Mutagenesis of Stel8, a putative GY subunit in the Saccharomyces cerevisiae pheromone response pathway1 MALCOLM WHITEWAY

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Eukaryotic Genetics Group, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, Que., Canada H4P 2R2 and Biology Department, McGill University, I205 Dr. Penfield Avenue, MontrPal, Que., Canada H3A IB1

DANIEL DIGNARD Eukaryotic Genetics Group, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal. Que., Canada H4P 2R2 AND

DAVIDY. THOMAS Eukaryotic Genetics Group, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, Que., Canada H4P 2R2 and Biology Department, McGill University, 1205 Dr. Penfield Avenue, MontrPal, Que., Canada H3A 1BI Received March 12, 1992 WHITEWAY, M., DIGNARD, D., and THOMAS,D. Y. 1992. Mutagenesis of Stel8, a putative GY subunit in the Saccharomyces cerevisiae pheromone response pathway. Biochem. Cell Biol. 70: 1230-1237. The yeast STEI8 gene product has sequence and functional similarity to the Y subunits of G proteins. The cloned STEI8 gene was subjected to a saturation mutagenesis using doped oligonucleotides. The populations of mutant genes were screened for two classes of STE18 mutations, those that allowed for increased mating of a strain containing a defective STE4 gene (compensators) and those that inhibited mating even in the presence of a functional STEI8 gene (dominant negatives). Three amino acid substitutions that enhanced mating in a specific STE4 (GP) point mutant background were identified. These compensatory mutations were allele specific and had no detectable phenotype of their own; they may define residues that mediate an association between the GP and GY subunits or in the association of the G f l subunit with other components of the signalling pathway. Several dominant negative mutations were also identified, including two C terminal truncations. These mutant proteins were unable to function in signal transduction by themselves, but they prevented signal transduction mediated by pheromone, as well as the constitutive signalling which is present in cells defective in the GPAI (Ga) gene. These mutant proteins may sequester GP or some other component of the signalling machinery in a nonfunctional complex. Key words: yeast, G protein, STE18, mutagenesis, pheromone response.

M., DIGNARD,D., et THOMAS,D. Y. 1992. Mutagenesis of Stel8, a putative GY subunit in the WHITEWAY, Saccharomyces cerevisiae pheromone response pathway. Biochem. Cell Biol. 70 : 1230-1237. Le produit du gtne STE18 de la levure a une sequence et une fonction similaires a celles des sous-unites Y des proteines G. Le gene STE18 clone a Cte mute par saturation en utilisant des oligonuclCotides partiellement dtgenkres. Les populations de gtnes mutes ont ett criblees afin de trouver deux categories de mutations STE18, celles permettant un accroissement de l'accouplement d'une souche contenant un gtne STE4 defectueux (mutations compensatoires), et celles inhibant l'accouplement mCme en presence d'un gtne STE18 fonctionnel (mutations ntgatives dominantes). Trois substitutions d'acides arnints qui augmentent l'accouplement de souches contenant une mutation ponctuelle spkcifique dans le gene STE4 (GB) ont it6 identifiks. Ces mutations compensatoires sont specifiques d'un alltle et n'engendrent pas par elles-mCmes de phenotype detectable. Elles pourraient coder des rksidus qui interviennent dans l'association des sous-unites GO et GY ou dans l'association de la sous-unite G f l avec d'autres tlkments du mecanisme de signalisation. Plusieurs mutations negatives dominantes ont egalement t t t identifiees, dont deux o i les ~ extrtmitts C sont tronquees. Ces protkines mutantes sont incapables elles-m&mesde participer a la transduction du signal, mais elles emptchent la transduction du signal engendrk par une pheromone et la signalisation constitutive obsente dans les cellules ayant une deficience du gtne GPAI (Ga). Ces protkines mutantes pourraient skquestrer GP ou un autre eltment du mecanisme de signalisation en formant un complexe non fonctionnel. Mots cles : levure, proteine G, STEI8, mutagentse, rtponse A une pheromone. [Traduit par la redaction]

