Molecular Determinants of Bioactivity of the Saccharomyces

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Aug 5, 2015 - nant of a-factor potency. Analyses on truncated a-factors suggest that sequential removal of NH2-terminal resi- dues leads to a gradient of ...
Vol. 269,No. 31,Issue of August 5, PP. 19817-19826,1994 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMJSTRK 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc

Molecular Determinantsof Bioactivity of the Saccharomyces cereuisiae Lipopeptide Mating Pheromone* (Received for publication, February 16, 1994, and in revised form, April 21, 1994)

Guy A. CaldwellS, Shu-Hua WangO, Chu-Biao XueO, Ying Jiangg, Hui-Fen Lul, Fred NaiderO, and Jeffrey M. BeckerSn From the program inCellular; Molecular; and Developmental Biology and Department of Microbiology, The University of Tennessee, Knoxville, Tennessee 37996-0845 and the §Department of Chemistry, College of Staten Island, City University of New York, Staten Island, New York 10301

The a-factor of Saccharomyces cerevisiae (YIIKGVFWDPAC(Farnesy1)-OCH,) is a peptide pheromone in which post-translational modification with a farnesyl isoprenoid and carboxyl methyl group is required f o r export and bioactivity. Truncated and carboxyl-terminal modified analogs of the a-factor were synthesized in order to determine the effect ofsuch modifications on bioactivity. Bioactivity studies on carboxyl-terminal analogs in which the chirality, the cysteine thioether, and the carboxyl ester were varied in an attempt to s t u d y the influence of topology on a-factor activity indicate that the hydrophobicity c o n f e r r e d b y the farnesyl moiety and not its specific spatial orientation is a k e y determinant of a-factorpotency. Analyses on truncated a-factors suggest that sequential removal of NH2-terminalresidues leads to a gradient of potency loss, with some amino acids exhibiting a slightly greater contribution to bioactivity than others. Random oligonucleotide-targeted mutagenesis of the gene encoding a-factor was coupled to a biological screen to identify altered a-factor peptides which are secreted y e t exhibit a loss of a-factor bioactivity.Transformants exhibiting this phenotype w e r e examined to identify codon changes presumably responsible f o r the altered phenotype, thus indicating residues that m a y contribute significantly to a-factor bioactivity.

The yeast Saccharomyces cerevisiae has served as a model organism for the analysisof the molecular events thatoccur in receptor-mediated signaltransduction (1). Mating between haploid yeast cells to form diploids is controlled by the reciprocal action of peptide mating pheromones, termed a-factor and a-factor, which are secreted by either a-cells or a-cells, respectively. These peptides bind to theirrespective receptor proteins on cells of the opposite mating type and subsequently induce growth arrest, morphological change, and mating (1, 2). Both pheromones are ligands for receptors that aremembers of the G protein-coupled receptor family, which includes the rhodopsin and adrenergic receptors. Analyses of this protein family has revealed that its members share the common structural organization of seven transmembrane domains although their amino acid sequences are not similar(3). This also appears to be the case for the yeast pheromone receptors: the Ste2p or a-factor receptor and the Ste3p or a-factor receptor (4-6).

Although the two yeast pheromones appear functionally equivalent, they are dissimilar in their structure, secretion, and biosynthesis. The a-factor is an unmodified 13-amino acid peptide (WHWLQLKPGQPMY) that is secreted through the classical yeast secretory pathway involving the endoplasmic reticulum, Golgi, and secretory vesicles (7). In contrast, the a-factor isa 12-amino acid lipopeptide, processed from either a 36- or 38-residue precursor, that is post-translationally modified by the addition of a C,, farnesyl isoprenoid and a carboxylterminalmethyl group (YIIKGVF’WDPAC(Farnesy1)-OCHJ (8). Furthermore, a-factor is exported via a mechanism that requires theaction of the yeastSTE6 gene product, a homolog of the mammalian multidrug resistance P-glycoprotein (9, 10). Extensive studies have been performed on the structural aspects of a-factor bioactivity and receptor binding (11-131, whereas relatively few studies on a-factor bioactivity have been published (14, 15). It has been previously shown that isoprenylation of a-factor is a prerequisite for its export and bioactivity (16-18). Removal of both the COOH-terminal farnesyl and methyl ester groups on a-factor results in a 100,000-fold drop in bioactivity (14). The presence of the isoprenyl moiety on a-factor may play a role in either the direct interaction of the pheromone with the Ste3preceptor or indirect presentation of the amino acid portion of a-factor to the receptor. Isoprenylation has been implicated in the membrane localization and function of various proteins including a-factor,nuclear lamins, rab vesicular trafficking proteins, and the oncogene product RAS (17-21). In fact, RAS-mediated cellular transformation is dependent upon membrane localization via farnesylation (20, 22, 23). Several recent studies indicate that farnesylation of proteins directly enhances their ability to interact withspecific targets. For example, the attachment of a farnesyl group t o RAS increases its ability to bind and activateadenylyl cyclase, even in the absence of membranes (24). Similarly, the interaction between the a subunit and Py subunits of transducin is highly favored specifically when the y subunit is farnesylated and carboxyl methylated (25). However, the function of protein prenylation remains tobe fully elucidated and very little is known about factors affectingthe interactionof lipopeptides withtheir recognition proteins (26). In this study, we utilize a combined approach of molecular biology and synthetic peptide biochemistry to analyze the biological effects of amino acid truncations, substitutions, and carboxyl-terminal modifications within afactor as a model system for the analysisof isoprene-mediated peptide-protein interaction.

