated to p H 4.0 by the addition of HC1 as indicated. See Table 1 for a description o f the yeast strains used.

Table 1. Yeast strains

Strain

Genotype

Source

S150-2B RC1689 XUM-20D XMWI-18A MC84 stel 1 MC84 stel2 7680-8-1 7680 ste50 MG8-B

a leu2-3, 112 ura3-52 trpl-289 his3-1

J. Ernst J. Ernst W. Duntze W. Duntze M. Ciriacy M. Ciriacy C. Jackson This work This work

a leu2 ura3-52 trpl his4,7 met2 tup7 ct leu2-3, 112 ura3-52 trpl his6 thr4 ss11-7 a lysl his6 ssl 2-1 leu2 ade2 ste11 6: : URA3 TRP1 leu2 ade2 ste12,1: : URA3 trpl a cry1 his4-580 lys2 trpl ura3-52 tyrl ° cyh2 sst2-1 SUP4-3 ~ 7680-8-1 ste50-2:: URA3 cL ade2 trpl tyrl ° his3/4? sst2-1 ste50-2: : URA3

Isogenic strains and their derivatives W303-1A a leu2-3, 112 ura3-1 trpl his3-11 ade2 canl-lO0 W303-1B et leu2-3, 112 ura3-1 trpl his3-11 ade2 canl-lO0 W303 a/ct leu2-3, 112 ura3-1 trpl his3-11 ade2 canl-lO0 WA-d2U a W303-1A, ste50-2: : URA3 WB-d2U ct W303-1B, ste50-2: : URA3 WD-d2U ste50-2 : : URA3 a/a W303-1,

R. Rothstein R. Rothstein R. Rothstein This work This work This work

WA-d200H WB-d200H WD-d200H

ste50-1 :: HIS3

This work This work This work

STE50 ste50-1 :: HIS3

This work

ste50-1 : : HIS3 ste50-2 : ."URA3

This work

ste50-2 : : URA3 ste50-1 :: HIS3

This work

ste50-2 : : URA3 scgl : : LEU2

M. Whiteway

STE50

WD-dd2H WD-dd2U WD-d2HU D 111

M325-1B WA-ste2 WD-d2sg WD-d0sg WA- 1Bd2

a W303-1A, ste50-1 : : HIS3 a W303-1B, ste50-1 : : HIS3 a/c~ W303-1, a/a W303-1, a/c~ W303-1, a/a

W303-1,

a/ct W303-1, a a

SCG1 W303-1A, (GAL1-STE4) HIS3 + W303-1A, ste2::LEU2 ste50-2 : : URA3

aft Dill, a/a Dill,

a

STE50 ste50-1: : HIS3

STESO M235-1B, ste50-2: : URA3

M. Whiteway M. Whiteway This work This work This work

147

Yeast transformation. Yeast transformation was performed according to the method described by Klebe et al. (1983) and modified by Dohmen et al. (1991): cells were grown in YEPD or SD media to 00600 = 0.6, harvested and resuspended in 1 M sorbitol, 10 mM Bicine, 3% ethylene glycerol pH 8.35, and glycerol was added to a final concentration of 15%. The cells were frozen in 200 gl aliquots at - 70 ° C in which form they can be kept for several months. For transformation, 1-10 gg D N A was added directly to frozen cells. After 5 rain of vigorous shaking at 37 ° C, 1 ml of 40% PEG 4000 in 200 mM Bicine pH 8.35 was added, and incubation continued at 30 ° C for another 60 min. Cells were then washed with 0.15M NaC1, 10mM Bicine pH 8.35 and spread on selective media. By this procedure about 10 transformants per gg of D N A were obtained with integrating plasmids and more than 1000 transformants per gg DNA with 2-gm based or CEN plasmids.

A

q

CEN3

I~--t--Z-//--* HIS4

FUS 1

EcoRI

I

~.

