Mol Gen Genet (1992) 235:122-130

OIGXI

© Springer-Verlag 1992

Functional conservation between Schizosaccharomycespombe ste8 and Saccharomyces cerevisiae STEll protein kinases in yeast signal transduction Unnur Styrkfirsd6ttir, Richard Egel, and Olaf Nielsen Institute of Genetics, University of Copenhagen, Oster Farimagsgade 2A, DK-1353 Copenhagen K, Denmark ReceivedApril 10, 1992 / AcceptedJune 2, 1992

Summary. In fission yeast (Schizosaccharomyces pombe), the marl-Pro gene, which is required for entry into meiosis, is expressed in response to a pheromone signal. Cells carrying a mutation in the ste8 gene are unable to induce transcription of marl-Pro in response to pheromone, suggesting that the ste8 gene product functions in the signal transduction pathway. The ste8 + gene encodes a 659 amino acid putative protein kinase, which is identical to the previously identified byr2 suppressor of the rasl defect. Furthermore, ste8 + is highly homologous to the Saccharomyces cerevisiae S T E l l gene, which functions in signal transduction in budding yeast. Expression of the S. cerevisiae S T E l l gene in S. pombe ste8 mutants restores the ability to transcribe marl-Pro in response to pheromone. Also, such cells become capable of conjugation and sporulation. When matl-Pm is artifically expressed from a heterologous promoter, ste8 mutant cells will enter meiosis. This demonstrates that the meiotic defect of ste8 mutants is due to the absence of the matl-Pm gene product. Key words: Schizosaccharomyces pombe Saccharomyces cerevisiae - Signal transduction Sexual differentiation - Protein kirlase

Introduction Unicellular fungi provide an attractive system for studying eukaryote signal transduction. Both the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe undergo differentiation during which cells of opposite mating type communicate by diffusible peptide pheromones. Because of the ease with which molecular genetic techniques can be applied to these organisms, it is possible to reveal the principal Offprint requests to: U. Styrk~rsd6ttir, Institute of Genetics, University of Copenhagen, Oster Farimagsgade 2A, DK-1353 Copenhagen K, Denmark. Phone: 453311 5446, Fax: 4533140375.)

features of the signal transduction pathway, beginning with pheromone/receptor binding and eventually leading to a change in the physiological state of the cell. In S. cereviseae, pheromone communication is essential for the process of mating, which ensures cell diploidization. Haploid cells of budding yeast can exhibit either of two complementary mating types, a or c~. Each cell type secretes a unique signaling peptide, a-factor or efactor (Thorner 1981), and displays at its surface a receptor that can bind the pheromone produced by the other cell type (Burkholder and Hartwell 1985; Nakayama et al. 1985; Hagen et al. 1986). The generation of a diploid a/e cell is the consequence of the bidirectional pheromone communication between a and ~ cells. The effect of a pheromone signal on cell physiology is brought about by transcriptional induction of a number of pheromone-responsive genes (Trueheart et al. 1987; McCaffrey et al. 1987). Apart from the pheromone-specific receptors themselves, this signal transduction pathway appears to be identical in a and c~cells (Bender and Sprague 1986; Nakayama et al. 1987). Both receptor types are coupled to the same heterotrimeric G protein, which is presumed to become activated by a conformational change induced by pheromone-receptor interaction (Dietzel and Kurjan 1987; Miyajima et al. 1987; Nakafuku etal. 1987; Jahng etal. 1988; Whiteway etal. 1989). The immediate target of the activated G protein has not been identified, but four different protein kinases, encoded by the genes STE7, S T E l l , KSS1 and FUS3, as well as the STE5 gene product are required for signal transmission (Teague et al. 1986; Nakayama et al. 1988; Courchesne et al. 1989; Rhodes et al. 1990; Elion et al. 1991). The protein kinases appear to constitute a phosphorylation cascade, which eventually phosphorylates and activates the STE12 transcription factor, thus completing the signalling pathway by allowing transcriptional induction of pheromone-responsive genes (Dolan and Fields 1990; Song et al. 1991). Cells of the distantly related fission yeast S. pombe also exhibit one of two complementary mating types, P or M. These two cell types can communicate by the