* This work was supported by Grant GM-46520 from the National Institutes of Health. The costs of publication of this article were deMATERIALS AND METHODS frayed in part by the payment of page charges. This article must thereYeast Strains and Methods-The following yeast strains were used: fore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. SM1229 (MATa Amfal::LEU2Amfa2::URAS trpl his4 canl leu2 ura3) 1 To whom correspondence should be addressed. (27) and RC757 (MATa sst2-1 rme his6 met1 canl cyh2) (28). Trans19817

Biological

19818

a-Factor

Activity

TABLE I Characterization of a-factor analogues No.

acid

PeptideAmino

Mr calculated

analysis

%%-

Yield

R,"

RP

%

I I1 I11 IV V VI VI1 VI11 IX X

XI XI1 XI11

a-Factor [des-Yl-a-Factor [des-Y.I.1-a-Factor [des-Y-I-11-a-Factor [des-Y.I.I.K.1-a-Factor

1629.9 1466.3 1353.9 1240.7 1112.6 1055.8 [des-Y.I.I.K.G.1-a-Factor [des-Y.I.I.K.G.V.1-a-Factor 956.0 [des-Y.I.I.K.G.V.F.1-a-Factor 809.0 Asp-Pro-Ala-Cys(Far)OMe 623.5 1629.9 [~-cys'~]-a-Factor 1629.9 [Cys'2(CH,)OFarl-a-Factor [~-Cys'~(CH,)OFarl-a-Factor 1629.9 [Ile6,Leusl-a-Factor 1571.0 [Ile6,Lys81-a-Factor 1586.0

1466.8 0.24 30 1353.8 0.1727 1240.7 21 1112.60.4929 1055.6 42 30 956.5 36 809.4 0.46 623.3 0.2728 1629.7 0.1547 1630.0 46 1630.0 0.1649 1571.8 32 1586.2 0.1030

0.64 0.61 0.16 0.61 0.69 0.55 0.69 0.58 0.69 0.71 0.64 0.70 0.16 0.70 0.75 0.18 0.71 0.67

Eluent = 1-butano1:aceticacid:water (4:l:l).

* Eluent = 1-butano1:aceticacid:water:pyridine (15:3:8:10). e

ND, not determined. These peptides were made from the same decapeptide as a-factor and we therefore did not carry out amino acid analysis.