Introduction A wide of eukar~oticsignal h2llsduction pathways commence at the cell surface with a receptor protein which is coupled to an intracellular transducer molecule termed a G protein (Simon et 1991). The G protein is composed ABBREVIATIONS: SDS, sodium dodecyl sulfate; YEPD, yeast extract - petone dextrose; bp, base pairs. 'NRC 33646. Printed in Canada / Imprime au Canada

of three different subunits, designated Ga, GP, and GY. The Ga subunit can bind the guanine nucleotides GDP or GTP, and G~ subunits, hence the name protein, while the which appear to always remain tightly associated, function essentiallyas a single subunit (Fung 1983; Neer and Clapham 1988). The association of a specific ligand with the receptor leads to a conformational change in the G protein complex; G D P , which is bound to the Ga subunit in the unactivated

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TABLE1. Strains used in this study Strain

Genotype

M200-6C M200-6C ste4-25 M200-6C ste4-14 M200-6C ste4-19 M200-6C ste4-114 M200-6C stel8::ura3 M325-1B W303-1A stel8::LEU2 Dl11

adel ura3 ilv3 sstl sst2 a adel ura3 ilv3 sstl sst2 ste4-25 a adel ura3 ilv3 sstl sst2 ste4-14 a adel ura3 ilv3 sstl sst2 ste4-19 a adel ura3 ilv3 sstl sst2 ste4-114 a adel ura3 ilv3 sstl sst2 stel8::ura3 a ade2 ura3 leu2 his3 trpl can1 [HIS3 GALI::STE4]a ade2 ura3 leu2 his3 trpl can1 stelb::LEU2 a ade2 ura3 leu2 his3 trpl can1 scgl::LEU2 a ade2 ura3 leu2 his3 trpl can1 + a his3 ura3 leu2 stel8::LacZ a his1 a

S11 DC17

state, is exchanged for GTP, and the GOYsubunits dissociate from the Ga subunit. This dissociated, activated G protein serves to stimulate a second message generating system such as adenylyl cyclase, which in turn leads to the modulation of a specific intracellular activity (Gilman 1987). The components of mammalian G proteins are known to take part in many interactions. As noted, the GO and GY subunits always remain tightly associated with each other, and this complex interacts in a reversible manner with the G a subunit (Neer and Clapham 1988). In addition, GY subunits undergo a posttranslational isoprenylation at the carboxyl terminus, which increases their hydrophobicity and may facilitate association with the plasma membrane (Simonds et al. 1991). The G protein complex also associates with the receptor molecule and this association modifies the receptor's affinity for its ligand (Gilman 1987). The activated G protein also interacts with effector enzymes such as adenylyl cyclase and cGMP phosphodiesterase to modulate their activity. Finally, there may be as yet unidentified proteins that associate with the G protein complex. RAS and other small GTPases have been found to be regulated by a variety of proteins that control the rates of GTP hydrolysis and guanine nucleotide exchange (Evans et al. 1991); similar regulatory activities may exist for heterotrimeric G proteins. In the yeast Saccharomyces cerevisiae the mating of a and a haploid cell types to create a diploid cell type is orchestrated by the extracellular peptides a and a pheromone which are secreted by each cell type. These pheromones serve to arrest cells of the opposite mating type in the G, phase of the cell cycle and to induce a series of gene products which prepare the cell for mating (Kurjan and Whiteway 1990; Marsh et al. 1991). The facility with which yeast can be genetically manipulated and the fact that mating is not essential for vegetative growth have made possible the identification of many of the genes involved in this process. The cloning and sequencing of these genes, together with physiological evidence, have shown that the response of yeast to mating pheromones involves a signal transduction pathway (Kurjan and Whiteway 1990; Marsh et al. 1991). This pathway includes cell surface receptors for the a or a peptide pheromones; these receptors are structurally similar to the mammalian receptor proteins that are found coupled to G proteins (Burkholder and Hartwell 1985; Nakayama et al. 1985; Hagen et al. 1986). Yeast cells also contain the components of a heterotrimeric G protein: the GPAI (SCGI) gene codes for the G a subunit (Dietzel and Kurjan

Ref. Whiteway et Whiteway el Whiteway et Whiteway et Whiteway et This work This work This work J . Kurjan

al. al. al. al. al.