123 two diffusible pheromones, P factor and M factor (Fukui et al. 1986; Leupold 1987). Since S. pombe is essentially a haploid organism, mating activities are induced only under conditions of nutritional starvation, which also promotes meiosis of diploid cells (Egel 1971, 1973). A consequence of this co-regulation of mating and meiosis is that not only the process of mating but also entry into meiosis requires a pheromone signal in fission yeast (Leupold et al. 1989). This is because the m a t l - P m gene, which is required for entry into meiosis, is transcribed in response to a pheromone signal (Nielsen et al. 1992). This provides the first example of a pheromone-controlled gene in S. pombe. Certain components of the fission yeast pheromone communication system have been molecularly characterized, and they appear to resemble those found in budding yeast. The M factor is a modified peptide with an overall chemical structure similar to that S. cerevisiae a factor (Davey 1992). The structure of the P factor is presently unknown. The mam2 gene, which encodes the P-factor receptor, shares sequence homology with the budding yeast S T E 2 gene encoding the a-factor receptor (Kitamura and Shimoda 1991). Recently, gpal, a gene encoding an S. pombe homolog of mammalian and budding yeast G~ proteins was identified (Obara et al. 1991). The S. pombe G~ subunit has a positive function in the signal transmission process. This is opposed to the S. cerevisiae Ga, the G P A l - e n c o d e d protein, which acts in a negative fashion (Miyajima et al. 1987; Jahng et al. 1988). A homolog of the K S S 1 and FUS3 protein kinases, encoded by the spkl, gene, has also been identified in fission yeast (Toda et al. 1991). In an attempt to identify additional components of the S. pombe signal transduction pathway, we have isolated the ste8 gene. The ste8 + function is required for pheromone-directed mat-Pm transcription, demonstrating that mutant cells are defective in the pheromone response pathway. The ste8 gene is capable of encoding a 659 amino acid putative protein kinase that shares extensive sequence homology with the S. cerevisiae S T E l l protein kinase. Since the S T E l l gene can complement the ste8 defect, this signal transduction kinase function appears to be conserved in evolution.

Materials and methods Yeast strains, media and genetic methods. The yeast strains used in this study are listed in Table 1. Standard genetic procedures were according to Moreno et al. (1991). Strains were grown either in complete medium (YEA/YEL) or minimal medium MSA, for detection of mating and sporulation, or PM and PM-N for physiological experiments. The ste8.': ura4 +/ste8 and ste8.':ura4+/ste3 strains were constructed by protoplast fusion of strain Eg569 with strains JM86 and JM66, respectively. Protoplast fusions were essentially as described (Nadin-Davis and Nasim 1990). The homozygous ste8 diploid strain, harbouring the pmatl-Pm plasmid, was isolated from plates containing phloxin B. The A m a t 2 , 3 : : L E U 2 + deletion was described by Klar and

Table 1. Schizosaccharomyces pombe strains

Strain

Genotype

Source/Reference

L968 JM66

h~

JM86

h 9° ste8 l e ul

JY736

h 9° s t e l l - 2 9 l e ul a d e 6 - M 2 1 0

SP329 SP330 Eg325

m a t l - M A m at 2,3: : L E U 2 + leu1.32 rnat-P A m at 2,3: : L E U 2 + l e ul .32 h 9° ura4-D18

Eg460 Eg467 Eg468 Eg569

h9° ura4-D18 ade6 h9° ste8 ura4-D18 h9° ste8 h9° ste8: :ura4 + ura4-D18 ade6

U. Leupold Michael and Gutz 1987 Michael and Gutz 1987 Sugimoto et al. 1991 A. Klar A. Klar Nielsen et al. 1992 This study This study This study This study

h9°ste3~ul

Miglio (1986) and the ura4-D18 deletion by Grimm et al. (1988). Cloning o f the ste8 gene. The ste8 gene was isolated by transformation of the S. pombe strain Eg467 (h 9° ste8 ura4-D18) with a partial Sau3AI digested of S. pombe genomic D N A cloned into pON163 (Weilguny etal. 1991), selecting for Ura + transformants. Approximately 50000 transformants were screened for the iodine-positive phenotype indicating formation of spores and, thus, complementation of the chromosomal ste8 mutation. Three complementing transformants were obtained. The plasmids recovered from these transformants all contained the same 2.59 kb insert. For further analysis, the complementing insert was cloned into the fission yeast vector pDW232 (Weilguny et al. 1991), generating the ste8 + plasmids pUS71 and pUS72, pUS71 and pUS72 contain ste8 + on a PstI fragment inserted in opposite orientations. Nucleotide sequence determination and analysis. A series

of nested deletions were created with exonuclease III (Henikoff 1984) in the plasmids pUS71 and pUS72. Nucleotide sequencing was performed with the dideoxy chain-termination method on double-stranded D N A (Sanger et al. 1977; Hattori and Sakaki 1986) using a Sequenase kit (United States Biochemical). Primers for the T7 and SP6 polymerase sites in these plasmids were used for sequencing. The complete sequence of the 2.59 kb complementing insert was determined in both directions. Gene disruption. For construction of a ste8 null allele, a HindIII-EcoRV ste8 + fragment was cloned into t h e HindIII-SmaI sites of pGEM3 (Promega). A 281 bp XbaI fragment was removed from the open reading

frame (ORF), and replaced by a 1.8 kb fragment containing the ura4 + gene (Grimm et al. 1988). A linear HindIII-EcoRI fragment, containing the disrupted ste8 allele, was used to transform the homothallic haploid strain Eg460 (h 9° ura4-D18 ade6). Most of the stable Ura + transformants were sterile. Precise replacement of

124

the wild-type allele by the disrupted gene was confirmed by Southern analysis of several Ura + transformants.