formation of yeast strains was performedby the LiAc method of Gietz et al. (29). Growth arrest or "halo" assays for measuring the bioactivity of synthetic a-factor and a-factor analogs was done by testing serial dilutions of peptides on lawns of supersensitive a-strain RC757, as previously described(14). Growth arrest assays for the selection of bioactive a-factor producing colonies under galactose-induced gene expression control were carried out either in the presence of 2% galactose, 2% raffinose (induced condition), or with 2% glucose only(repressed condition), as described (16). Mating restoration assays were performed by the exogenous addition of serial dilutions of a-factor or a-factor analogs to a mixture of a-strain RC757 and a-strain SM1229 (Amful Amfa2) on media selective for growthof diploid coloniesof these strains, as previously outlined (14). Synthesis and Characterization of Zkuncated and Carboxyl-terminal Modified a-Factors-Analogs of the a-factor in which the peptide portion of the molecule was systematically truncated (I-VIII; Table I) or where the topology of the carboxyl terminus was modified(E-XI; Table I) were synthesized using a combination of solid-phase and solutionphase procedures (30-32). In general our strategy involves the synthesis of protected peptide fragments on phenylacetamidomethyl-resins, cleavage of the protected fragments and condensation of these fragments with suitably derivatized dipeptides (Fig. 1, Scheme 1).This approach was successful forpeptides I-V, E-XI,XII, and XI11 (Table I). The amino terminus and side chains of all fragments were protected with Fmoc' and OFm protecting groups to avoid exposing the trans, trans-farnesyl moiety to acid. All amino acids are of the t-configuration except for Cys which can be D or L as indicated. The hydrophobicity of the protected fragment increased significantly as the size decreased making it very difficult to separate the fully protected and farnesylated pheromone fromthe protected amino-terminal fragment. To circumvent this problem we prepared peptide methyl esters beginning with cystine methyl ester using either a backing off procedure (Fig. 1, Scheme 2) or a fragment condensationprocedure (Fig. 1, Scheme 3). Using these approaches final products were obtained after regiospecific farnesylation of the SH group using acid catalysis in the presence of zinc (33). Peptides subjected to bioassay were isolated byHPLC and were >98%homogeneous (see Fig. 2) on reversed-phase C,, pBondapak columns (Waters Inc.) using both CH,CN/H,O/CF,COOH and CH,OW H,O/CF,COOH gradients where the methanol or acetonitrile content at the end of the gradient was typically 100% (0.025% CF,COOH). Yields of the purified lipopeptides varied from 21 to 49%. These yields reflect the low solubility of the products in aqueous media which resulted in significant problems with precipitation on the column during HPLC purification. It was, therefore, often necessary to purify several times to obtain the required homogeneity. Amino acid analysis was carried out at The abbreviations used are: Fmoc, 9-fluorenylmethoxycarbonyl; BOP, benzotriazol-l-yl-oxy-tris-(dimethylamino)phosphonium hexafluorophosphate;DMF, dimethylformamide;EtOAc, ethyl acetate; Far, farnesyl; HOAc, acetic acid HOBt, 1-hydroxybenzotriazole; HPLC, high performance liquid chromatography; MeOH, methanol; OFar, farnesol ester; OFm, 9-fluorenylmethyl ester; HMG, 3-hydroxy-3-methylglutaryl.