ste50-2 [ ste50-1

J

URA3

m [

HIS3

0.5 kb

Recombinant DNA manipulation and sequencing. Recombinant D N A methods followed standard protocols (Sambrook et al. 1989). The YIp5 plasmid-based clone C1G was obtained from S.G. Oliver (Manchester) and was derived initially from a D N A bank of mapped clones assigned to yeast chromosome III, originally constructed by C. Newlon (New Jersey). A 4.2 kb EcoRI fragment contained the complete STE50 gene. This fragment was cloned into the SmaI site of YEp351 (Hill et al. 1986) by blunt-end ligation to create a LEU2 STE50 high copy number plasmid (YEp351-BIG2). The same fragment was inserted into the EcoRI site of the pFL38-HIS (gift from F. Lacroute) to generate a STE50 centromerebased plasmid (pFL38H-BIG2). For the construction of the STE50-LacZ fusion plasmid (pBIG2zl), the 2.5 kb BglII fragment, which contains the whole 5" flanking region and the N-terminal 68 codons of the STE50 gene, was inserted into the BamHI site of plasmid YEp363(LEU2) (Myers et al. 1986). The plasmid YIF200H was constructed by inserting the HIS3 gene sequence into the BgllI and NheI sites, deleting 898 nucleotides of the STE50 gene (Fig. 1). The ste50-1 mutation was created by gene replacement with plasmid YIF200H. This mutation deleted all but the first 68 amino acids of the Ste50 protein. An internal fragment of the STE50 gene (position + 205, relative to the first ATG codon to position + 723) was cloned into YIP5 to obtain the integrating plasmid YIF2. The ste50-2 mutation was introduced into strains W303-1A, W303-1B and W303D by internal fragment disruption. The plasmid YIF2 was linearised at the unique KpnI site within the STESO internal fragment and used to transform a-, a- and a/a mating type strains, creating a duplicated, disrupted ste50 gene: one copy is truncated at the 3' end eliminating 105 amino acids from the C-terminus of the protein, and the other is truncated at the 5' end and lacks sequences upstream of position + 205 of the STE50 gene (Fig. 1 and 2). All experiments involving the ste50-2 mutants were conducted in selective media to ensure the continued presence of the duplicated disruption. The gene disruptions were then confirmed by complementation of the defects with the STE50 CEN

~,

. ~

J

Fig. 1A, B. A Restriction map of the STE50 region. The STE50 gene is located on chromosome III adjacent to HIS4. B structure of the

disruption alleles ste50-1 and ste50-2 plasmid (pFL38H-BIG2), by Southern analysis and by tetrad analysis. The plasmid pMG44-1 is plasmid YEp351 plus a 1.1 kb PvuII fragment of the C-terminally deleted STE50 gene from our library of deletions. The plasmid pMG10 contains the FUS1-LacZ fusion gene and the yeast LEU2 gene for selection. It was constructed by inserting the 4.5 kb NcoI fragment of plasmid pSB234 (gift from J.A. Brill and G.R. Fink) into the BamHI site of the 2 ginbased plasmid YEp 13. This FUS1-LacZ fusion gene contains the promoter region and the N-terminal 180 codons of the FUS1 gene. DNA sequences were determined by the dideoxy chain termination method (Sanger et al. 1977) using [3SS]dATP and T7 DNA polymerase. The sequence was established from both D N A strands. The sequence analysis of 11.9 kb of C1G clone was performed as part of the European Community programme to sequence yeast chromosome III (Ramezani Rad et al. 1991). Matin 9 assay. Overnight cultures were diluted 1:1 in either YEPD or synthetic medium and grown for another 2 h. Approximately 3 x 106 cells of each parent were mixed, resuspended in a small volume, plated on YEPD and then incubated at 30 ° C. At different times the cells were fixed with formaldehyde by the method described in Elion et al. (1990). Estimation of the efficienty of zygote formation, described in the legend to Table 3, was based upon the method of Sena et al. (1973). The peak value obtained for zygote formation was used to indicate the efficiency of zygote formation in each reaction. Quantitative mating assays were performed essentially as described by Elion et al. (1990). Agglutination was assayed as described by Michaelis and Herskowitz (1988). Pheromone sensitivity assay. Short-term exposure: cells were grown in YEPD or selective medium to a n 0 0 6 o o

148 of 0.2-0.4, pelleted and resuspended in YEPD or selective medium (adjusted to pH 4.0 with HC1 prior to use) containing 5 gM a-factor (Sigma). For the control, the same amount of the cells was resuspended in the same media without a-factor. Samples were incubated at 30 ° C, then fixed as above and photographed. Long-term exposure: a halo assay was used as described by Elion et al. (1990).

1 TCACGTCATTCGATTCGTCACTCJ~TT TTCTTGCTCTTATTCCAGTACTGCGTCAGTACAA 61

TGTTTCTTCT CCCTGGAAAAGTCCTGCGTACJ~GTTATAACGTACAGCCTGCTCCACTTAT

122 TCATTTTTTGATATTGCTTCCTTTCTCCCTTTATTTA.4ACTATCATCGGCGACATATT TC 181

CCAATAGG~GCTGAJLk, AATACTATCGCTAATTATAACAAAGAAGCTAGGTCGAAGGA

241

CTC~GACAGAGGTCGTACTAGCAGAGATAGCAAATCAGAT GGAGGACGGTAAACAG

301

GCCATCAATGAGGGATCAAACGATGCTTCGCCGGATCTGGACGTGAAT GGCJ~CAATATTG

14 E D

7 A

Assays of pheromone production. The amount of pheromone produced by the cells was determined semi-quantitatively with pheromone-supersensitive cells. Cell lawns were prepared as described by Elion et al. (1990). Cells to be tested were grown to early log phase and spotted onto filter discs (Whatmann 3MM), which were then placed upon the supersensitive cell lawn, and incubated for 40 h at 28 ° C. fl-Galactosidase assay. Strains harbouring a LacZ vector were grown in synthetic medium to select for the presence of the vector. Pheromone induction and measurement of [3-galactosidase were performed as described (Trueheart and Fink 1989) using 2 gm-based plasmids containing the STE50-lacZ fusion gene (pBIG2zl) or the FUS1lacZ fusion gene (pSB234; pMG10).

