Proc. NatI. Acad. Sci. USA Vol. 87, pp. 4363-4367, June 1990 Biochemistry

f8 and y subunits of a yeast guanine nucleotide-binding protein are not essential for membrane association of the a subunit but are required for receptor coupling (peptide hormone/Saccharomyces cerevsiae/signal transduction/guanosine

5'-[r-thio]triphosphate)

KENDALL J. BLUMER* AND JEREMY THORNERt Division of Biochemistry and Molecular Biology, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720

Communicated by Daniel E. Koshland, Jr., March 8, 1990 (received for review January 6, 1990)

In Saccharomyces cerevisiae, oligopeptide mating pheromones, a-factor and a-factor, activate a G protein-linked signal-transduction pathway that culminates in conjugation between haploid MATa and MATa cells (19, 20). Genetic data suggest that the a- and a-factor receptors, encoded respectively by the STE3 (21, 22) and STE2 (21, 23-26) genes, activate a G protein whose a, 13, and fy subunits are encoded by the GPA) (SCG)) (27, 28), STE4 (29), and STE18 (29) genes, respectively. The py complex apparently modulates downstream effectors because loss ofthe GPA) gene product provokes a cellular response (27, 28), whereas loss of either the STE4 or STEJ8 gene products prevents a cellular response (29), even when GPAI is absent (30). We developed assays to detect functional coupling between the a-factor receptor and its cognate G protein. These assays and immunological methods were used to determine the effects of mutations in the GPAI, STE4, and STE18 genes on receptor-G protein coupling and on localization of the GPA) gene product. Our results address the separate contributions of individual G protein subunits in coupling to receptor and in targeting of the G, subunit to the membrane.

Conditions were devised to demonstrate ABSTRACT GTP-regulated coupling between the yeast STE2-encoded receptor and its cognate guanine nucleotide-binding protein (G protein). Treatment of partially purified membranes with guanosine 5'-[v-thio]triphosphate (GTP[y-SJ) converted the receptor from a high-affinity state (Kd = 17 nM) to a much lower affinity state (Kd 150 nM), as judged by three independent criteria: rate of ligand (a-factor) dissociation, equilibrium binding, and antagonist competition. Expression of STE2 from the GAL) promoter in MATa/MATa diploids, which do not express GPAI (encoding G protein a subunit, G.), STE4 (encoding G protein 13 subunit, Gp), and STE18 (encoding G protein y subunit, Gy) but do express another G protein a subunit (product of GPA2), yielded a single class of low-affinity receptors that were GTP[y-S]-insensitive, indicating that STE2 gene product cannot couple productively with other G proteins, even in the absence of competition by its cognate G protein. By using gpal, STE4, and stel8 mutations, it was found that all three G protein subunits were required for functional coupling, as judged by the absence of high-affinity receptors when any of the three gene products was altered. This finding demonstrates that Gp and Gy subunits are essential for formation of a productive complex between a Ga subunit and its corresponding receptor. Wild-type STE4 and STE18 gene products were not essential for membrane localization of the GPAI gene product, as indicated by cell fractionation and immunological analyses, suggesting that Gpr and GY subunits interact with the receptor or make the G,, subunit competent to associate correctly with the receptor, or both.

MATERIALS AND METHODS Yeast Strains, Plasmids, and Media. JE112 (MATa leu2-3,112 ura3-52 trp) his3-Al sst)-A5) has been described (31). DJ1647-1 (MATa ste5-3 bar)-) cry) ade2-1 his4-580 lys2 trp) tyri SUP4-3), DJ730-2-3 (DJ164-7-1 scgl-7), DJ803-2-1 (MATa ade2-1 bar)-) can) cry) cyh2 his4-580 leu2 lys2 SUP4-3 steS-3 trp) ura3 scg)::lacZ6), and DJ736-34-1 (DJ164-7-1 STE4HPI) were provided by D. Jenness. KB112-18 (JE112 ste)8::LEU2) was constructed by integrative transformation (32) of JE112 with plasmid M65p5 (provided by M. Whiteway) cleaved with Pst I and HindIII. KBY1, constructed by mating between RK511-6B (31) and RK537-3B (24), was used for expression of STE2 from the GAL) promoter. Construction of plasmid pYKK13, in which the STE2 gene is placed under control of the GAL) promoter, will be described elsewhere. Rich medium (YP) or synthetic medium (33) containing 2% glucose (or 2% galactose and 0.1% raffinose for expression of STE2 from the GAL) promoter) were used for growth of cells. LSM medium (24) was used for labeling yeast cells with 4 Preparation of Subcellular Fractions, Equilibrium Binding, and Ligand Dissociation Assays. JE112 was grown at 300C. DJ164-7-1 and its isogenic derivatives were grown at 340C to inactivate the temperature-sensitive ste5-3 gene product,

