MOLECULAR AND CELLULAR BIOLOGY, Sept. 1990, p. 4439-4446 0270-7306/90/094439-08$02.00/0 Copyright © 1990, American Society for Microbiology
Vol. 10, No. 9
G Protein Mutations That Alter the Pheromone Response in Saccharomyces cerevisiae DAVID E. STONE AND STEVEN I. REED* Department of Molecular Biology, Research Institute of Scripps Clinic, 10666 North Torrey Pines Road, La Jolla, California 92037
Received 25 March 1990/Accepted 28 May 1990 The GPAI gene of Saccharomyces cerevisiae encodes a Ga protein that couples the membrane-bound pheromone receptors to downstream elements in the mating response pathway. We have isolated seven mutant alleles of GPAI that confer pheromone resistance: G50D (a glycine-to-aspartate change at position 50), G322E, G322R, E355K, E364K, G470D, and an E364K-G470D double mutant. All of the mutations lie within large regions that are highly conserved between Gpal and four other G. proteins; four of the changes are located in domains with proposed functions. On the basis of a gentic analysis, the pheromone-unresponsive GPAI alleles can be divided into two classes: those that encode constitutively activated proteins and those that encode proteins unable to respond to the upstream signal. Our results support the hypothesis that the activated form of Gpal stimulates adaptation to pheromone.
Like higher eucaryotic cells, the yeast Saccharomyces cerevisiae utilizes receptor-coupled G proteins to transduce environmental stimuli across the plasma membrane. The mating response, for example, is initiated by peptide pheromones, which are constitutively secreted by each haploid mating type and which bind to surface receptors on cells of the opposite type (6, 38). The components of the matingsignal transducer have been identified genetically, and the genes encoding them have been cloned and sequenced (7, 15, 25, 27, 41). On the basis of the primary structure of their products and loss-of-function phenotypes, GPAI, STE4, and STE18 are thought to encode the Ga, G^, and Gy subunits, respectively, of a heterotrimeric G protein (7, 15, 25, 27, 41). This G protein most probably interacts with Ste2 and Ste3, the pheromone receptors expressed by MATa and MATat cells, respectively (4, 29). Primary-structure analysis of both Ste2 and Ste3 predicts a topology similar to that of the 0-adrenergic/rhodopsin family of receptors, characterized by seven transmembrane domains (4, 8, 29, 30). By analogy to receptor-coupled G proteins in mammalian cells (11, 36), the inactive form of Gpal is expected to bind GDP as well as a heterodimer composed of the STE4 and STE18 gene products. Interaction of Gag3y-GDP with the activated receptor stimulates the exchange of GDP for GTP, causing a conformational switch in Ga and the release of Gw. GaM catalyzed hydrolysis of GTP returns the system to its initial state.
Genetic analysis of the yeast mating reaction indicates that G.-Y activates the pheromone response and that Ga acts as a negative regulator (1, 5, 7, 13, 15, 18, 25, 28, 42). In contrast, the primary signal in most receptor-coupled transduction pathways is generated by the Ga and not the G,3^ moiety (31). However, the pheromone response pathway clearly provides an excellent model system, since the physiology of G protein function appears to be highly conserved and the biology of signaling is genetically accessible in S. cerevisiae. To further elaborate the relationship between the structure and function of Ga proteins, we sought dominant mutations in GPA1 that uncoupled the pheromone response. The * Corresponding author. 4439
isolation and characterization of seven pheromone-unresponsive GPA1 (GPAJPhUn) alleles are described below. MATERIALS AND METHODS Mutagenesis and sequence analysis. A plasmid containing the GPAJ coding region fused to the GAL] promoter (5) was mutagenized with hydroxylamine as previously described (21). Plasmids that conferred pheromone resistance were purified from S. cerevisiae, and the putative GPA1 mutant alleles were subcloned to the sequencing vector, T3T718U (Pharmacia, Inc.). The sequence of 17 independently isolated GPAJ mutant alleles (called GPAJPhUM alleles) was determined by priming synthesis from single-strand templates with five evenly spaced oligomers by the method of Sanger et al. (33). Yeast strains, plasmids, and media. The strains used in the growth inhibition were constructed as follows. IVY-D2, a MATa leu2 GPA1 ura3/MATot leu2 gpal:: URA3 ura3 strain, was transformed with the GPAJPhun and the GPA1 wild-type single-copy vectors, marked by the LEU2 gene. The insertional mutant allele gpal:: URA3 is nonfunctional and is referred to as gpal null throughout the