the Wistar Institute and at the Brigham and Women's Hospital. Fast atom bombardment-mass spectrometry was performed by the Mass Spectrometry facility at theUniversity of Tennessee. All peptides had the expected amino acidratios and molecular ions, which are reported as monoisotopic masses to the nearest 0.1 dalton. The structures of the farnesol ester peptides (X and X I ; Table I) were confirmed using 400 MHz 'H NMR spectroscopy. Synthesis of peptides containing farnesol esters have not been previously reported in the literature. In the following paragraphs we report details on the synthesis of [Cys'2(CH,)OFarl-a-factor ( X Table I). The corresponding D-analog was prepared using identical procedures. Fmoc-L-CysfCHJ-OH-To a solution of HCl L-CYS (3.1520g,mmol) in 20 ml ofwater and 30 ml of dioxane wereadded Na,CO, (4.8 g, 45mmol) and methyl iodide (1.25 ml, 20 mmol). The solution was stirred overnight, then cooled in an ice bath, and to it was added Fmoc-Cl(5.17 g, 20 mmol) batchwise. Four hours later when TLC indicated the disappearance of Cys(CH,), the solution was diluted with water and extracted with ether threetimes. The aqueous solution was acidified with 12 N HC1 to pH 3 andextracted with EtOAc three times. The combined organic phase was washed with water, dried (MgSO,), and concentrated. The crude product was purified on a silica column and crystallized frommethanouether to give 5.1 g(72%)of pure product (m.p. 202 (dec.);R, 0.56 (Et0Ac:MeOH:HOAc= 50:lO:l)). NMR (dimethyl sulfoxide-d,) S 7.89 (d, 2H); 7.71 (d, 2H); 7.36 (m, 5H); 4.27 (m, 3H); 3.92 (m, 1H); 2.95 (dd, 1H); 2.71 (dd, 1H); 2.0 (s, 3H). Fmoc-L-CysfCHJ-OFar-To a solution of Fmoc-L-Cys(CH,)OH (1.79 g, 5 mmol) in 10 ml of DMF cooled in an ice bath were added farnesol (1.27 ml, 5 mmol), BOP (2.21 g, 5 mmol), and 3 ml of pyridine. The solution was stirred for 2 h, EtOAc was added and the organic phase was washed with citric acid, water, NaHCO,, and water, dried (MgSO,), and concentrated. The crude product was purified on a silica gel column to give 3.4 g (82%) of oily product. R, 0.59 (Et0Ac:PE = 1:4). NMR (dimethyl sulfoxide-d,) 6 7.89 (d, 3H); 7.72 (d, 2H); 7.37(m, 4H); 5.27(t, 1H);5.04 (t, 2H); 4.58 (d, 2H); 4.53 (m, 1H); 4.26 (m, 3H); 2.84 (dd, 1H); 2.73 (dd, 1H); 2.06 ( s , 3H); 1.98 (m, 8H); 1.64 (s, 3H); 1.62 (s, 3H); 1.54 ( s , 6H). Fmoc-Ala-L-CysfCHJ-OFar-Fmoc-L-Cys(CH,)-OFar (1.68 g, 3 mmol) and 4-dimethylaminopyridine(1.10 g, 9 mmol) were dissolved in 5 ml of DMF and the solution was stirred overnight. WhenHPLC indicated the complete deprotection of the Fmoc group, the solution was cooled in an ice bath, and Fmoc-Ala (933 mg, 3 mmol) and BOP (1.33 g, 3 mmol) were added. After stirring for 1 h, EtOAc was added and the organic phase was washed with citric acid, water, NaHCO,, and water, dried (MgSO,), and concentrated. The crude dipeptide was observedby reversed-phase HPLC to be one major product. Since dipeptide diasteromers are generally resolved during reversed-phase chromatography it is unlikely that significant racemization occurred during synthesis of the farnesyl ester. Purification on a silicagelcolumngave a white powder. Yield 1.5 g (78%); m.p. 74-76"C; R, 0.44 (Et0Ac:PE = 13. NMR (dimethyl sulfoxide-d,) 6 8.34 (d, 1H); 7.89 (d, 2H); 7.73 (d, 2H); 7.56 (d, 1H);7.37 (m, 4H); 5.27 (t, 1H);5.06 (t, 2H); 4.57(d, 2H); 4.45(m, 1H); 4.24 (m, 3H); 4.14 (m, 1H); 2.80(m, 2H); 2.07 (s, 3H); 2.00 (m, 8H); 1.65 (s, 3H); 1.63 (s, 3H); 1.55 ( 8 , 6H); 1.23 (d, 3H).