361

1

N

E

G S

N D

A

S

P

D

L

D

N

G T

;

Q

L

ATGAATAATGAAGACTTTTCCCAGTGGTCGGTTGATGATGTGATAACTTGGTGTATATCC

27 N N N E D F S Q W S V D D V 421

l

T M C l

S

ACGCTGGAGGTGGAAGAJU~CCGATCCATTATGTCAGAGACTGCGAGAJUUtTGATATTGTA

47

T L E V E E T D P L C (I R L

481

R E N D % V

GGAGATCTTTTGCCGGAATTGTGCTTGCAAGATTGCCAGGACTTGTGTGACGGTGATTTG

67 G D L 541

L P E L

C L Q D C Q D L

C D G D L

AATAAGGCCATA/~kATTCAAGATACTGATCAATAAGATGAGAGACAGCAAGTT GGAGTGG

87

N K k

601

l

K F K

1 L

l

N K N R D S K L E ;/

AAGGACGACAAGACT CAAGAGGACATGATAACGGTACTGA/UUUtCTTGTACACTACTACA

107 K D D K T 0 661

E D 14 1 T V L K N L Y T T T

TCTGCGAAATTGCAAGAATTTCAATCGCAGTACACAAGGCTGAGGATGGATGTCTTGGAC

127 S A K L 0 721

E F Q S Q Y T R L R N D y

I. ID

GTAATGAAGACCAGCTCAAGCTCTTCTCCGATTAACACACATGGAGTGTCCACTACGGTA

147 V H K T S S S S S P I 781

V

G g

N T M q V ~

T T ¥

CCTTCTTCAAACAACACAATTATACCCAGTAGTGACGGTGTGTCTCTTTCACAAACAGAC

167 P S S N N T

l

l

P S S D G V S I.

~; Q T ID

Nucleotide sequence accession number. The sequence of the STE50 locus has been deposited in the EMBL Data Library (accession number Z 11116).

84,1 TATTTCGACACAGTTCATAACCGACAATCACCGTCAAGGAGAGAAT CCCCGGTAACGGTA

207

F R Q P S L

Results

961

CCCCAAATAT CTACAAACCAATCTCACCCATCTGCCGTTTCAACAGCGAACACACCGGGG

187 Y F P T V 1t I~ R g 901

S P S R R E S P y

T V

TTTAGGCAACCCAGTCTTTCCCACTCAAAATCTTTGCACAAGGATAGCAAAAACJUUtGTA

227 P Q I

S H S K S L

S T N (I

p K p

~; K N K V

S N P S A V ~ T A ~1T

p Q

Isolation and characterisation of the STE50 9ene

1021 CCATCACCTAACGAGGCGTTAAAACAGTT GCGTGCATCTAAAGAAGACTCCTGCGAACGG

During work on the EC yeast genome sequencing project the gene STE50 was isolated and sequenced (Fig. 1 and 2). STE50 is located distal to the HIS4 gene on chromosome III. Upstream of the coding sequence there are a number of poly(dA) and poly(dT) stretches, which may act to promote constitutive transcription (Struhl 1985). The open reading frame encodes a polypeptide of 346 amino acids that exhibits no significant similarity to protein sequences currently in the data banks (EMBL; GenBank; NBRF-Nucleic; NBRF-Protein; SwissProt; Yeast-Nucleic; Yeast-Protein). The sequence of the gene and the encoded polypeptide do not suggest a function for STE50.

1081

247 P S P N E A L K Q L

267

R A S K E D S C E R

ATCTTGAkJL~CGCAATGAAAAGACATAACTTAGCAGATCAGGATTGGAGACAATATGTC I

L K N A M K R H M L A D 0 D W R Q Y V

1141 TTGGTCATTTGCTATGGGGATCAAGAGAGGCTGTTAGAATTGAACGAAAAGCCTGTGATC 287

L V

!