Certain peptides (1-5), neurotransmitters (6, 7), hormones (8, 9), olfactants (10), and light (11) elicit specific cellular responses by acting through receptors coupled to guanine nucleotide-binding protein (G protein)-linked signal-transduction systems. G proteins are a/3y heterotrimers, and receptor coupling specificity is determined primarily by the type of a subunit (Ga) present (12, 13). Although isolated Ga subunits bind guanine nucleotides, they fail to couple productively with receptors (14, 15); /B3y complexes must also be present. It has been proposed that the jry complex promotes receptor coupling by anchoring the Ga subunit to the membrane (16). An obligatory membrane-anchoring function seems unlikely because both the a subunit and the /3y complex of the rhodopsin-coupled G protein, transducin, are soluble (14). It also has been suggested from indirect evidence that the f3y complex promotes coupling by interacting with receptors (17, 18). Specific roles for the individual 18 and y subunits in receptor coupling are ill-defined because their separation in active form generally has not been achieved.

Abbreviations: Nle, norleucine; GTP[y-S], guanosine 5'-[y-thio]triphosphate; GDP[13-S], guanosine 5'-[,8-thio]diphosphate; G protein, guanine nucleotide-binding protein. *Present address: Department of Cell Biology and Physiology, Washington University School of Medicine, Saint Louis, MO 63110. tTo whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

4363

4364

Proc. Natl. Acad Sci. USA 87 (1990)

Biochemistry: Blumer and Thorner

which is required at a step in the pheromone response pathway downstream of the receptor and G protein (30, 34). Subcellular fractions were prepared from labeled or unlabeled cells as described (24), except that the buffer was 10 mM Tris acetate/1 mM magnesium acetate/0.1 mM EDTA/ 10% (wt/vol) glycerol, pH 8.0. Equilibrium binding assays using purified 35S-labeled a-factor (35S-a-factor) and partially purified plasma membrane fractions were performed essentially as described (24), except that the buffer was 50mM Tris acetate/500 mM potassium acetate/i mM magnesium acetate/0.1 mM EDTA, pH 8.0. Competition binding assays included 35S-a-factor at a concentration (20 nM) near its Kd for receptor binding and various concentrations of an a-factor antagonist, des-Trpl-[Ala3, Nle'2]a-factor, where Nle is norleucine (35) (provided by J. Becker). Rates of 35S-a-factor dissociation from the receptor were measured as follows. Membrane fractions (500 ,ug of protein) were incubated at 220C for 30 min in the presence of a saturating concentration (100 nM) of 35S-a-factor (50 Ci/mmol; 1 Ci = 37 GBq). Samples were diluted 1:100 in the presence of 10 ILM unlabeled synthetic a-factor (Peninsula Laboratories) with or without various concentrations of different nucleotides (Pharmacia LKB). Aliquots were removed at intervals, filtered, washed, and analyzed by liquid scintillation spectrometry as described (24). Generation of GPA1-Specific Antibodies, Detection of GPA1 Protein, and Detergent Treatment of Membrane Fractions. A trpE-GPAI gene fusion was constructed as follows. A 910base-pair (bp) Pvu II/EcoRI fragment of yeast genomic DNA encoding the carboxyl-terminal 201 residues of the GPAI gene product was made blunt-ended and ligated with Sma I-cut pATH11 DNA (36). Nucleotide sequencing across the vectorinsert junction of the resultant plasmid (pATH11-GPA1) and the production in E. coli of a trpE-GPA1 fusion protein of the expected size (59 kDa) confirmed that the trpE and GPAJ sequences were joined in a continuous reading frame. Purification of the trpE-GPA1 fusion protein from the inclusion body fraction of E. coli, immunization of rabbits, and affinity purification of GPA1-specific antibodies were performed as described (37). GPA1-specific antibodies were used to immunoprecipitate the GPA1 product from soluble and particulate fractions derived from cells labeled with 35SO2- as described (24). Detergent extraction of membrane fractions (400 ,g of protein) were performed at 4°C for 1 hr in the presence of 1% (wt/vol) Triton X-100. Extracts separated into detergentsoluble and detergent-insoluble fractions by centrifugation for 20 min at 100,000 x g were analyzed by immunoprecipitation using GPA1-specific antibodies.