text. Transformants were sporulated, asci were dissected, and segregants were scored for leucine and uracil prototrophy, as well as mating type, to isolate MATa GPA1 and MATa gpal:: URA3 strains carrying the various plasmid-borne Ga alleles. For measurement of FUS1 expression, representative MATa gpal:: URA3 GPAlPhun strains were spread onto plates containing 1 ,ug of 5-fluoroorotic acid per ml to select for uracil auxotrophs (2). The 5-fluoroorotic acid-resistant and ura derivatives were then transformed with pSB231 (40), a plasmid containing a FUSI-lacZ hybrid gene and the URA3 gene as a selectable marker. The mutant GPA1 alleles were rejoined to the GPAJ promoter as follows. An XhoI-SphI fragment cut from YCp5O (A14-1) (15) and containing the sequence from base pairs (bp) -1750 to +350 relative to the ATG of GPAJ was subcloned to the polylinker of T3T718U. By using the polymerase chain reaction, the GPAJ promoter region was amplified with a BamHI site inserted 6 bp 5' to the ATG of GPA1 and an XbaI site inserted at bp -1750. The 1.75 kilobases (kb) of sequence upstream of the GPAJ coding
4440
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region was moved to the multiple cloning site of the centromeric vector, YCplaclll (10), as a BamHI-XbaI fragment. BamHI-SacI restriction fragments containing the mutant and wild-type GPAI alleles (with 8 and 298 bp flanking the 5' and 3' ends of the coding sequence, respectively) were then moved from the sequencing vector (T3T718U) to the centromeric GPAJ promoter vector. All media were based on synthetic complete SD medium (34). For induction of GPAI in cells containing the GALJpGPAI plasmid, galactose (2%) was substituted for glucose as the carbon source. Growth inhibition zone assays. Strains were tested for pheromone-induced growth inhibition in standard halo assays, as previously described (5). After growth to midlogarithmic phase, approximately 105 cells were diluted into 6.5 ml of soft agar and spread onto plates. Various doses synthetic a-factor were then dotted onto the surface of the plates in 4-,l aliquots. The plates were incubated for 2 days at 30°C prior to photographing. Leucine was omitted from all media to select for plasmid maintenance. I-Galactosidase assays. (-Galactosidase activity was determined as described previously (35). The optical density of the growing yeast culture was measured at 600 nm, and a measured volume (v, in milliliters) was pelleted in a siliconized 1.5-ml microcentrifuge tube. The supernatant was removed, and the cell pellet was frozen in a dry-ice-ethanol bath (-70'C). For the enzymatic assay, the cell pellets were vigorously suspended in 800 ,u of Z buffer (22)-20 ,ul of 0.1% sodium dodecyl sulfate-20 lI of chloroform. After the suspension had been incubated for 5 to 10 min at 300C, the reaction was initiated by the addition of 160 RI of onitrophenyl-p-D-galactoside (4 mg/ml) in A buffer (22). The reaction was terminated by the addition of 400 pl of 1.0 M Na2CO3, and the incubation time (t, in minutes) in the presence of o-nitrophenyl-p-D-galactoside was noted. Cleavage of o-nitrophenyl-p-D-galactoside produces a colored, o-nitrophenol, that can be quantified by determining the A420. The cell debris were then pelleted for 1 min in a microcentrifuge, and the A420 of the supernatant was determined with a spectrophotometer. Units of 3-galactosidase activity were defined as (A420 x 1,000)/(optical density at 600 nm x t x v). RESULTS Identification of mutations in GPAI that confer dominant pheromone resistance. A plasmid containing the GPAI coding region fused to the GAL] promoter was mutagenized with hydroxylamine (21), and the resulting library was used to transform a MATa GPAI strain of S. cerevisiae. Transformant colonies were replica plated to media containing various concentrations of a-factor (2 to 20 ,ug/ml) and either galactose to induce expression of the plasmid-borne GALJpGPAI alleles or glucose to repress GALJp-GPAJ expression. We screened for colonies that were resistant to pheromone on galactose but not glucose-that is, those whose insensitivity to a-factor was dependent on GALJ-GPAI expression. Since the GAL] promoter is highly active when cells are grown in galactose-based medium, the plasmidborne alleles were expressed at many times the level of the native GPA1. Plasmids carried by pheromone-resistant colonies were purified, and the GPAI alleles were sequenced. From a collection of 17 independently isolated GALJp-GPAI plasmids, we identified seven pheromone-unresponsive (GPAJPhun) alleles (Fig. 1): G50D (a glycine-to-aspartate change at position 50), G322E, G322R, E355K, E364K,
MOL. CELL. BIOL.