Biological

a-Factor

Activity

19819

tion was added dropwise to 50 ml of ether and the precipitate was filtered, dissolved in methanollwater, and purified using reversed-phase HPLC. The yields, fast atom bombardment-mass spectrometry, and amino acid ratios are given in Table I. Random Oligonucleotide-targeted Mutagenesis oftheMFal Gene-A 1.6-kilobase BamHI DNA fragment containing the MFul gene of S. cereoisiae (34),which encodesthe primary precursor of the a-factor, was R I = Far, R2 = M e (I - V. IX, XI. XIII) used as the template for oligonucleotide-targeted mutagenesisof a speR I = M e ; R2 = Far (X, XI) cific region of this geneby a variation (35) of the method of Kunkel(36). This fragment was subcloned into phagemid vector pTZ19U (37) at the BamHI site of the polylinker and the resultingvector used as a source R', of single-stranded DNA for subsequent in vitro mutagenesis primedby a specific pool of mutagenic syntheticoligonucleotides (16,35). This pool Val-Phc-Trp-Asp-Pro of oligonucleotides was prepared by "doping" or contaminating each of GI?-Val-Phe-Trp-Asp-Pro LysCly-Val-Phe-Trp-Asp-Pro the nucleoside phosphoramidites used in the synthesis of the singleIlc-Lys-Gly-Val-Phe-Trp-Asp-Pro stranded DNA with a specific percentage of a n equimolar mixtureof all Ile-llc-LysGly-Val-Phc-Trp-Asp-Pro four phosphoramidites prior t o synthesis of the target DNA region of Tyr-lie-llf-Lys-Gly-Val-Phe-Trp-Asp-Pro interest. An oligonucleotide specifically corresponding to the region of Tyr-lle-llc-LysCly-Ile-Phc-~u-Asp-Pro Tyr-lle-lle-Lys-Gly-lle-Phe-L?s-Asp-Pro the MFulgene that encodes the mature, secretedform of the a-factor, with the exception of the COOH-terminal cysteine (Fig. 3), was synthesized using nucleoside phosphoramidite mixtures that were contamiScheme 1 nated at a level of 6.0% equimolar mixture. This predetermined level of doping was designed to approximate the introduction of 1.5 random base substitutions per oligonucleotide calculated on the basis of the procedure of McNeil and Smith (38). PhenotypicScreening for a-FactorsLacking Biological ActivityMutagenized M F u l containing plasmidDNA was isolated from an amplified poolof several thousand Escherichia coli colonies and a 1.0kilobase EcoRI- BamHI gene fragment containing the a-factor coding sequence, but lacking its promoter, was isolated and subcloned into a yeast expression vector. This plasmid, pRS316GU (a gift from Phil Hieter), contains the galactose-inducibleGAL1 promoter in the KpnI site of the polylinker of vector pRS316 which carries TRPI as the selectable marker (39). The EcoRI site in the MFul gene waspreviously introduced by site-directed mutagenesis, as described (16). Again,over 1000 colonies containing the ligatedM F a l gene fragment werepooled, amplified, and their plasmid DNA isolated. This pool of randomly mutated a-factor encoding expression vectors was transformed into yeast strain SM1229 (Amful AmfaZ). Close to 1000 tryptophan-selected transformants were individually transferred into microtiter plates containing media selective for growth of the yeast and plasmid maintenance. The yeast in these plates weregrown to high density and 25-pl portions were removedand spotted onto lawns of a-strain RC757 in the presence of either galactoseor glucose. Plates were incubated overnight a t 30 "C and scored the following day for evidence of growth arrest on a) DIEAIDCCNOBI. b) TFNCH2CI2DM.5; c) I)TFNCH~CIZDMS; 2) ZnJ 90% HOAc in H2O: 3) Farnesylatlon; d) PiperidineDMF both glucose and galactose. Individual colonies of yeast cells that did not exhibit a-factor bioactivity were identified and used as the source of Scheme 2 DNA from which part of the MFal gene was amplified and sequenced using a procedure developed for the rapid isolation and sequencing of plasmid DNA directly from yeast colonies (40). Generation and Purification of a-Factor Antibodies-Preparation of antiserum was performed according t o the procedure of Sterne (41). Briefly, New Zealand White rabbits were immunized with an insoluble aggregate of a synthetic peptide corresponding t o the peptidyl portionof a-factor (YIIKGVFWDPAC)suspended in phosphate-buffered saline (15 mM NaPO,, pH 7.5,150mM NaCl) at 1mgiml. Injections were performed using 0.5 ml of this suspension emulsified initially with an equal volume of complete Freund's adjuvent. Subsequent injections employed Freund's incomplete adjuvent as an emulsifier. All injections were pera) TFNCH2CI2DMS:b) DIEA/BOP, c) I ) Zn/HOAc(9U% in H20);Z) Farnesylalton. d) PiperidlneDMF formed both intraperitoneal and subcutaneously on a weekly basis for a period of 3 months. Rabbits were bled 3-5 days following injection of Scheme 3 antiserum and reactivity with both the antigen and synthetic a-factor was verified by enzyme-linked immunosorbent assay. Fractionation of FIG.1. Synthetic s c h e m e s f o r a - f a c t o r analogs. Synthesis and coupling of peptide fragments was performed as described under "Ma- IgG was performedby combining 20 ml of this antiserum with20 ml of 36% Na,SO, slowly, and stirring at 22 "C for 2 h. The antibody was terials and Methods." pelleted by centrifugation at 500 x g for 15 min a t 22 "C. The superna[Cy~'~(CHJOFarl-a-Factor (xi-This peptidewassynthesized by tant was discarded and the pellet resuspended in 20 mlof phosphatecondensing Fmoc-Tyr-Ile-Ile-Lys(Fmoc~-Gly-Val-Phe-Tp-Asp~OFm~buffered saline. This suspension was subjected to a second round of Pro and Ala-~-Cys(CH3)0Far according Fig. to 1, Scheme1(Fig. 1). To precipitationwith36% Na,SO, andthendialyzedagainstseveral a solution of Fmoc-Ala-L-Cys(CH,)OFar (50 mg, 79 pmol) i n 2 ml of washes of phosphate-buffered saline for several daysat 4 "C. This fracacetonitrile was added 0.2 ml of piperidine, and thesolution was stirred tion was filtered through a 0.2 p~ Gelman filter, aliquoted, and stored at room temperature for 20 min and lyophilized. This material was a t -20 "C until use. directly used for coupling to Fmoc-Tyr-Ile-Ile-Lys(Fmoc)-Gly-Val-Phe- In Vivo Labeling and Immunoprecipitation of Extracellular Trp-Asp(OFm)-Pro(100ml53.1 qmol) usingthe BOP reagent (23.5mg, a-Factor-The immunoprecipitation of extracellular a-factor was per53.1 pmol) in 2 mlof DMF containing 5% diisopropylethylamine. The formed using a procedure adaptedfrom that of Hrycyna et al. (42) and entire reaction mixture wascooled i n a nice bath. After 1 h, the crude takes advantageof the previous observationthat approximately 95%of peptide was isolated by precipitation and the protecting groups were secreted a-factor adherest o the walls of culture tubes due to its hydroremoved using 10% piperidine in DMF for 30 min. The resultantsolu- phobic nature and relative insolubility in aqueous solutions(41).Cul-