1988b 1988a 1988a 1988a 1988a

Finegold et al. 1990 Whiteway et al. 1987

1987; Miyajima et al. 1987), the STE4 gene codes for the GO subunit, and the STE18 gene encodes the GY subunit (Whiteway et al. 1989). The same heterotrimeric G protein is functionally coupled to the cell type specific pheromone receptors encoded by the STE2 and STE3 genes (Kurjan and Whiteway 1990; Marsh et al. 1991). Much of our current understanding of the interactions of the G protein subunits with each other and with other components of signal transduction pathways have come from direct biochemical studies of mammalian G protein subunits (Gilman 1987). However, evidence for proteinprotein interactions and identification of specific regions and residues involved in those interactions can also be obtained through mutagenesis. If mutagenesis of one component of an interaction leads to a measurable phenotype, then isolation of compensating mutations may identify other participants in the interaction. The advantage of the yeast system for such an approach is that the consequences of the interaction and its alteration can be studied in vivo. As the G protein involved in mating is the only heterotrimeric G protein system identified in yeast, the results of such studies might be expected to yield unequivocal answers about the interacting components. Therefore, we have initiated a genetic investigation into the interactions of the components of the yeast G protein complex. In this paper we have used saturation mutagenesis of the cloned GY subunit to study its role in the mating pheromone signal transduction process. We demonstrate that mutations which define its interaction with the GO subunit and other mutants which define interactions with other components of the signal transduction pathway can be isolated. Materials and methods Yeast strains The strains used in this study are listed in Table 1. Standard procedures were used for strain construction and identification (Sherman et al. 1982). Strain M325-IB contains a galactose inducible STE4 gene stably integrated into the yeast genome at the HIS3 locus. Plasmid M135p3 contains a GAL1 promoter driving expression of the STE4 gene (Whiteway et al. 1990) cloned as an EcoRI to SalI fragment into the polylinker of plasmid pRS303 (Sikorski and Heiter 1989). This integrating plasmid was targeted (OrrWeaver et al. 1981) to the HIS3 locus of strain W303-1B by partial cleavage with BstXI and HIS3 + colonies that arrested in response to growth on galactose were selected. One such colony was crossed to the isogenic W303-1A strain to create diploid DM325 and meiotic products containing the GALl::STE4 construct were isolated after sporulation and tetrad dissection.

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Strain M200-6C stel8::ura3 was constructed in two steps. First a Hind111 to SphI fragment containing a URA3 disruption of the STEI8 gene was transformed into strain M200-6C and sterile, URA3 transformants were selected. Then a ura3 - derivative of this strain was selected on plates containing 5-fluoro-orotic acid (Boeke et al. 1984). +