coupling between the a-factor receptor and a G protein. In the absence of exogenously added guanine nucleotide, ligand-receptor complexes dissociated at a characteristic rate (til, 2 60 min) (Fig. 1). Addition of 1 juM GTP[y-S] caused bound a-factor to dissociate from receptor nearly 10-fold more rapidly (til,2 8 min). GDP[If-S] was significantly less effective at promoting a-factor dissociation, whereas ATP[S] and ADP[J3-S] had no detectable effect. Conversion of the a-factor receptor from a high- to a low-affinity state was, therefore, specific for guanine nucleotides. The results of titration experiments indicated that the relative efficacy of guanine nucleotides in promoting a-factor dissociation was: GTP[yS] > 5'-guanylyl imidobisphosphate (GMP-P[NHIP) = GTP > 5'-guanylyl methylenebisphosphate (GMP-

P[CH2]P) = GDP[f-S]. Quantitative measurements of receptor affinity were obtained by two different equilibrium binding methods. First, in the absence of guanine nucleotide, a homogeneous class of a-factor binding sites (Kd = 17 nM) was observed by Scatchard analysis (Fig. 2A). In the presence of 100 j.M GTP[yS, receptor affinity for ligand was reduced by a factor of at least 8 (Kd 150 nM, although a Bm, could not be determined accurately). Second, a ligand competition assay using an a-factor antagonist, des-Trpl-[Ala3, Nle12]a-factor (35), was used to obtain an independent estimate of the change in receptor affinity caused by guanine nucleotide. In good agreement with the equilibrium binding results, the IC50 values for displacement of authentic 35S-a-factor from the >

1 02

w

None 0.01 gM 0.1 gIM

i

ii

None 1 gM 10

1

jIM

gM

10

gM

c1

0

20

10

102

0

None

1ijM

102

10

20

None 1 FM 10 gM

-

IlojM

RESULTS a-Factor Receptor-G Protein Coupling in Vitro. Two hallmarks of functional coupling between receptors and G proteins are agonist-promoted binding and hydrolysis of GTP by Ga subunits and a decrease in agonist binding affinity of the receptor in the presence of guanine nucleotides. To detect coupling between the a-factor receptor and its G protein, we first attempted to measure a-factor-dependent GTP binding and GTPase activities in partially purified yeast plasma membrane preparations, as well as equilibrium binding of 35S-a-factor in the presence and absence of guanine nucleotides. Under conditions known to promote high-affinity binding of a-factor to its receptor in whole cells or isolated membranes (pH 6.0, low ionic strength) (23, 24), these methods failed to yield evidence of a guanine nucleotideregulated interaction (data not shown). To determine whether receptor-G protein coupling could be detected under other reaction conditions, we developed a convenient method to measure the rate of dissociation of a-factor from its receptor. Such assays, when carried out at pH 8.0 and higher ionic strength, gave clear evidence of

101

I

0

.

10

time (min)

I I

20

101

I

a

0

10

II

20

time (min)

FIG. 1. Dissociation of 35S-a-factor from its receptor is promoted 35Sspecifically by guanine nucleotide. Rate of dissociation of from a-factor prebound to receptor in partially purified membranes JE112 was determined as described. Receptor occupancy was calculated as the fraction of binding sites remaining (in percent of initial). Initial receptor occupancy was measured before ligand dissociation was initiated. Nonspecific binding observed in a 500-fold excess of unlabeled a-factor was 2-4 fmol/mg of protein. Values given are the averages of the specific binding (200-250 fmol/mg) obtained from assays performed in triplicate. (Upper) Guanine nucleotides: guanosine 5'-[ythioltriphosphate (GTP[y-S]) (Left) and guanosine 5'-[f3-thio]diphosphate (Right). (Lower) Adenineandnucleoadentides: adenosine 5'-[rthio]triphosphate (ATP[yS]) (Left) osine 5'-[1J-thio]diphosphate (ADP[J3-S]) (Right). The concentrations of nucleotides employed are on the right.