G470D, and an E364K-G470D double mutant. These mutations all lie within large regions that are highly conserved between Gpal and four other Ga proteins (Fig. 1), and all six result in a change in charge. By analogy with ras and Gs models of structure and function (3, 14, 19), four of the GPAI mutations are located in domains with proposed functions. The Gly-50-to-Asp-50 mutation is in the phosphate box, a region that is thought to interact with GDP and GTP and to affect GTP hydrolysis. This mutation is analogous to that of the activating (oncogenic) ras mutation, Asp-12 (39). G322E and G322R mark the second position of the five-residue S box, thought to affect the conformational switch induced in Ga proteins by the binding of GTP (3, 14, 23). The Gly-to-Asp change at position 470 is in the putative receptor-binding domain, and the remaining mutations, E355K and E364K, are in conserved regions of unknown function. Five of the six GPAIPh"n mutations are in or are near the putative guanine-nucleotide-binding pocket (Fig. 2). To compare the pheromone sensitivity of cells expressing the various GALJp-GPA1Phu" alleles, we performed growth inhibition zone, or halo, tests (16, 32). The primary responses of yeast cells to mating pheromone include arrest in the G1 phase of the cell cycle, induction of mating-specific transcripts, and morphological changes. The halo test is based on the observation that mating pheromone prevents responsive cells from forming colonies. With the exception of G470D, each of the mutant alleles conferred complete resistance to a 20-p.g point source of a-factor mating pheromone when expressed at high levels in a wild-type GPAI background (data not shown). When the same cells were challenged with large doses of pheromone in liquid medium, we saw no evidence of even a transient G1 arrest or the morphological changes associated with the response. In contrast, cells carrying the G470D allele formed halos about one third smaller than those formed by wild-type control cells (data not shown). Over a period of 2 to 3 days, the mutant halos gradually filled in, while the control halos remained clear. The GPA1PhU" alleles fall into two classes. If one relies on the model of G protein function in mammalian cells, there are several means by which a defective GPAI molecule might block the mating response. The affinity of the mutant protein for G,3 might be increased; the GPAI protein might be unable to interact with the activated receptor; or the mutant Ga might be able to receive the receptor signal but fail to undergo the appropriate conformational switch. In all of these cases, Gy would not be released as readily and signal transduction would be inhibited. Alternatively, the pheromone resistance conferred by the GPAI alleles might depend not on a failure to transduce the signal, but on a tendency to turn it off. It is known that when yeast cells are continually exposed to pheromone, they eventually resume growth (5, 24, 26). Therefore, the mating response pathway of S. cerevisiae, like analogous signaling systems of higher eucaryotes, can adapt to a continuous signal. Based on the behavior of cells carrying the G5OV allele described by Miyajima et al. (24), it is likely that Gpal is involved in this adaptive response and that mutations in GPAI can augment this function. Since the stoichiometry of G protein subunits is important for proper function (5, 42), we were concerned that overexpression of the mutant proteins might obscure the mechanisms that underlie the pheromone resistance conferred by GPAJPh"n mutations. A large excess of mutant protein might, for example, render mutations that confer pheromone insensitivity indistinguishable from those that stimulate ad-
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PHEROMONE RESPONSE IN S. CEREVISIAE
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Vol. 10, No. 9
G Protein Mutations That Alter the Pheromone Response in Saccharomyces cerevisiae DAVID E. STONE AND STEVEN I. REED* Department of Molecular Biology, Research Institute of Scripps Clinic, 10666 North Torrey Pines Road, La Jolla, California 92037