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Yeast transformation The lithium acetate transformation protocol of Ito et al. (1983) was used. Media Standard media were used (Whiteway et al. 19886). Pheromone response Two procedures were used to measure pheromone responsiveness. In the plate assay, plates containing appropriate media were spread with 50 pL of a 100 pg/mL solution of synthetic a-factor (Sigma Chemical Corp., St Louis, Mo.) dissolved in 90% methanol. Strains to be tested were spread or replica plated on these plates and the cell response of growth or no growth was scored. In the spot, assay cells were embedded in suitable solid media. Aliquots of 10 pL of the 100 pg/mL cr-factor solution were spotted on the surface of the agar and the zone of growth inhibition was used as a measure of the pheromone responsiveness. ~ h STE18 g gene was cloned as an Ssp1 to PstI fragment into the PvuII to PstI sites of pVT100-U (Vernet et al. 1987), a yeast Escherichia coli shuttle vector with a single-stranded phage origin of replication. Three contiguous regions of the STEI8 gene were subjected to mutagenesis. Three 66-mer oligonucleotides encompassing amino acids 24-46 (A), 47-69 (B), and 70-92 (C) were synthesized using doped nucleotides (Ner et al. 1988) on an Applied Biosvstems 380A synthesizer and ~urifiedas described (Sanchez~ e s c a d o and r ~ r d e - a1984). Each nucleotide mix containkd 98.5% of the correct nucleotide and 0.5% each of the other nucleotides (McNeil and Smith 1985). These oligonucleotides were used in a standard site-directed mutagenesis procedure, using single-stranded M70p2 DNA grown in E. coli strain CJ236 using M13K07 as the helper phage (Kunkle 1987). The mutagenized DNA was transformed into E. coli strain MC1061, the transformants were pooled, and total plasmid DNA was extracted using the alkaline-SDS protocol (Maniatis et al. 1982) to generate libraries of mutant genes. DNA from each of the three pools (A, B, and C) was transformed into the yeast strain S11, which contains a null mutation in the STE18 gene. Approximately 10% of the plasmids in each mutagenized pool were not able to confer significant mating ability to strain S11, and thus, these plasmids had a mutant STE18 gene. Mutant isolation Two separate screenings of the mutant libraries were performed. In the first, plasmids from the three mutagenized pools were transformed into the mating defective strain M200-6C ste4-25. Plasmids which conferred mating enhanced beyond that produced by the wild-type STE18 gene on plasmid M70p2 were identified by patch matings of the transformants to a MATa tester strain, DC17. Plasmids were isolated from candidate colonies and retested in the strain M200-6C ste4-25. Plasmids that conferred mating above the background level are referred to as having compensating mutations. In the second screen, STEI8 mutants that conferred a dominant interference with the pheromone response pathway were detected by transforming the pheromone-supe&ensitive strain M200-6C and screening for colonies which exhibited enhanced resistance to the mating pheromone a-factor. Transformants were patched to - ura plates and these patches were then replica plated to - ura plates spread with 5 pg of synthetic a-factor (Sigma). Plasmids were isolated from colonies that exhibited enhanced pheromone resistance and were retested in strain M200-6C.

FIG. 1. Enhancement of mating of ste4-25 mutant strain. Strain M200-6C ste4-25 was transformed with various pVT100-U based plasmids and the transformants were mated to strain DC17 overnight on YEPD plates. The mating mixtures were then replica plated to minimal plates to detect prototrophic diploid formation. Transformants containing the vector pVT100-U gave few prototrophic diploids, while transformants containing the unmutagenized STEI8 gene on plasmid M70p2 mated at a higher level. Point mutations of the STE18 gene further enhanced the mating level; C6 and C8 (both T71I), C7 (V80E), and B5 (S65L) all showed a higher level of prototroph formation than that conferred by the wild-type STEI8 gene. Plasmid stability measurements Colonies grown on selective medium were inoculated into YEPD medium and grown to saturation overnight (approximately six doublings). Suitable dilutions were then spread on YEPD plates and after 2 days of growth, they were replica plated in -ura medium. Plasmid stability was measured as the proportion of the total colonies that had retained the URA3+ plasmid. Sequencing Mutant plasmids were sequenced by the dideoxynucleotide chaintermination method (Sanger et al. 1977) directly using alkaline denaturation (Chen and Seeburg 1985).

Results Isolation of compensating mutations Strain M200-6C ste4-25 has a very low mating efficiency because of a specific amino acid change (N81Y) in the STE4 gene product. This defect in mating is partially overcome by increased expression of the STE18 gene product (Clark et al. 1993; see Fig. 1). We screened mutagenized populations of the STE18 gene and looked for mutations in the STEl8 gene that would further enhance the mating ability of the ste4-25 mutant strain beyond that simply because of STEl8 overexpression. No enhanced mating was detected from 900 pool A transformants. One of 700 pool B transformants enhanced mating, as did 3 of 1400 pool C transformants. Plasmids were isolated from these transformants and retransformed into strain M200-6C ste4-25, and the mating levels were assessed. As can be seen in Fig. 1, plasmids C6, C7, C8, and B5 all allowed a higher level of mating than plasmid M70p2 alone. Thus four compensatory mutations were identified from a total of approximately 3000 colonies analyzed. Characterization of compensating mutations The STE18 gene from each of the four compensatory plasmids was sequenced. In each case, a single nucleotide change leading to a single amino acid change was identified relative to the wild-type STE18 gene. These changes were found within the regions targeted for mutagenesis. The B5

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TABLE2. Compensation of Ste4 mating defects by Stel8 mutant proteins Matinga