19820

FIG.2. HPLC of farnesyl ester afactor analogs. Panels and A B, [~-Cys'~(CH,)OFar]-a-factor;panels C and D,[~-Cys'~(CH,)OFarl-a-factor; panels E and F, [~-Cys'~l-a-factor. A linear gradient from 80% water (0.025% trifluoroacetic acid) to80% acetonitrile (0.025% trifluoroacetic acid) over 30 min was used for panelsA, C , and E . A linear gradient from 80%water (0.025% trifluoroacetic acid) to 100% methanol (0.025%trifluoroacetic acid) over 40 min was used forpanels B, D,and F. Chromatography was performed ona pBondapak CIScolumn (3.9 x 300 mm).

a-Factor Biological Activity

l

:

0

;

;

:

;

l

20

10

0

30

10

20

30

40

10

0

20

30

(A)

5 N

2 8 ,/ /'

1 0

;

: ' 10

'

,

20

'

I

30

I

1

40

0

10

20

30

0

10

20

30

40

Retention Time (rnin) *tures (5 ml) of SM1229 or SM1229 transformed with either the wildtypeormutagenized MFal gene subcloned inexpressionvector pRS314GU weregrown in minimal medium containing amino acid supplements overnight toa density ofA,,, = 0.7. Experiments involving M F a l gene induction or repression were performed in media containing either 2% galactose, 2% raffinose (induced) or 2% glucose (repressed). Cells were harvested and resuspended in a microcentrifuge tube with 0.5 ml of fresh medium. Following the addition of 120 pCi of [36Slcysteine (DuPont NEN, >600 Cilmmol), these cultures were incubated a t 30 "C for 2 h. Labeling was stopped by the addition of 0.5 ml of an ice-cold stop solution(40 m~ cysteine, 40 mM methionine, 20mM NaN,, 500 pg/ml bovine serum albumin). This mixture was removed from the culture tubes and the tubes washed with H,O. 400 p1 of 1-propanol was added to the washed tubes and mixed by vortexing t o elute the adhered a-factor from the walls of the tubes. At this stage a 20-pl aliquot of 1-propanol was removedfor use in TLC analysis; the remaining l-propanol solution was taken to dryness in a vacuum centrifuge. Samples were resuspended in 25 pl of 1 x Laemmli buffer (2 x = 20% glycerol, 10% 2-mercaptoethanol, 4.3% SDS, 0.125 mM Tris-CI, pH 6.8, 0.2% bromphenol blue) then added toa microcentrifuge tube containing 1.3 ml of immunoprecipitation buffer (1% Triton X-100, 150 m~ NaCl, 5 mM EDTA, 50 mM Tris-C1, pH 7.5)and heatedfor 3 min at 100 "C. Insoluble debris was removedfrom samples by rapid centrifugation for 1 min i n a microcentrifuge and the supernatants were transferred to a new microcentrifuge tube. Purified IgG (10pl) was added to each sample and

MFal

5'

3'

1 I

NH~.MQPSTATAAPKEKTSSEKKDN~YIIKGVFWDPA~CVIA-COOH

w

target region of random mutagenesis

Tyr-Ile-lle-Lvs-Gly-Val-Phe-Trp-Asp-Pro-Ala-C s OCH3

7-

s

.