Received 25 March 1990/Accepted 28 May 1990 The GPAI gene of Saccharomyces cerevisiae encodes a Ga protein that couples the membrane-bound pheromone receptors to downstream elements in the mating response pathway. We have isolated seven mutant alleles of GPAI that confer pheromone resistance: G50D (a glycine-to-aspartate change at position 50), G322E, G322R, E355K, E364K, G470D, and an E364K-G470D double mutant. All of the mutations lie within large regions that are highly conserved between Gpal and four other G. proteins; four of the changes are located in domains with proposed functions. On the basis of a gentic analysis, the pheromone-unresponsive GPAI alleles can be divided into two classes: those that encode constitutively activated proteins and those that encode proteins unable to respond to the upstream signal. Our results support the hypothesis that the activated form of Gpal stimulates adaptation to pheromone.
Like higher eucaryotic cells, the yeast Saccharomyces cerevisiae utilizes receptor-coupled G proteins to transduce environmental stimuli across the plasma membrane. The mating response, for example, is initiated by peptide pheromones, which are constitutively secreted by each haploid mating type and which bind to surface receptors on cells of the opposite type (6, 38). The components of the matingsignal transducer have been identified genetically, and the genes encoding them have been cloned and sequenced (7, 15, 25, 27, 41). On the basis of the primary structure of their products and loss-of-function phenotypes, GPAI, STE4, and STE18 are thought to encode the Ga, G^, and Gy subunits, respectively, of a heterotrimeric G protein (7, 15, 25, 27, 41). This G protein most probably interacts with Ste2 and Ste3, the pheromone receptors expressed by MATa and MATat cells, respectively (4, 29). Primary-structure analysis of both Ste2 and Ste3 predicts a topology similar to that of the 0-adrenergic/rhodopsin family of receptors, characterized by seven transmembrane domains (4, 8, 29, 30). By analogy to receptor-coupled G proteins in mammalian cells (11, 36), the inactive form of Gpal is expected to bind GDP as well as a heterodimer composed of the STE4 and STE18 gene products. Interaction of Gag3y-GDP with the activated receptor stimulates the exchange of GDP for GTP, causing a conformational switch in Ga and the release of Gw. GaM catalyzed hydrolysis of GTP returns the system to its initial state.
Genetic analysis of the yeast mating reaction indicates that G.-Y activates the pheromone response and that Ga acts as a negative regulator (1, 5, 7, 13, 15, 18, 25, 28, 42). In contrast, the primary signal in most receptor-coupled transduction pathways is generated by the Ga and not the G,3^ moiety (31). However, the pheromone response pathway clearly provides an excellent model system, since the physiology of G protein function appears to be highly conserved and the biology of signaling is genetically accessible in S. cerevisiae. To further elaborate the relationship between the structure and function of Ga proteins, we sought dominant mutations in GPA1 that uncoupled the pheromone response. The * Corresponding author. 4439
isolation and characterization of seven pheromone-unresponsive GPA1 (GPAJPhUn) alleles are described below. MATERIALS AND METHODS Mutagenesis and sequence analysis. A plasmid containing the GPAJ coding region fused to the GAL] promoter (5) was mutagenized with hydroxylamine as previously described (21). Plasmids that conferred pheromone resistance were purified from S. cerevisiae, and the putative GPA1 mutant alleles were subcloned to the sequencing vector, T3T718U (Pharmacia, Inc.). The sequence of 17 independently isolated GPAJ mutant alleles (called GPAJPhUM alleles) was determined by priming synthesis from single-strand templates with five evenly spaced oligomers by the method of Sanger et al. (33). Yeast strains, plasmids, and media. The strains used in the growth inhibition were constructed as follows. IVY-D2, a MATa leu2 GPA1 ura3/MATot leu2 gpal:: URA3 ura3 strain, was transformed with the GPAJPhun and the GPA1 wild-type single-copy vectors, marked by the LEU2 gene. The insertional mutant allele gpal:: URA3 is nonfunctional and is referred to as gpal null throughout the text. Transformants were sporulated, asci were dissected, and segregants were scored for leucine and uracil prototrophy, as well as mating type, to isolate MATa GPA1 and MATa gpal:: URA3 strains carrying the various plasmid-borne Ga alleles. For measurement of FUS1 expression, representative MATa gpal:: URA3 