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STE4 allele

STE18 allele

(x

~nhancement~

'The mating ability ( X lo-') of the indicated STE4 mutants harboring each of the STEl8 alleles on a high copy plasmid is shown. b ~ h enhancement e in mating ability conferred by the indicated STE18 allele, relative to the wild-type STE18, is shown for each STE4 mutation.

mutation was a C-T transition, which changed serine 65 (TCG) to leucine (TTG). The C7 mutation was a T-A transversion, which changed valine 80 (GTA) to glutamic acid (GAA). Both C6 and C8 contained the same change; a C-T transition which switched theronine 71 (ACA) to isoleucine (ATA). The ability of the STE18 mutants to compensate for the mating defect of different mutations in the STE4 gene was quantified. The mating level of strains containing various STE4 alleles transformed with the mutant or wild-type STE18 plasmids was determined by quantitative matings with a MATa tester strain. As can be seen in Table 2, the compensatory effect was confined to the ste4-25 allele. Other STE4 alleles did not exhibit significant enhancement in mating beyond that caused by the presence of the high copy STEl8 plasmid M70p2. We assessed the ability of the STEl8 mutants to replace the wild-type STEl8 gene. Strain M200-6C stel8::ura3, which has a mutant ura3 gene inserted in the STEl8 gene, provides a sensitive assay for STEl8 function. Plasmid M70p2 and the four mutant derivatives of M70p2 were transformed into this strain and the pheromone responsiveness was assessed by spot assays. In each case, the pheromone responsiveness conferred by the mutant gene was indistinguishable from the wild-type gene (Fig. 2). Plasmid stability Because increased copy number of the wild-type STE18 gene in the ste4-25 mutant strain leads to an increase in mating, we assessed whether the STEl8 point mutations had enhanced the mitotic stability of the plasmids. After overnight growth in nonselective medium, suitable dilutions of the transformants were plated on rich medium and the plasmid stability was measured by determining the frequency of plasmid-containing cells. The plasmids containing the

FIG. 2. Activity of the STEI8 compensatory mutations. Strain M200-6C stel8::urajl was transformed with either M70p2 or the mutant derivatives. The transformants were embedded in agar and 10 pg of a-factor in 90% ethanol was spotted on top of the plates. The halo of growth arrested cells formed by the pheromone is a measure of the complementation of the STEI8 defect. In all cases the plasmid-borne STE18 allele was capable of efficiently complementing the STEI8 null mutation. Panel A shows the response of M200-6C stel8::urajl transformed with M70p2 carrying the wildtype STEI8 gene, panel B shows the response of a transformant carrying the B5 mutant of STE18, panel C shows the response of the C6 mutant of STEI8, and panel D shows the response of the C7 mutant of STE18. Colonies within the zone of growth inhibition represent sterile mutations arising in the culture; the frequency of these mutants is variable from experiment to experiment.

TABLE 3. Mitotic stability of plasmids carrying different STE18 alleles Plasmid

Total colonies

URA' colonies

Stability (%)

132 119

67 69 22

-

M70p2 C6 C7 C8 B5

198

171 202 174

186

45 77 84

44 45

mutant STE18 alleles did not show enhanced stability relative to the wild-type-containing plasmid M70p2; in fact, the plasmids containing mutations C7, C8, and B5 showed somewhat reduced stabilities (Table 3). Isolation of dominant negative mutations The mutant libraries were screened for dominant negative mutations as described in Materials and methods. Such dominant negative mutations will reduce pheromone response, even in the presence of the wild-type STE18 allele, and will create a pheromone-resistant transformant. Approximately 400 transformants were screened for each pool. From these screens, one strongly interfering mutant was identified from each of pools A and B, and five were identified from pool C. These mutants were retransformed into strain M200-6C; as can be seen in Fig. 3, all the plasmids conferred significant pheromone resistance to this strain. The transformants were also subjected to a more sensitive spot assay for pheromone responsiveness. In each case, the presence of the mutant gene on a high copy plasmid modified the pheromone response