Biochemistry: Blumer and Thorner 0

Proc. Natl. Acad. Sci. USA 87 (1990)

20

0 1

-O.

co a)

0E2r .

1 0 U-C 7 E0 Im .1. LL co

- 5

U-

6

O. 0

50

100 150 200 250 a-Factor Bound

300

350

(f-mol/mg)

C

L)

co

°=

0

0

,

0' -9.0

-7.0 I

-8.0

' -6.0 a

-5.0

-4.0

log [ antagonist (M) ] FIG. 2. Effect of GTP[y-S] on equilibrium binding of 35S-a-factor to its receptor. Equilibrium binding to membranes from JE112 was conducted at pH 8.0 and high ionic strength as described. (A) Scatchard analysis of equilibrium binding of 35S-a-factor to receptor in isolated membranes (24) in the absence (E) and presence (*) of added 100 AM GTP[,y-S]. Bmax in the presence of GTP[y-S] could not be determined with complete confidence because the high concentrations of a-factor required gave high levels of nonspecific binding. (B) Competitive displacement of bound 35S-a-factor by the a-factor antagonist des-Trpl-[Ala3, Nle12]a-factor in the absence (El) and presence (*) of 100 ,uM GTP[y-S]. Receptor occupancy was calculated as described in the legend to Fig. 1. Apparent IC50 values for the displacement of 35S-a-factor by the antagonist in the absence and presence of GTP[y-S] were 12.5 AM and 1.8 ,uM, respectively.

receptor by the antagonist were shifted to about a 7-fold lower concentration when 100 ,uM GTP[y-S] was present (Fig. 2B). Because similar decreases in affinity were observed by either the kinetic or equilibrium measurements, the primary effect of guanine nucleotides is to lower the receptor affinity for a-factor by enhancing its rate of dissociation. Coupling in Vitro Requires Functional GPAI, STE4, and STE18 Gene Products. To determine whether the a-factor receptor is functionally coupled to the G protein subunits thought to function in the pheromone response pathway, ligand dissociation assays were performed by using membrane fractions prepared from strains carrying wild-type or mutant copies of the GPAJ (SCGI), STE4, and STE18 genes. The scgl-7 (gpal) allele that was used (28) is a missense mutant (see below), and the stel8::LEU2 allele is a lossof-function insertion mutation (29). Mutants lacking Gg (ste4 null alleles) expressed such a low level of receptor that analysis of a-factor binding was not technically feasible

4365

(K.J.B., unpublished data); therefore, we used a dominant STE4HPl allele. Because this mutation can lead to a cellular response even in the absence of pheromone (34), it appears to encode a Gp3 subunit that is unable to associate with the Ga subunit and, thus, is able to interact constitutively with downstream effectors (34). Cell response was prevented in such strains and in scgl-7 mutants by the steS-3 mutation. Measurements of a-factor dissociation rates indicated that even in the absence of guanine nucleotide, membrane fractions prepared from the scgl, STE4HPI and stel8 mutants contained receptors that exist in a low-affinity state (Fig. 3). Two lines of evidence indicated that this behavior represented uncoupled receptors. First, a rate of a-factor dissociation similar to that seen with the membranes from the mutant cells (in the absence of added nucleotide) was observed when membranes from wild-type cells were treated with GTP[y-S]. Second, upon treatment with GTP[y-S], membranes derived from the scgl-7 mutant reproducibly displayed a slight enhancement in a-factor dissociation rate, as expected if this missense allele encodes a Ga subunit that retains partial function. In contrast, GTP[y-S]-treated membranes derived from the STE4HP1 and stel8 mutants failed to display any detectable enhancement in the rate of a-factor dissociation. These in vitro results suggest that a-factor receptor-G protein coupling absolutely requires functional forms of the STE4 and STE18 gene products (Gp and Gy, respectively) as well as the GPAI (SCGI) gene product (Ga). To test whether the a-factor receptor is capable of coupling with other yeast G proteins, we expressed the a-factor receptor (STE2 gene product) from the inducible GAL] promoter in MATa/MATa diploids. These cells do not express GPAJ (Ga), STE4 (Ga9), and STE18 (G.) but do express other homologs of Ga subunits-for example, GPA2 (38). Equilibrium binding methods indicated that receptors expressed in MATa/MATa diploid cells bound a-factor with low affinity (Kd 150-200 nM; data not shown) in both the presence and the absence of guanine nucleotide. Thus, even when competition from its cognate G protein was eliminated, the a-factor receptor did not couple productively with other yeast G proteins. Possible Roles of j3 and y Subunits. One essential role of the STE4 and STE18 gene products in receptor coupling might be to provide an anchor to retain the Ga (GPA1) subunit at the membrane (39, 40). To test this possibility, we generated GPA1-specific antibodies and used them to determine the distribution of the GPAJ gene product between soluble and membrane fractions prepared from the wild-type and mutant cells. GPA1-specific antibodies immunoprecipitated a 49-kDa polypeptide from the particulate fraction of 35S-labeled wildtype MATa cells (Fig. 4), a species close to the size predicted from the GPAI gene sequence (54 kDa). This 49-kDa species represents the GPAI gene product because it comigrated with the product of in vitro translation of GPA1 mRNA (data not shown) and because it was absent in immunoprecipitates of membrane fractions prepared from cells carrying a gpal: :lacZ null mutation (Fig. 4). The soluble fraction of all cells examined possessed an antigenically related protein of 47-48 kDa, which is not the GPAI gene product because it was also found in the fraction from the gpal:: lacZ insertion mutant. Cell fractionation and immunoprecipitation indicated that the GPAJ gene product was present in the particulate fraction, even in the scg-7 (gpa1), STE4HPI, and ste18::LEU2 mutants (Fig. 4). These results suggest that the GPA1 protein (Ga) can associate with the membrane when G, is absent, G is functionally altered, or Ga itself is inactive. It was possible, however, that the GPA1 protein was found in the particulate fraction because, in the absence of a functional fBy complex, Ga,, subunits aggregate or adsorb to other insoluble material. Native Ga subunits are generally released =