GPAlPhun strains were spread onto plates containing 1 ,ug of 5-fluoroorotic acid per ml to select for uracil auxotrophs (2). The 5-fluoroorotic acid-resistant and ura derivatives were then transformed with pSB231 (40), a plasmid containing a FUSI-lacZ hybrid gene and the URA3 gene as a selectable marker. The mutant GPA1 alleles were rejoined to the GPAJ promoter as follows. An XhoI-SphI fragment cut from YCp5O (A14-1) (15) and containing the sequence from base pairs (bp) -1750 to +350 relative to the ATG of GPAJ was subcloned to the polylinker of T3T718U. By using the polymerase chain reaction, the GPAJ promoter region was amplified with a BamHI site inserted 6 bp 5' to the ATG of GPA1 and an XbaI site inserted at bp -1750. The 1.75 kilobases (kb) of sequence upstream of the GPAJ coding
4440
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region was moved to the multiple cloning site of the centromeric vector, YCplaclll (10), as a BamHI-XbaI fragment. BamHI-SacI restriction fragments containing the mutant and wild-type GPAI alleles (with 8 and 298 bp flanking the 5' and 3' ends of the coding sequence, respectively) were then moved from the sequencing vector (T3T718U) to the centromeric GPAJ promoter vector. All media were based on synthetic complete SD medium (34). For induction of GPAI in cells containing the GALJpGPAI plasmid, galactose (2%) was substituted for glucose as the carbon source. Growth inhibition zone assays. Strains were tested for pheromone-induced growth inhibition in standard halo assays, as previously described (5). After growth to midlogarithmic phase, approximately 105 cells were diluted into 6.5 ml of soft agar and spread onto plates. Various doses synthetic a-factor were then dotted onto the surface of the plates in 4-,l aliquots. The plates were incubated for 2 days at 30°C prior to photographing. Leucine was omitted from all media to select for plasmid maintenance. I-Galactosidase assays. (-Galactosidase activity was determined as described previously (35). The optical density of the growing yeast culture was measured at 600 nm, and a measured volume (v, in milliliters) was pelleted in a siliconized 1.5-ml microcentrifuge tube. The supernatant was removed, and the cell pellet was frozen in a dry-ice-ethanol bath (-70'C). For the enzymatic assay, the cell pellets were vigorously suspended in 800 ,u of Z buffer (22)-20 ,ul of 0.1% sodium dodecyl sulfate-20 lI of chloroform. After the suspension had been incubated for 5 to 10 min at 300C, the reaction was initiated by the addition of 160 RI of onitrophenyl-p-D-galactoside (4 mg/ml) in A buffer (22). The reaction was terminated by the addition of 400 pl of 1.0 M Na2CO3, and the incubation time (t, in minutes) in the presence of o-nitrophenyl-p-D-galactoside was noted. Cleavage of o-nitrophenyl-p-D-galactoside produces a colored, o-nitrophenol, that can be quantified by determining the A420. The cell debris were then pelleted for 1 min in a microcentrifuge, and the A420 of the supernatant was determined with a spectrophotometer. Units of 3-galactosidase activity were defined as (A420 x 1,000)/(optical density at 600 nm x t x v). RESULTS Identification of mutations in GPAI that confer dominant pheromone resistance. A plasmid containing the GPAI coding region fused to the GAL] promoter was mutagenized with hydroxylamine (21), and the resulting library was used to transform a MATa GPAI strain of S. cerevisiae. Transformant colonies were replica plated to media containing various concentrations of a-factor (2 to 20 ,ug/ml) and either galactose to induce expression of the plasmid-borne GALJpGPAI alleles or glucose to repress GALJp-GPAJ expression. We screened for colonies that were resistant to pheromone on galactose but not glucose-that is, those whose insensitivity to a-factor was dependent on GALJ-GPAI expression. Since the GAL] promoter is highly active when cells are grown in galactose-based medium, the plasmidborne alleles were expressed at many times the level of the native GPA1. Plasmids carried by pheromone-resistant colonies were purified, and the GPAI alleles were sequenced. From a collection of 17 independently isolated GALJp-GPAI plasmids, we identified seven pheromone-unresponsive (GPAJPhun) alleles (Fig. 1): G50D (a glycine-to-aspartate change at position 50), G322E, G322R, E355K, E364K,
MOL. CELL. BIOL.