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- ura

a-factor

FIG. 3. Pheromone resistance conferred by dominant negative mutations. Strain M200-6C was transformed either with plasmid M70p2 or with plasmids containing dominant negative mutant derivatives of M70p2. These transformants were grown on - ura plates and then replica plated to YEPD plates containing a-factor. All the transformants containing the dominant negative mutant STE18 plasmids could grow on the a-factor containng plate, while the transformant containing M70p2 was unable to grow in the piesence of pheromone. TABLE4. Suppression of scgl::LEU2 inviability by STEl8 dominant negative alleles Plasmid

Tetrads

LEU2+ spores0

M70p2

6 7 7 7 5 7 7 7

0

A1 B1

C1 C2 C10 C11 C5 1

70, 1992

8

13 4 8 7

12 10

FIG.4. Mating ability of strains containing dominant negative mutant alleles of STEl8. Strain S11, which contains a disruption of the STEl8 gene and thus lacks STEl8 function, was transformed with plasmid M70p2, as well as the seven derivatives of M70p2 containing the dominant negative alleles of STE18. These transformants were tested for mating as in Fig. 1. Only the M70p2 transformant was able to mate; all the dominant negative alleles were unable to supply the STEl8 function. the SCGI gene. Dissection of the untransformed diploid or of diploids containing the wild-type STEI8 gene generated only two healthy spores per tetrad, both of which were leu2 - . However, transformants containing any of the interfering mutant plasmids frequently generated viable LEU2' spore colonies (Table 4) and these colonies were always URA3+ as well. Thus the presence of the interfering allele on a high copy plasmid was sufficient to interfere with the constitutive arrest signal generated by the loss of SCGI .

Functionality of the STE 18 alleles The dominant negative mutants were tested for their function as STE18 alleles by transformation into strain W303-1A stel8::LEU2, which carries a disruption of the STEI8 gene. None of the plasmids carried a functional STE18 gene, as no mating was detected for any of the transformants (Fig. 4). The plasmids were also transformed into the potentially supersensitive pheromone-resistant strain M200-6C stel8::uraJ. These transformants were tested for pheromone responsiveness using the spot assay and in all cases the cells remained insensitive to the pheromone (data not shown).

Interference with Ste4 overproduction Overproduction of the STE4 gene product also leads to constitutive expression of the pheromone response pathway (Whiteway et al. 1990). Strain M325-1B contains an integrated copy of the STE4 gene under control of the galactose-inducible promoter GALI. When this strain is grown on galactose-containing medium, the induction of the STE4 gene leads to cell cycle arrest and the formation of large, morphologically aberrant cells. We assessed whether the presence of the interfering alleles of STEI8 interfered with cell cycle arrest created by Ste4 overproduction. As is shown in Fig. 5, the plasmids containing the B1, C1, C2, C 10, C11, and C5 1 mutations were able to suppress the cell cycle arrest caused by the galactose-induced overexpression of STE4. However, mutation A l , which was efficient at suppressing pheromone response and the constitutive signal generated by the scgl null mutation, was not effective in suppressing the STE4 overexpression phenotype.

Rescue of scgl null mutants The mutants were also tested for their ability to interfere with a constitutive signal generated by loss of the Ga subunit SCGI. This constitutive signal causes haploid cells to arrest their cell cycle at start and thus these cells cannot grow to form colonies. The plasmids were transformed into diploid strain Dl 11, which is heterozygous for a LEU2 insertion in

Sequencing STE18 dominant negative alleles The nucleotide sequences of four of the dominant negative alleles were determined. The A1 plasmid had two changes within the targeted region, a G to C transversion at position 101, which changed arginine 34 (AGA) to a threonine (ACA), and an A to T transversion at position 127, which changed arginine 43 (AGG) to tryptophan (TGG). The B1

'The number of LEU2' spore colonies observed among the segregants of the indicated number of dissected tetrads is presented. AU LEU+ spores were also

URA'.

of the transformed cells and allowed significant growth in the presence of the pheromone (data not shown).