4366

Biochemistry: Blumer and Thorner 1o00

Is

Proc. Natl. Acad. Sci. USA 87 (1990)

100

WT

scg1-7

80

80

60

60-

a

40

I

20 20

20 0' 0

2

8

6

4

,.W

2

4

8

6

~~~~~STE4Hp

10 0

h

0

10

10

stel 8::LEU2

810 610

40

200 0

4

2

8

6

v

10

0

2

,

DISCUSSION The a-factor receptor of Saccharomyces cerevisiae was shown to be functionally linked to a G protein in vitro as

FRACTION GPA1 ProductL

GPA1 scgl::aacZscgl-7 STEE4 S

P S

P

S

P

6

* W *18 0

time (min)

time (min)

from membranes by treatment with mild nonionic detergents (13), whereas protein aggregates typically are resistant to solubilization by such detergents. Accordingly, the membrane fractions derived from 35S-labeled wild-type, segl-7, STE4HPI, and stel8::LEU2 cells were treated with the nonionic detergent Triton X-100. The detergent extracts were fractionated by ultracentrifugation, and the resultant pellet and supernatant fractions were analyzed by immunoprecipitation with GPA1specific antibodies, followed by gel electrophoresis. A similar analysis of Triton X-100 extracts of membrane fractions from unlabeled cells was also performed by immunoblotting after gel electrophoresis. In every case (data not shown), the Triton X-100 treatment efficiently released GPA1 protein in a soluble form, indicating that Ga is found in the particulate fraction, even in the absence of functional and y subunits because it is membrane-associated.

GENOTYPE

4

S P S

csLel8: LEU2 P

4. W

FIG. 4. Immunological localization of the GPAJ gene product (Ga subunit). A set of strains (see Materials and Methods) were grown at 34WC to inactivate the thermolabile steS-3 gene product and were labeled with 35SOJ-. This procedure blocks the constitutive signal produced as a result of the scgl (gpal) and STE4HP1'mutations because the STE5 gene product is required for a step in the pheromone response pathway downstream of the receptor and G protein subunits (30, 34). Subcellular fractions prepared from the labeled cells were solubilized with SDS, immunoprecipitated with GPA1specific antibodies, and analyzed by SDS/PAGE and fluorography (24). For the wild-type cells and the scgl::lacZ6, scgl -7, and STE4HP1 mutants, immunoprecipitations were performed with soluble and membrane fractions derived from an equal number of cells. For the stel8::LEU2 mutant, four times as many cells were used because we observed that GPA1 protein was expressed in this strain at a somewhat lower level ("50% of wild type). The apparent molecular masses of immunoprecipitated proteins were determined relative to those of labeled marker proteins (24).

1

FIG. 3. Effect of mutations in the GPAI, STE4, and STE18 genes on the regulation of receptor coupling by guanine nucleotide. Measurement of the dissociation of 35S-a-factor from its receptor was performed in the absence (o) and presence (*) of 10 PM GTP[y-S] as before (see Fig. 1). The membrane fractions used were prepared from wild-type (WT) cells, cells

carrying loss-of-function mutations in

the Ga (scgl-7) or Gy (stel8::LEU2) subunit genes, or a mutation in the Go subunit gene (STE4HP) that is thought to eliminate interaction with the Ga subunit. Initial levels of receptor occupancy were: wild-type cells, 220 fmol/mg; scgl-7 cells, 56 fmol/mg; STE4HPI cells, 51 fmol/mg; stel8:: LEU2 cells, 22 fmol/mg. Values given are the average of three independent determinations (bars indicate the SEM).