C Y G D Q E R L

L E L M E K P V l

1201 ATATTCAAGAACTTA.~GCAACAGGGTTTGCACCCCGCCATTATGTTAAGAAGAAGAGGT 307

l

F K N L K Q Q G L

H P A

1 N L R R R G

1261 GATTTCGAAGAAGTAGCAATGATGAACGGAAGTGACAATGTCACCCCCGGTGGAAGACTC .327 D F E E V A N N N G S D N V T P G G R L 1"421 TAATGTGCAGTTGTCATGCACATCATCATAC rAAACTTACACGAATAGGATAACATGTAT 1,381 GCTAGCAGAATATATAT~~TTATTGATGCCTTTAAACTTATACTATTATA 1441 CTATATTATGTTATATTATATTATTAGTTTTATAGATATATTGAGATATGTTGAATATGA

Fig. 2. Nucleotideand aminoacid sequencesof the STE50 gene. The single-letter amino acid code is used. Regions enriched in S and T are underlined

ste50 mutants are defective in matin 9 Two disruption alleles ste50.':HIS3 (ste50-1) and ste50::URA3 (ste50-2) which encode the first 68 and 241 N-terminal amino acids, respectively (Materials and methods and Figs. 1 and 2), resulted in a defect in mating. Haploid strains M A T a and M A Ta containing either of these ste50 alleles were defective in zygote formation when crossed with a ste50 mutant, but formed diploids in crosses with wild-type cells at very low frequency, as measured by the zygote formation (Table 2) and diploid

selection assays (Table 3). All the crosses shown in Table 3, involving a ste50 mutant as one partner, generated viable zygotes at a low frequency (0.5 to 1.5%), while the ste50 x ste50 cross produced no zygotes. The control plasmid bearing the wild-type STE50 gene (Materials and methods) restored fertility to ste50 mutants. Tetrad analysis of diploid strains heterozygous for the ste50 mutations showed a 2:2 segregation of the mating defect.

149 Table 2. Efficiency of zygote formation in different crosses MAT~

STE50 ste50-2: m.c. STE50 ste50-2: s.c. STE50 ste50-2 ste50-1

MATa STE50

ste50-2 : m.c. STE50

ste50-2 : s.c. STE50

ste50-2

ste50-1

21 28.6

20.5 15.4

17.3 22.0

1.7 0.5

0.2 ND

20.8

18.9

9.9

0.2

ND

0.4 0.2

0.1 ND

0.1 ND

0.0 ND

0.0 0.0

The strains used are isogenic except for the mating type locus and the STE50 locus. The mating efficiencies were determined at 30° C and are expressed as the percentage of cells that had formed diploids after 4 h (see Materials and methods). In each cross only the

genotype at the STE50 locus is shown, s.c. STE50, single copy of STE50 gene carried by CEN pFL39H-BIG2; m.c.STE50, multiple copies of STE50 gene carried by YEP351-BIG2; ND, not determined

Table 3. Efficiency of diploid formation in crosses involving ste50 mutants as measured by a quantitative mating assay

MATa ste50-1 ceils underwent morphological changes (forming shmoos) in the presence of pheromone, but they exhibited a severe defect in mating as shown in Table 3 and 4 and they were twice as resistant to a-pherom o n e as the wild-type cells, as measured by the sensitive halo assay (data not shown). Mutants defective in known components of the signal transduction pathway ( S T E 2 , S T E 4 , etc.) affect both transcriptional and cell cycle responses to mating pheromones. The only phenotypes exhibited by the ste50-1 m u t a n t were those affecting sensitivity to pheromone-induced arrest and the ability to mate.

MATa

MATe

STE50 ste50-2

STE50

ste50-1

31.6 0.5

1.5 0.0

The numbers indicate the percentage of cells that had formed diploids after 4 h at 30

Table 4. Induction of FUSI-lacZ by a-pheromone MATa strain

STE50 ste50-1 ste50-2 STE50 (YEpSTE50)

Units of activity Uninduced

Induced

0.3 0.7 0.3 0.3

27 32 0.4 68.4

All strains are isogenic except at the STE50 locus and all harbour plasmid pMG10, a 2g-LEU2-based plasmid encoding the FUS1lacZ fusion gene obtained from pSB234. Cells were induced for 2 h with 5 /aM a-pheromone as described in Materials and methods

Haploid strains of a and a mating type bearing the ste50-2 m u t a t i o n both showed decreased levels of a- and

s - p h e r o m o n e production (data not shown), which is a phenotype c o m m o n l y associated with mutations that lead to a p h e r o m o n e response defect, whereas haploid strains bearing the ste50-1 mutation produce normal amounts of a- and a-factor. A standard test for normal induction o f matingspecific function is the level of induction of F U S 1 , a gene that is highly induced in the presence of p h e r o m o n e (Trueheart et al. 1987). When assayed with a Fusl-[3galactosidase fusion protein ( F u s l - L a c Z ) F U S 1 was induced to approximately normal levels by a - p h e r o m o n e in ste50-1 cells (Table 4).