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Glu

Gal

FIG. 5. Dominant negative suppression of cell cycle arrest due to overexpression of the STE4 gene product. Strain M325-lB, containing an integrated galactose-inducibleSTE4 gene, was transformed with plasmid M70p2 and the derivatives of M70p2 containing the dominant negative alleles of STEIS. These transformants were patched to - ura glucose plates and then replica plated to - ura galactose plates. The transformants containing both the A1 mutation and the wild-type allele of STE18 grew poorly on the galactose containing plates, and microscopic analysis revealed a high level of morphologically aberrant cells characteristic of cells overexpressing STE4. In contrast, all the other dominant negative alleles of STE18 suppressed this growth defect and the colonies contained predominantly morphologically normal cells.

mutation changed the G at position 166 to a T; this resulted in the change of alanine 56 (GCA) to serine (TCA) and also destroyed the NsiI site within the STEl8 gene. The two sequenced mutations in the C block region created the same protein by two different events. The C1 change was a 1-bp deletion of nucleotide 248; this caused a frameshift which resulted in serine 83 (TCA) becoming a TAA stop codon. The C10 mutation also replaced the serine 83 (TCA) with a stop codon (TGA); this occurred through a C to G transversion of nucleotide 248 which changed serine 83 to TGA. Discussion The STEl8 gene of Saccharomyces cerevisiae encodes the Y subunit of the G protein involved in the pheromone response pathway (Whiteway et al. 1989). Such GY proteins take part in known associations with other cellular components. In yeast, these components include both the GO subunit which is encoded by the STE4 gene (Clark et al. 1993), as well as the plasma membrane through a posttranslationally added isoprenoid lipid (Finegold et al. 1990). Furthermore, all three G protein subunits are required to generate a high affinity binding receptor through association with the STE2 gene product (Blumer and Thorner 1990). In addition, the yeast BY element associates with the Gpal (Ga) subunit to deactivate the response pathway (Whiteway et al. 1990; Cole et al. 1990; Nomoto et al. 1990) and with some as yet unidentified downstream component to activate the response pathway. We have used saturation mutagenesis as a first step in the investigation of the roles of the STE18 gene in the pheromone response pathway of yeast. We mutagenized the region of STE18 from codon 24 to codon 92 by means of three pools of oligomers each of 66 nucleotides in length that were synthesized in the presence of low levels of incor-

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rect nucleotides (Ner et al. 1988). This stretch of amino acids did not include the amino terminal region, which contains a polyglutamine tract, and the carboxyl terminal CAAX box motif, which has previously been targeted by classical sitedirected mutagenesis (Finegold et al. 1990). This random mutagenesis was highly efficient, as 10% of the transformants contained a readily detected defect in Stel8 function. Mammalian P"I subunits are tightly associated and appear to function as a single element (Neer and Clapham 1989; Fung 1983), and the STE4 and STEl8 gene products of yeast have been shown to interact by an in vivo protein association assay (Clark et al. 1993). We asked whether functional defects in one subunit could be compensated by changes in the other subunit. We have found that a specific point mutant in Ste4, an asparagine to tyrosine change at position 81, leads to a significant reduction in the signal transduction capability of the Ste4 protein, and creates an almost sterile phenotype in strain M200-6C. This defect is partially compensated by overproduction of the STE18 gene product (Clark et al. 1993; see Fig. 1) and this compensation can be enhanced by further point mutations in the STE18 gene itself. These compensatory mutations occurred at a number of sites in the Stel8 protein (serine 65, theronine 71, and valine 80), but seemed allele specific in their interaction with Ste4, as they did not significantly enhance the mating ability of other STE4 alleles tested. Allele-specific suppression between two genes is often genetic evidence of a physical interaction between the proteins encoded by the genes (Jarvik and Botstein 1975). In the case of STE4 and STE18, there is direct evidence of a physical interaction between the gene products (Clark et al. 1993), and thus the sites of the interacting mutations may define residues that are important in the association of Stel8 (7) with Ste4 (P). Alternatively, because both STE4 and STE18 are necessary for signal transduction (Whiteway et al. 1989), the sites of mutation may define residues that interact with a downstream component in the signalling pathway. These two models are not readily distinguishable genetically and biochemical assessment of the affinities of the wild-type and mutant versions of the Ste4 and Stel8 proteins will be the most conclusive way to distinguish these possibilities. We also identified dominant negative alleles of the STE18 gene which, when overexpressed, could interfere with signal transduction even in the presence of the wild-type STE18 gene product. These dominant negative mutations also likely affect the interactions between the STEl8 gene product and other components of the signal transduction pathway. Overproduction of a misfunctional component of a multisubunit complex can lead to the inactivation of the entire complex even in the presence of the normal complement of the wildtype protein (Herskowitz 1987). This occurs when the misfunctional protein interacts with other subunits of the complex, but the resulting complex is nonfunctional. Thus the functional elements become sequestered into nonfunctional complexes. The majority of the dominant negative mutations were located in the region mutagenized by oligo(C). Two independent mutations in this region were sequenced and both had acquired termination codons that created identical truncated proteins. These truncated proteins lacked the carboxyl terminus of Stel8, including the CAAX box which serves as a target for isoprenylation. Isoprenylation is believed to be important in localizing proteins and anchoring