evidenced by a dramatic guanine nucleotide-dependent decrease of its ligand-binding affinity. Surprisingly, this receptor-G protein coupling was detected at pH 8.0 but not at pH 6.0. At pH 8.0, even when guanine nucleotide is absent, the affinity of receptor for a-factor was reduced by a factor of about 5-10 compared with the values we obtained previously at pH 6.0 (24, 26, 31). Because pH similarly influences a-factor binding to receptor in membranes from MATa/ MATa diploids, where all three G protein subunits are absent (27-29), we conclude that pH affects receptor-ligand interaction, rather than receptor-G protein coupling. The pH dependence of this effect suggests that ligand recognition by the receptor involves a histidine residue. Indeed, there is a histidine residue at position 2 of a-factor, and this residue is essential for biological activity of the pheromone (41, 42). Protonation of His-2 of a-factor may stabilize the conformation of the peptide most appropriate for binding (43), or extracellular pH may directly affect the structure of the receptor itself. It has been proposed that ionic interaction between ligands and charged residues within the third transmembrane segment are critical for activation of the f3adrenergic receptor (44) and rhodopsin (45, 46). Not surprisingly, guanine-nucleotide-modulated receptorG protein coupling in vitro required a functional Ga subunit (GPAI gene product). However, receptor-G protein coupling in vitro also required functional forms of the Gp subunit (STE4 gene product) and G., subunit (STE18 gene product). Thus, our results and earlier genetic data (27-29, 34) strongly imply that the GPAI, STE4, and STE18 gene products associate and interact directly with the a-factor receptor. Because mutations in GPAI, STE4, or STE18 effectively eliminated receptor-G protein coupling in vitro, and because expression of STE2 in MATa/MATa diploids generated only low-affinity guanine nucleotide-insensitive binding sites, the a-factor receptor fails to interact to a significant extent with other yeast G proteins, including the GPA2 gene product (38). Thus, in contrast to muscarinic acetylcholine receptors, which can interact with either G, (47) or Go (15), the a-factor receptor seems to couple effectively with only a single type of yeast G protein. Studies of mammalian signaling systems indicate that Ga subunits mediate interaction with specific receptors, although ,By complexes are also required. In yeast, Ga subunits are also likely to be a major determinant of coupling specificity. Genetic evidence suggests that mammalian Go asso-

Biochemistry: Blumer and Thorner ciates with the yeast STE4 and STEJ8 gene products but fails to couple with the a-factor receptor (28). The mechanism by which B]y promotes Ga-receptor inter-