Overexpression o f STE50 causes increased pheromone sensitivity

The effects of overexpression of the S T E 5 0 gene in haploid cells were tested by measuring the sensitivity to a-pheromone, and the level o f induction of a matingspecific gene, F U S 1 . The wild-type strain W303-1A ( M A T a S T E 5 0 ) or an ste50 m u t a n t strain (WAd2U) containing S T E 5 0 on a high copy n u m b e r plasmid showed a higher sensitivity to a-pheromone-induced cell cycle arrest than the wild-type strain containing only vector (Fig. 3). A similar increase in p h e r o m o n e sensitivity for division arrest was found by monitoring the accumulation of unbudded cells in liquid cultures that had been treated with varying concentrations of a - p h e r o m o n e (data not shown). Overexpression of S T E 5 0 had no effect on the basal level of F U S 1 expression as judged by the Fusl-[3-galactosidase assay, but it led to a two-fold increase of the induction level of F U S 1 in the presence of a - p h e r o m o n e (Table 4). F r o m these results, it was very clear that high copy numbers of S T E 5 0 increased sensitivity to a-pherom o n e and induction of cell type-specific transcription. The results also confirmed the conclusion that S T E 5 0 is required for activation of the mating response.

150

A oe f a c t o r

WT

B ste50-2

1/zg

1 /zg

C ste50-2 s.c.STE50 1 #g

Fig. 3A-E. The effects of multiple copies or disruption of the STE50 gene on a-pheromone sensitivity. Quantitation of growth inhibition on solid medium was tested by a-pheromone halo assay of lawns of MATa strains. Cells were plated in top agar on solid selective media and filter discs containing different amounts (in 4 gl aliquots) of synthetic a-factor (Sigma) were placed on the nascent lawn of

STE50 expression is not cell-type specific The STE50 gene was constitutively transcribed during vegetative g r o w t h o f M A T a, M A T a a n d M A T a / M A Ta cells. Its expression was relatively unaffected by pherom o n e s in M A Ta and M A T a cells as s h o w n by [3-galactosidase assay in STE50-lacZ gene fusion tests (Table 5) and R N A d o t - b l o t analysis (data n o t included). This response is different f r o m that o f m o s t genes k n o w n to function in the G protein-linked signal t r a n s d u c t i o n pathway.

D m.c.STE50

E m.c.STE50

1/zg

0.032/~g

cells and incubated at 30° C for 48 h. The amounts of synthetic a-factor added to each of the discs are indicated. Yeast strains: A WT, W303-1A; B ste50-2, WA-d2U; C ste50-2 s.c.STE50, WAd2U with the STE50 gene on CEN plasmid; D, E m.c.STE50 W303-1A with the STE50 gene on multicopy plasmid

a-Factor

~

-t-

WT

ste50-2

Further characterisation of the C-terminal truncation mutant ste50-2 Unlike the ste50-1 mutants, the C-terminal t r u n c a t i o n m u t a t i o n (ste50-2) affected b o t h transcriptional and cell cycle responses to m a t i n g factors. The ste50-2 m u t a n t s failed to induce FUS1 expression even in the presence o f high c o n c e n t r a t i o n s o f p h e r o m o n e (Table 4) a n d they did n o t u n d e r g o m o r p h o l o g i c a l changes in the presence o f p h e r o m o n e (Fig. 4). M A T a STE50 a n d M A T a ste50-1 strains arrest in G1 as uninuclear, u n b u d d e d cells when trated with high concentrations o f a - p h e r o m o n e . In con-

ste50-2 s.c. STE50

J

m.c.STE50 Table 5. Expression of STE50-1acZ

Strain

MA~ MA~ MA~/a MA~/a

STE50 and ste50-2 cells. Morphological changes of the exponentially growing yeast cells with different genotypes at the STE50 locus in response to 5 gM pheromone are shown. Ceils were fixed with formaldehyde and photographed with phase contrast optics (Zeiss). Yeast strains: WT, W303-1A; ste50-2, WA-d2U; ste50-2 s.c. STE50, WA-d2U with single copy of the STE50 gene carried on CEN plasmid pFL38H-BIG2; m.c. STE50, W303-1A with multiple copies of the STE50 gene carried on YEp351-BIG2 Fig. 4. a-pheromone induction of G1 arrest in

Units of activity Uninduced

Induced

1.2 1.2 0.7 0.7

1.3 1.4 0.8(apheromone) 0.7(a-pheromone)

The strains used are isogenic except for their mating type. All strains harbour plasmid pBIG2zl. The a-type cells were induced with 5 pM a-factor in the medium (pH 4.0). The a-type cells were induced with the medium in which the wild-type a cells had been grown. After 2 h most of the cells had arrested either as shmoos or unbudded cells