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WHITEWAY ET AL.

Ner, S.S., Goodin, D.B., and Smith, M. 1988. A simple and efficient procedure for generating random point mutations and for codon replacements using mixed oligonucleotides. DNA, 7: 127-134. Nomoto, S., Nakayarna, N., Arai, K.-I., and Matsumoto, K. 1990. Regulation of the yeast pheromone response pathway by G protein subunits. EMBO J. 9: 691-696. Orr-Weaver, T.C., Szostak, J., and Rothstein, R.J. 1981. Yeast transformation: a model system for the study of recombination. Proc. Natl. Acad. Sci. 78: 6354-6358. Sanchez-Pescador, R., and Urdea, M.S. 1984. Use of unpurified synthetic deoxynucleotide primers for rapid dideoxynucleotide chain termination sequencing. DNA, 3: 339-343. Sanger, F., Nicklen, S., and Coulson, A.R. 1977. DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 74: 5463-5467. Sherman, F., Fink, G.R., and Hicks, J.B. 1982. Methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Sikorski, R.S., and Heiter, P. 1989. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics, 122: 19-27. Simon, M.I., Strathmann, M.P., and Gautam, N. 1991. Diversity of G proteins in signal transduction. Science (Washington, D.C.), 252: 802-808. Simonds, W.F., Butrynski, J.E., Gautam. N., Unson, C.G., and Spiegel, A.M. 1991. G-protein f l :membrane targeting requires

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subunit coexpression and intact Y C-A-A-X domain. J. Biol. Chem. 266: 5363-5366. Vernet, T., Dignard, D., and Thomas, D.Y. 1987. A family of yeast expression vectors containing the phage f l origin of replication. Gene, 52: 225-233. Whiteway, M., Freedman, R., Van Arsdell, S., Szostak, J.W., and Thorner, J. 1987. The yeast ARDl gene product is required for repression of cryptic mating-type information at the HML locus. Mol. Cell. Biol. 7: 3713-3722. Whiteway. M., Hougan, L., Dignard. D., Bell, L., Saari, G., Grant, F., O'Hara, P., MacKay, V.L., and Thomas, D.Y. 1988a. Function of the STE4 and STEIB genes in mating pheromone signal transduction in Saccharomyces cerevisiae. Cold Spring Harbor Symp. Quant. Biol. 53: 585-590. Whiteway, M.S., Hougan, L., and Thomas, D.Y. 1988b. Expression of MFa1 in MATa cells supersensitive to a-factor leads to self-arrest. Mol. Gen. Genet. 214: 85-88. Whiteway, M.S., Hougan, L., Dignard, D., Thomas, D.Y., Bell, L.. Saari, G.C., Grant, F.J., O'Hara, P., and MacKay, V.L. 1989. The STE4 and STEl8 genes of yeast encode potential P and 7 subunits of the mating factor receptor-coupled G protein. Cell, 56: 467-477. Whiteway, M., Hougan, L., and Thomas, D.Y. 1990. Overexpression of the STE4 gene leads to mating response in haploid Saccharomyces cerevisiae. Mol. Cell. Biol. 10: 217-222.

Mutagenesis of Ste18, a putative G gamma subunit in the Saccharomyces cerevisiae pheromone response pathway.

The yeast STE18 gene product has sequence and functional similarity to the gamma subunits of G proteins. The cloned STE18 gene was subjected to a satu...
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