action in mammalian cells is still unknown. Even for the well-characterized rhodopsin-transducin system, TY has been reported to be either dispensable (48) or necessary (49) for receptor coupling. Our finding that the STE4 and STE18 gene products are absolutely required for a-factor receptorG protein coupling raises the possibility that mammalian 8 and y subunits serve a similar essential function. Our immunological and cell fractionation studies indicate the yeast f3 and y subunits are not essential for association of the Ga subunit with the membrane fraction. Although the plasma membrane-bound a-factor receptor could be uncoupled when G. is absent or Gp is functionally altered simply because GPA1 protein associates inappropriately with another membrane, we currently favor the idea that yeast /y subunits contribute to the productive interaction between the Ga subunit and the receptor by causing a conformational change in the Gal subunit or by directly contacting the receptor itself, or both. It is not clear how yeast Ga is localized to the membrane. GPA1 protein does contain consensus signals for N-terminal myristoylation (50) and appears to be myristoylated (J. Reid and K. Matsumoto, personal communication). However, specific, yet unproductive, association with the membranebound receptor could also promote attachment of Ga to the membrane. We thank Jeff Becker, Duane Jenness, and Malcolm Whiteway for generously providing research materials; Ignacio Aguigui for purifying the anti-GPA1 antibodies; and Karl Klose for constructing the GAL1-STE2 expression plasmid. This work was supported by American Cancer Society Postdoctoral Fellowship PF2632 to K.J.B. and by National Institutes of Health Research Grant GM21841 to J.T. 1. Masu, Y., Nakayama, K., Tamaki, H., Harada, Y., Kuno, M. & Nakanishi, S. (1987) Nature (London) 329, 836-838. 2. Jackson, T. R., Blair, L. A. C., Goedert, M. & Hanley, M. (1988) Nature (London) 335, 437-440. 3. McFarland, K. C., Sprengel, R., Phillips, H. S., Kohler, M., Rosemblit, N., Nikolics, K., Segaloff, D. L. & Seeburg, P. (1989) Science 245, 494-499. 4. Paramentier, M., Libert, F., Maenhaut, C., Lefort, A., Gerard, C., Perret, J., Van Sande, J., Dumont, J. E. & Vassart, G. (1989) Science 246, 1620-1622. 5. Yokota, Y., Sasai, Y., Tanaka, K., Fujiwara, T., Tsuchida, K., Shigemoto, R., Kakizuka, A., Ohkubo, H. & Nakanishi, S. (1989) J. Biol. Chem. 264, 17649-17652. 6. Kubo, T., Fukuda, K., Mikami, A., Maeda, A., Takahashi, H., Mishina, M., Gafa, T., Ichiyama, A., Kangawa, K., Kojima, M., Matsuo, H., Hirose, T. & Numa, S. (1986) Nature (London) 323, 411-416. 7. Julius, D., MacDermott, A. B., Axel, R. &Jessell, T. M. (1988) Science 241, 558-564. 8. Dixon, R. A. F., Kobilka, B. K., Strader, D. J., Benovic, J. L., Dohlman, H. G., Frielle, T., Bolanowski, M. A., Bennett, C. D., Rands, E., Diehl, R. E., Mumford, R. A., Slater, E. E., Sigal, I. S., Caron, M. G., Lefkowitz, R. J. & Strader, C. S. (1986) Nature (London) 321, 75-79. 9. Kobilka, B. K., Kobilka, T. S., Daniel, K., Regan, J. W., Caron, M. G. & Lefkowitz, R. J. (1988) Science 240, 13101316. 10. Jones, D. T. & Reed, R. R. (1989) Science 244, 790-795. 11. Nathans, J. & Hogness, D. S. (1984) Proc. Nati. Acad. Sci. USA 81, 4851-4855. 12. Neer, E. J. & Clapham, D. E. (1988) Nature (London) 333, 129-134. 13. Freissmuth, M., Casey, P. J. & Gilman, A. G. (1989) FASEB J. 3, 2125-2131. 14. Fung, B. K.-K. (1983) J. Biol. Chem. 258, 10495-10502.

Proc. Natl. Acad. Sci. USA 87 (1990)

4367