151 trast, M A Ta strains of the ste50-2 genotype continued to bud and all stages of the mitotic division cycle were present in cultures (Fig. 4). The continued budding of ste50-2 cells in the presence of pheromone was also observed in the halo assay for G1 arrest by a-pheromone (Fig. 3). While wild-type cells were arrested by 1 gg a-pheromone, ste50-2 cells efficiently formed colonies on plates containing 10 times more (10 ~tg) a-pheromone. Nevertheless, resistance of ste50-2 cells to a-pheromone appeared to be the result of hyperadaptation. In the halo assay, after 18 h at 28 ° C, the ste50-2 cells appeared to have begun to fill in the halo at all, indicating that the cells initially responded to pheromone but then became desensitised. These results indicated that the ste50-2 product interfered with the pheromone signal transduction pathway and presumably ste50-2 promoted cellular recovery, independently of the known G proteinlinked signal transduction pathway (see Discussion). We tested the interference properties of the ste50-2 product when overexpressed in wild-type cells. Plasmid pMG44-1 (YEpste50-2) was transformed into a wildtype strain, and the phenotype of the resulting transformants was checked. M A T a or M A T a cells carrying multicopy ste50-2 in the genetic background of STE50 were able to mate, but efficiency of zygote formation was in all cases lower than in wild-type cells; M A T a STE50 x M A Ta STE50 YEPste50-2 crosses resulted in a 50 % lower efficiency of zygote formation in comparison with wild type; in MA Ta STE50 YEPste50-2 x M A T a STE50 YEPste50-2 crosses, zygote formation reached 25% of the wild-type level. The halo assay showed a two-fold reduction in sensitivity of wild-type cells carrying the multicopy ste50-2 plasmid in comparison with isogenic wild type cells without the plasmid (results not shown). These findings suggested that the presence of normal Ste50 proteins establishes a condition that largely overcomes the mating pheromone response defect of the C-terminal truncation mutant ste50-2.

The ste50-2 mutation suppresses the growth arrest of cells overproducing STE4 (G0)

We tested whether the ste50-2 mutation, which eliminated normal response to mating pheromone, could also eliminate the galactose-induced arrest seen in strain M325-1B (Whiteway et al. 1990), which contains the STE4 gene under G A L l control. The URA3 disruption mutation ste50-2 was introduced into M325-1B and URA + transformants were tested for viability and fertility on SM galactose medium. The transformants were not arrested on galactose medium, they formed healthy colonies with cells exhibiting a normal morphology. The cells were defective in mating. By comparison the original M235-1B strain exhibited growth arrest on galactose medium and formed tiny colonies which contained primarily abnormally-shaped large cells (Fig. 5). Thus the ste50-2 mutation suppressed the phenotypic effects of overexpression of STE4. We then asked whether the ste50-2 mutation could also eliminate the growth arrest of scgl (gpal). Diploid

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Fig. 5. Survival of ste50-2 GAL1STE4 in galactose medium. Cultures of strains were grown on glucose to mid-log phase, then shifted to YEPGal media. The plates were then incubated at 30° C for 36 h. For photographythe cells were treated as in Fig. 3. Yeast strains: ste50-2 WA-d2U; GAL1-STE4, M325-1B; ste50-2 GAL1STE4, WA-1Bd2

D l l l cells (SCG1/scgl::LEU2; Dietzel and Kurjan 1987) were transformed with the appropriate fragments of plasmid YIF2, for the ste50::URA3 gene disruption (ste50-2), and of plasmid YIF200H, for the ste50: :HIS3 gene replacement (ste50-1). Haploid cells from tetrads of the URA3 transformants and HIS3 transformants were analysed. In all cases two spores in each tetrad produced normal colonies (the SCG1 phenotype), and the two other spores produced abnormally pear-shaped cells (corresponding to the scgl phenotype). Thus STE50 mutations do not suppress the growth defect of a SCG1 disruption mutant. Overexpression of STE50 had no effect on strains carrying disruptions of STE2, STE4, S T E l l or STE12 (data not shown). Furthermore the ste50-1 and ste50-2 mutations had no effect on constitutive growth arrest phenotype of overexpressed STE12 (data not shown). Since the ste50-2 mutation could not suppress the constitutive mating response phenotype of scgl strains, the ability of ste50-2 to suppress the constitutive mating response of G~ (STE4) overexpression must be dependent on SCG1 function. Functional interaction between STE50 and SST2

Several genes have been identified the products of which permit pheromone-treated yeast cells to recover from the effects of pheromone and enter into a new division cycle. These include SST2, which is required in both M A T a and M A T a cells (Chan and Otte 1982; Kurjan and Dietzel 1987). Yeast M A T a cells that carry the sst2-1 mutation cannot grow on plates containing a-pheromone because once arrested the cells do not recover, leading to an apparent supersensitive phenotype (Otte and Chan 1982). The SST2 gene product is proposed to act on the G~ (SCG1) subunit of the G protein (Miyajima et al. 1989; Kurjan et al, 1991). To determine the epistatic relationship between SST2 and STESO in the response to