1

2

3

4

5

6

Southern analysis. Restriction enzyme digested genomic D N A was electrophoresed on a 0.7% agarose gel and blotted onto a nylon membrane (Hybond-N). Hybridization was done with a DIG-dUTP-labeled E c o R V ste8 + D N A fragment according to the Boehringer-Mannheim protocol. Detection of hybridized fragments was done with the chemiluminescent substrate AMPPD (Boehringer Mannheim). Northern analysis. R N A was prepared from exponentially growing S. pombe cells (at a density of 5 x 106/m1-1 x 10V/m1) according to the method for Sherman et al. (1986), modified for S. pombe by Nielsen and Egel (1990). Cells were grown in PM medium at 30°C to a density of 5 x 106/ml. R N A was prepared from half of this culture while the other half was transferred to P M - N medium and grown for a further 4-6 h before R N A preparation. R N A samples (10 gg) were run on 1.0% or 1.5% formaldehyde gels and blotted onto Hybond-N membranes (Amersham). The filters were hybridized to single-stranded R N A probes as described (Nielsen et al. 1992). The m a t l - P m specific probe has been described previously (Nielsen and Egel 1990). A single-stranded ste8 + specific probe was obtained by in vitro transcription with SP6 R N A polymerase of pUS70, which contains a HindIII-PstI subclone of pUS71 in pGEM3 (Promega). pUS70 was linearized with SalI prior to transcription. Construction o f p m a t l - P m . The m a t l - P m ORF was fused to the inducible nmt promoter (Maundrell 1990). The nmt promoter is under thiamine-mediated transcriptional control. In the presence of thiamine, transcription from the promoter is repressed, while absence of thiamine results in a large increase in transcription. Thiamine was added to a concentration of 2 gM when required. The m a t l - P m coding sequence was isolated from the plasmid pART6-Piadh (Kelly et al. 1988). A N d e I SacI fragment, containing the m a t l - P m ORF, was cloned into the NdeI-SacI sites in the plasmid pREP2 (K. Maundrell, personal communication) to give pmatlPm.

4

matl -Pm !!

Fig. 1. Northern blot analysis of the effect of ste8 on transcriptional induction of m a t l - P m . Lanes 1 and 2, h 9° wild type strain (L968); lanes 3 and 4, h 9° ste8 mutant strain (Eg468); lanes 5 and 6, h 9° ste8 mutant strain (JM86) harbouring the Saccharomyces cerevisiae pSTE11.1 plasmid. Odd-numbered lanes, vegetatively growing cells; even-numbered lanes, cells starved for nitrogen. The hybridization probe was a 32p-labeled m a t l - P m specific probe. The two upper bands present in all six lanes represent unspecific hybridization to 25S and 18S ribosomal RNA. Equal loading to RNA was confirmed by ethidium bromide staining of the gel (data not shown)

A

tl 90

B hg°ste8

C

hg°ste8 + pSTE11.1

D h9°ste8/h9°ste8 + pmatl-Pm

Results

ste8 mutants are not able to induce transcription of matl-Pm Several different S. pombe ste mutants have been characterized, which are completely defective in the process of sexual differentiation (Thuriaux et al. 1980; Girgsdies 1982; Michael and Gutz 1987; Kitamura et al. 1990; Leupold and Sipiczki 1991). Of 11 mutants tested (stets t e l l ) all, expect stelO, are unable to respond morphologically to mating pheromones (Leupold et al. 1991), suggesting that their phenotype might be caused by a defect in the signal transduction pathway. Recently, it

Fig. 2A-D. Functional complementation of ste8. Cells were grown on MSA plates and micrographs were taken after 2 days (A, B, C) or 4 days (D) of incubation at 30° C. A L968; B Eg468; C JM86 harbouring the S. cerevisiae pSTEll.1 plasmids; D the ste8/ ste8 diploid strain Eg467-2n, carrying the pmatl-Pm plasmid grown without thiamine. Photographs were taken with Nomarski optics