15. Florio, V. A. & Sternweis, P. C. (1989) J. Biol. Chem. 264, 3909-3915. 16. Sternweis, P. C. (1986) J. Biol. Chem. 261, 631-637. 17. Kelleher, D. J. & Johnson, G. L. (1988) Mol. Pharmacol. 34, 452-460. 18. Halpern, J. L. , Chang, P. P., Tsai, S. C., Adamik, R., Kanaho, Y., Sohn, R., Moss, J. & Vaughan, M. (1987) Biochemistry 26, 1655-1658. 19. Herskowitz, I. (1988) Microbiol. Rev. 52, 536-553. 20. Cross, F., Hartwell, L. H., Jackson, C. & Konopka, J. B. (1988) Annu. Rev. Cell Biol. 4, 429-457. 21. Nakayama, N., Miyajima, A. & Arai, K. (1985) EMBO J. 4, 2643-2648. 22. Hagen, D., McCaffrey, G. & Sprague, G. F., Jr. (1986) Proc. Natl. Acad. Sci. USA 83, 1418-1422. 23. Jenness, D. D., Burkholder, A. C. & Hartwell, L. H. (1983) Cell 35, 521-529. 24. Blumer, K. J., Reneke, J. E. & Thorner, J. (1988) J. Biol. Chem. 263, 10836-10842. 25. Marsh, L. & Herskowitz, I. (1988) Proc. Natl. Acad. Sci. USA 85, 3855-3859. 26. Yu, L., Blumer, K. J., Davidson, N., Lester, H. A. & Thorner, J. (1989) J. Biol. Chem. 264, 20847-20850. 27. Miyajima, I., Nakafuku, M., Nakayama, N., Brenner, C., Miyajima, A., Kaibuchi, K., Arai, K., Kaziro, Y. & Matsumoto, K. (1987) Cell 50, 1011-1019. 28. Dietzel, C. & Kurdan, J. (1987) Cell 50, 1001-1010. 29. Whiteway, M., Hougan, L., Dignard, D., Thomas, D. Y., Bell, L., Saari, G. C., Grant, F. J., O'Hara, P. & MacKay, V. L. (1989) Cell 56, 467-477. 30. Nakayama, N., Kaziro, Y., Arai, K.-I. & Matsumoto, K. (1988) Mol. Cell. Biol. 8, 3777-3783. 31. Reneke, J. E., Blumer, K. J., Courchesne, W. E. & Thorner, J. (1988) Cell 55, 221-234. 32. Rothstein, R. (1983) Methods Enzymol. 101, 202-211. 33. Sherman, F., Fink, G. R. & Hicks, J. B. (1986) Methods in Yeast Genetics (Cold Spring Harbor Lab., Cold Spring Harbor, NY). 34. Blinder, D., Bouvier, S. & Jenness, D. D. (1989) Cell 56, 479-486. 35. Raths, S. K., Naider, F. & Becker, J. M. (1988) J. Biol. Chem. 263, 17333-17341. 36. Sauter, M., Boos, H., Hirsch, F. & Mueller-Lantzsch, N. (1988) Virology 166, 586-590. 37. Harlow, E. & Lane, D. (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Lab., Cold Spring Harbor, NY). 38. Nakafuku, M., Obara, T., Kaibuchi, K., Miyajima, I., Miyajima, A.; Itoh, A., Nakamura, S., Arai, K., Matsumoto, K. & Kaziro, Y. (1988) Proc. Natl. Acad. Sci. USA 85, 1374-1378. 39. Nakayama, N., Arai, K.-I. & Matsumoto, K. (1988) Mol. Cell. Biol. 8, 5410-5416. 40. Schafer, W. R., Kim, R., Sterne, R., Thorner, J., Kim, S.-H. & Rine, J. (1989) Science 245, 379-385. 41. Naider, F. & Becker, J. M. (1986) Crit. Rev. Biochem. 21,

225-247. 42. Masui, Y., Tanaka, T., Chino, N., Kita, H. & Sakakibara, S. (1979) Biochem. Biophys. Res. Commun. 86, 982-987. 43. Jelicks, L. A., Broido, M. S., Becker, J. M. & Naider, F. R. (1989) Biochemistry 28, 4233-4240. 44. Strader, C. D., Sigal, I. S., Candelore, M. R., Rands, E., Hill, W. S. & Dixon, R. A. F. (1988) J. Biol. Chem. 263, 1026710271. 45. Sakmar; T. P., Franke, R. R. & Khorana, H. G. (1989) Proc. Natl. Acad. Sci. USA 86, 8309-8313. 46. Zhukovsky, E. A. & Oprian, D. D. (1989) Science 246, 928930. 47. Kurose, H., Katada, T., Haga, K., Haga, K., Ichiyama, A. & Ui, M. (1986) J. Biol. Chem. 261, 6423-6428. 48. Yamazaki, A., Tatsumi, M., Torney, D. C. & Bitensky, M. (1987) J. Biol. Chem. 262, 9316-9323. 49. Fukada, Y., Ohguro, H., Saito, T., Yoshizawa, T. & Akino, T. (1989) J. Biol. Chem. 264, 5937-5943. 50. Towler, D. A., Gordon, J. I., Adams, S. P. & Glaser, L. (1988) Annu. Rev. Biochem. 57, 69-99.