152

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Fig. 6A, B. a-pheromone induction of G1 arrest and recovery after removal of pheromone by washing in sst2-1 and sst2-1 ste50-2 cells. A The recovery-promoting activity of the sst2-1 ste50-2 double m u t a n t in the presence of a-pheromone in the long term assay, and the effect of high copy numbers of STE50 on c~-pheromone response of sst2-1 cells. Quantitation of growth inhibition on solid medium was tested by the a-pheromone halo assay as described in the legend to Fig. 3. The amounts of synthetic c~ factor applied to each disk for sst2-1 and sst2-1 m.c. STESO strains were 0.8 ng (top zone), 4 ng (middle left), 8 ng (middle), 16 ng (middle right), 32 ng (bottom zone) and for the sst2-1 ste50-2 strain were 32 ng, 64 ng, 128 ng, 250 ng, 500 ng. Plates were incubated for 48 h. B Exponentially growing cells were incubated for 2 h in 5 g M a-pheromone at 30 ° C

sst2-1 ste50-2

and washed three times with three volumes of fresh medium, and then resuspended in 1 volume of fresh medium. The MATa sst2 cells showed 100% shmoos after pheromone removal and 10 h incubation in fresh medium, whereas 10% of the MATa sst2 ste50 cells had started the new cell cycle by this time. Open squares, untreated cells; closed circles, cells incubated in 2 gM c~-factor; open circles, a-pheromone-treated cells after washing and further incubation in fresh medium. Yeast strains: WT, W303-1A; sst2-1, 7680-8-1; sst2-1 steSO-2, 7680 ste50; sst2-1 m.c. STE50, 7680-8-I with STE50 gene on multicopy plasmid

mating pheromones, an sst2-1 Y E p S T E 5 0 strain was constructed. This was made by transforming plasmid Y E p S T E 5 0 into a sst2-1 strain. A double mutant M A T a sst2-1 steSO-2 strain was constructed by transplacing the ste50." .'URA3 deletion allele into the sst2-1 strain. Interestingly, the overexpression of STESO in the sst2-1 strains had no effect on the supersensitivity of these cells, as judged by the a-pheromone halo assay, but the sst2-1 Y E p S T E 5 0 cells generally grew better than the sst2-1 parental cells (Fig. 6A). The control experiment showed that overexpression of STE50 in wild-type cells caused a forty-fold increase in sensitivity to a-pheromone compared with the expression of a single copy of STE50 (Fig. 3). The steSO-2 sst2-1 double mutant was able to overcome pheromone-induced arrest and could resume growth, as judged by the increased turbidity of the halos (Fig. 6A); sst2-1 cells were not able to overcome arrest under the same condition. Using an additional assay for suppression of the ste50-2 mutation by sst2-1, we asked whether the double

mutant could mate. In contrast to the ste50-2 mutants, which were a- and a-pheromone resistant and are sterile, the M A T a sst2-1 steSO-2 double mutant (strain 7680 ste50) formed diploids in crosses with M A T a sst2-1 ste50-2 cells (strain MGS-B) at a rate ten times lower than that of wild-type cells, as shown by zygote formation assay and diploid selection assay (data not shown). This means that an sst2-1 mutation partially suppresses the mating defect of the ste50-2 mutation. The ste50-2 sst2-1 mutant was no longer supersensitive to a-pheromone-induced arrest after removal of pheromone by washing. In Fig. 6B it is show that all ste50-2 sst2-1 cells started a new cell cycle (budding) after removal of pheromone and 10 h incubation in fresh medium. In contrast, the sst2-1 strain remained division-arrested after the same treatment. Thus, the ste50-2 mutation suppressed the desensitisation resistance o f the sst2-1 mutant. These results support the hypothesis that the ste50-2 mutation promotes the recovery process through the action of the G~ subunit of the G protein.