125

has been established that transcription of the m a t l - P m gene requires a pheromone signal (Nielsen et al. 1992). Since this provides a much more specific assay for the identification of mutants that are defective in signal transduction, it was of interest to see whether cells carrying various ste mutations are able to induce transcription of the matl-Prn gene. We found that loss of the ste8 + function did indeed abolish the ability of homothallic strains to induce transcription of the m a t l - P m gene (Fig. 1). Since the M cells of the homothallic h 9° ste8 strain still produce the M factor (Leupold et al. 1991), the P cells should transcribe the marl-Pro gene, if their signal transduction pathway is intact. However, in contrast to the wild-type strain, this is not observed with the ste8 strain (Fig. 1). Hence, we conclude that ste8 mutants are defective in the pheromone response pathways. Expression of the m a t I - P m gene is required for entry into meiosis (Egel 1973; McLeod et al. 1987). We therefore considered the possibility of restoring meiosis in a diploid ste8/ste8 strain by artificially expressing the matI-Prn gene from a heterologous promoter. The matlPm gene was cloned under the control of the inducible nmt promoter (Maundrell 1990). This construct was transformed into a diploid ste8/ste8 strain and the nmtI promoter was activated by shifting the cells to medium without thiamine. This resulted in a measurable level of meiosis and sporulation in these cells (Fig. 2). This demonstrates that the defect in meiosis in the ste8 mutant is caused by a failure to induce transcription of the rnatl-Prn gene.

Isolation and characterization of the ste8 gene

We cloned the ste8 gene by screening an S. pombe genomic library for clones that were able to restore normal

A

1 kb

sexual differentiation in a ste8 mutant strain. Among approximately 50000 transformants, three complementing transformants were obtained. The plasmids recovered from these transformants all contained the same 2.59 kb insert (Fig. 3). The nucleotide sequence of the insert was determined. This revealed a major uninterrupted reading frame of 1977 bp, capable of coding for a 659 amino acid protein (Fig. 4). Comparison of this sequence with the sequence of the byr2 + gene (Wang et al. 1991), described earlier as capable of complementing the ste8 mutation, shows that this is indeed the same gene. A disrupted allele of ste8 was constructed by replacing a 281 bp fragment of the ste8 ÷ gene with the ura4 + marker gene. Because the ste8 function was not expected to be essential for cell viability, the disruption was carried out in a haploid strain. A DNA fragment containing the disrupted allele was transformed into the homothallic strain Eg460 (h 90 ura4-D18 ade6). Replacement of the wild-type ste8 gene by the disrupted fragment was confirmed by Southern analysis of several Ura + transformants (Fig. 3). The disrupted strain is completely sterile but otherwise has no phenotype. In order to demonstrate that the cloned gene was in fact ste8 +, the strain carrying the disrupted allele was protoplast-fused to the original ste8 mutant strain. The resulting ste8::ura4+/ste8 diploids were not able to sporulate. As a positive control, we fused the strain carrying the disrupted allele to a ste3 mutant strain (Michael and Gutz 1987), creating ste8:: ura4+/ste3 diploid cells. These sporulated upon nitrogen starvation and gave rise to h 90 progeny. These results demonstrate that the ste8 + gene had indeed been cloned, and not an unlinked suppressor. Furthermore, the byr2 gene described by Wang et al. (1991) is allelic to ste8. We shall refer to this gene as ste8, since this was its first designation (Michael and Gutz 1987).

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Fig. 3. A Restriction map of the ste8 gene. The extent and direction of the ste8 open reading frame is shown by an arrow. The cloned insert is indicated by a solid line, whereas the dashed line indicates genomic D N A of the ste8 locus not included in the cloned insert. The linear fragment used to disrupt the ste8 gene is shown at the bottom of the figure. Restriction enzymes : Ac, AccI; Av, AvaI; B, BclI; E, EcoRI; H, HindIII; R, EeoRV; S, SspI; X, XbaI. B Southern blot analysis of genomic D N A prepared from wild-type

strain EG325 (h 9° ura4-D18; lanes 1 and 2) and a ste8 disruptant strain Eg569 (lanes 3 and 4). D N A was digested with HindIII (lanes 1 and 3) and EeoRV (lanes 2 and 4). The hybridization probe was a DIG-dUTP labeled EcoRV ste8 + fragment. The 2.4 kb and 1.8 kb bands correspond to the intact ste8 + allele and the 4.2 kb, 1.9 kb and 1.4 kb bands derive from the disrupted allele. The filter was reprobed to an ura4-specific probe to confirm these results (data not shown)

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The S. cerevisiae gene STEI 1 complements the ste8 mutation It has already been noticed that the byr2/ste8-encoded protein shares h o m o l o g y with several serine-threonine

type protein kinases (Wang et al. 1991). We have compared the deduced amino acid sequence of the ste8-encoded protein with the sequences present in the data banks. This analysis revealed that the ste8 sequence is highly h o m o l o g o u s to the S. cerevisiae protein kinase

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Functional conservation between Schizosaccharomyces pombe ste8 and Saccharomyces cerevisiae STE11 protein kinases in yeast signal transduction.

In fission yeast (Schizosaccharomyces pombe), the mat1-Pm gene, which is required for entry into meiosis, is expressed in response to a pheromone sign...
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