G470D, and an E364K-G470D double mutant. These mutations all lie within large regions that are highly conserved between Gpal and four other Ga proteins (Fig. 1), and all six result in a change in charge. By analogy with ras and Gs models of structure and function (3, 14, 19), four of the GPAI mutations are located in domains with proposed functions. The Gly-50-to-Asp-50 mutation is in the phosphate box, a region that is thought to interact with GDP and GTP and to affect GTP hydrolysis. This mutation is analogous to that of the activating (oncogenic) ras mutation, Asp-12 (39). G322E and G322R mark the second position of the five-residue S box, thought to affect the conformational switch induced in Ga proteins by the binding of GTP (3, 14, 23). The Gly-to-Asp change at position 470 is in the putative receptor-binding domain, and the remaining mutations, E355K and E364K, are in conserved regions of unknown function. Five of the six GPAIPh"n mutations are in or are near the putative guanine-nucleotide-binding pocket (Fig. 2). To compare the pheromone sensitivity of cells expressing the various GALJp-GPA1Phu" alleles, we performed growth inhibition zone, or halo, tests (16, 32). The primary responses of yeast cells to mating pheromone include arrest in the G1 phase of the cell cycle, induction of mating-specific transcripts, and morphological changes. The halo test is based on the observation that mating pheromone prevents responsive cells from forming colonies. With the exception of G470D, each of the mutant alleles conferred complete resistance to a 20-p.g point source of a-factor mating pheromone when expressed at high levels in a wild-type GPAI background (data not shown). When the same cells were challenged with large doses of pheromone in liquid medium, we saw no evidence of even a transient G1 arrest or the morphological changes associated with the response. In contrast, cells carrying the G470D allele formed halos about one third smaller than those formed by wild-type control cells (data not shown). Over a period of 2 to 3 days, the mutant halos gradually filled in, while the control halos remained clear. The GPA1PhU" alleles fall into two classes. If one relies on the model of G protein function in mammalian cells, there are several means by which a defective GPAI molecule might block the mating response. The affinity of the mutant protein for G,3 might be increased; the GPAI protein might be unable to interact with the activated receptor; or the mutant Ga might be able to receive the receptor signal but fail to undergo the appropriate conformational switch. In all of these cases, Gy would not be released as readily and signal transduction would be inhibited. Alternatively, the pheromone resistance conferred by the GPAI alleles might depend not on a failure to transduce the signal, but on a tendency to turn it off. It is known that when yeast cells are continually exposed to pheromone, they eventually resume growth (5, 24, 26). Therefore, the mating response pathway of S. cerevisiae, like analogous signaling systems of higher eucaryotes, can adapt to a continuous signal. Based on the behavior of cells carrying the G5OV allele described by Miyajima et al. (24), it is likely that Gpal is involved in this adaptive response and that mutations in GPAI can augment this function. Since the stoichiometry of G protein subunits is important for proper function (5, 42), we were concerned that overexpression of the mutant proteins might obscure the mechanisms that underlie the pheromone resistance conferred by GPAJPh"n mutations. A large excess of mutant protein might, for example, render mutations that confer pheromone insensitivity indistinguishable from those that stimulate ad-
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