153 Discussion

In this report we describe the genetic characterisation of the STE50 gene found in the course of sequencing chromosome III of Saccharomyces cerevisiae. STE50 is a very interesting gene that is required for cell differentiation. The STE50 gene is located distal to HIS4 on chromosome III, and encodes a putative hydrophilic 346-residue protein, rich in serine and theronine residues, which displays no striking similarity with known proteins in the current data bases that might suggest its potential function. STE50 expression is not cell type specific and its expression in MATa and MATa cells is unaffected by pheromones. When present on a high copy number plasmid, STE50 causes supersensitivity to a-pheromone, and increases the level of a-pheromone-induced transcription of the mating gene FUS1 in haploid a cells. Mutants bearing either of two gene disruptions, ste50-1 or ste50-2, are sterile and have an altered sensitivity to a-pheromone. The ste50-1 mutant is only twice as resistant to a-pheromone-mediated G1 arrest as the wild type, and exhibits a defect in mating despite retaining an intact signal transduction pahtway. The ste50-2 mutation, which is a C-terminal truncation (ste50: :URA3, a duplicated disrupted ste50 gene deleting 105 out of 346 amino acid residues plus N-terminal deletion) of the STE50 gene, interferes with the mating signal transduction pathway and results in a pheromone response defect. The level of the mating defect observed (0.2 to 1.5%) when both ste50-1 and ste50-2 are crossed with wild-type cells (Tables 2 and 3) suggests that, at least initially, the defect in ste50-2 signalling does not greatly diminish mating ability. Recently a mutation has been created (ste50-3) by insertion-deletion which has removed the entire open reading frame. This allele (ste50-3) mimics the behaviour of ste50-2 as measured by sensitivity to a-pheromone and zygote formation assay (Xu and Ramezani Rad, unpublished results). The primary defect of the C-terminal truncation allele of STE50 appears to be a defect in the early response to a-pheromone (e.g. induction of FUS1). It is possible that this defect of ste50-2 may be rescued in the ste50-1 mutant by a related function. The fact that there is no significant difference between the ste50-1 and ste50-2 alleles in the mating assay is more consistent with a lesion in communication and polarisation of mating partners than in signalling. This is also observed for mutants that allow mating without receptors (Jahng et al. 1989; Jackson et al. 1991). Further analysis will show if there is any interaction between ste50 mutants and the ste2 (receptor) mutants, which define the other component of this system. Yeast cells exposed to mating factors recover from G1 arrest after a period of time and resume growth. Thus arrest is transient. This adaptation response appears to function by several independent pathways and to involve the receptor, G~, G~ and the SST2 gene. Miyajima et al. (1989) have proposed that the activated G~ subunit (G~-GTP) provokes a recovery process. Cole et al. (1991) recently reported that the Ste4 protein (G~) is rapidly phosphorylated after treatment of a cells with a-factor.

This phosphorylation appears to play a role in adaptation based on the observation that deletion of a segment of STE4 eliminates pheromone-induced phosphorylation and causes cells to become hypersensitive to mating factors. It has been further shown that an intact G~ subunit is required for this phosphorylation. The results of the a-pheromone halo test (Figs. 4 and 6) indicate that the ste50-2 cells may have a defect in adaptation (hyperadaptation), rather than in the pheromone signalling pathway, and the low expression level of FUSI-lacZ after 2 h may be the result of hyperadaptation to apheromone. Thus ste50-2 cells are not just defective in mating, the adaptation response may also be affected (hyperadaptation). This interaction might be at the level of modification of G protein function. In this study we have shown that ste50-2 suppressed the constitutive mating response phenotype associated with STE4 overexpression, and promoted cellular recovery; however, in cells defective in the GPA1 (SCG1) gene, the presence of the ste50-2 does not suppress the constitutive mating response phenotype. It is possible that the negative effects of the C-terminal truncation allele of STE50 on signalling require GPA1, suggesting a possible interaction between Ste50 and the G~ subunit of the G protein. We have also demonstrated that the ste50-2 mutation partially suppresses the desensitisation defect of the sst2-1 mutation, whereas the sst2-1 mutation partially restores fertility and pheromone sensitivity to ste50-2 cells. The SST2 gene product is proposed to act on G~ or some other G protein subunit since certain G~ mutant alleles are epistatic to sst2 mutations (Miyajima et al. 1989; Kurjan et al. 1991). The finding that sst2-1 and ste50-2 mutually suppress each other suggest that both may act on G p a l (Scgl). An example of such dual control is known in the case of CDC25 and IRA both of which act on the Ras protein (Tanaka et al. 1989). Acknowledgements. We are grateful to S.G. Oliver, P.P. Slonimski, F. Lacroute, M. Ciriacy, J. Ernst, G. Fink, R. Roggenkamp, J. Heinisch, W. Duntze, C.L. Jackson, D.Y. Thomas, M. Whiteway, and S. Fields for providing plasmids and strains. R. Wells, R. K611ing and U.J. Santore are acknowledged for their critical reading of the manuscript and D.Y. Thomas for comments on the manuscript. This work was supported by the EC BAP programme and the Bundesminister ffir Forschung und Technologie. References

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STE50, a novel gene required for activation of conjugation at an early step in mating in Saccharomyces cerevisiae.

A new gene, STE50, which plays an essential role in cell differentiation in Saccharomyces cerevisiae was detected and analysed. STE50 expression is no...
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