. .

FIG.3. Representation of the primary gene product of the afactor gene, the region of this gene targeted for random mutagenesis, and the mature a-factorpheromone following intracellular processingand post-translational modification. tubes were incubated overnighta t 4 "C. 45 p1of Protein A-Sepharose CL-4B beads (Pharmacia Biotech Inc.), resuspended in a 1:3 ratio of beads:immunoprecipitation buffer, wereaddedtoeachsampleand samples were mixed by inversion at 4 "C for 90 min. Beads were pel-

a-Factor Biological Activity

19821

TABLE I1 Bioactivity of a-factor truncation and topology analogs ~

~~

Peptide

Growth arrest",b

Mating restoration"' ng

Truncation analogs 0.25 ( 1 ~ ) ~ 0.025 ( 1 ~ ) ~ YIIKGVFWDPAC(Far)-OCH, >2,500 (>lO,OOOx) YIIKGVFWDPAC 2,500 (100,000~) l(4X) 1(40x) IIKGVFWDPAC(Far)-OCH, 1(4x) 10 (400x) IKGVFWDPAC(Far)-OCH, 2 (8x1 10 (400x1 KGVFWDPAC(Far)-OCH, 4 (16x) 10 (400x1 GVFWDPAC(Far)-OCH, 31 ( 1 2 5 ~ ) 100 (4,000~) VFWDPAC(Far)-OCH, 31 ( 1 2 5 ~ ) 250 (10,000~) FWDPAC(Far)-OCH, 250 (1,000~) 1,000 (40,000~) WDPAC(Far)-OCH, >1,000(>40,OOOx) >1,000 (>40,OOOx) DPAC ( Far ) -OCH, Topology analogs YIIKGVFWDPA-[o-C(Far)-OCH,] 4.0 0.5 (20x1 (16x) 0.1 (4x) 0.5 (2x) YIIKGVFWDPA-[o-C(CH,)-OFar] 0.1 (4x) (8x1 2.0 YIIKGVFWDPA-[L-C(CH,)-OF~~] Growth arrest assay performed using supersensitive a-strain RC757. The amount listed in the table represents the smallest amount of peptide giving a response in the growth arrest assay. * The values represented in the table are theaverage of at least threedeterminations with each test yielding a result variantby not more than one 2-fold dilution. Mating restoration assay performed by selecting for diploidconstellations between a-strain RC757 and a-factor gene deletion a-strain SM1229 (Amfal Amfa2) in the presence of exogenously added peptide. The amount listed in the table represents the smallest amount of peptide giving a response in the mating restoration assay. Number in parentheses represent fold change in activity compared to wild-type a-factor. leted by centrifugation for 10 s, the aqueous layer was removed, and beads were washed with 1 mlof immunoprecipitation wash buffer A (0.1% Triton x-loo, 0.O2% SDS, 150 mM NaC1, mM EDTA, 50 mM Tris-C1, pH 7.5). This wash was repeated three times. The beads were washed Once with immunoprecipitation washbuffer B (150 mM NaC1, 5 EDTA, 50 mM WS-c1,PH 7.5), resuspended in 30 pl of2 x Laemmli buffer, heated for 3 min at 100 "C,and clarified by rapid centrifugation in a microcentrifuge. This supernatant was transferred to scintillation fluid to determine immunoprecipitated counts. Thin Layer Chromatography of in Vivo Labeled a-Factor-Portions of the propanol eluate removed from microcentrifuge tubes following in vivo labeling of a-factor with [36Slcysteine,as above, wereseparated by thin layer chromatography (TLC) accordingto the procedure of Marcus et al. (43). Samples were resolved on Silica Gel 60 F-254 TLC plates (Bodman Chemical) in a mixture of 1-butano1:acetic acid:water (4:l:l) for 1.5-2.0 h, dried using a blow dryer, and exposed directly to Kodak XAR-5 film for 5-7 days.

-

assays suggest that sequential loss of specific amino acid residues negativelyaffectspheromone activity (Table 11). Removal ofthe NH,-terminal tyrosineresidue reduces bioactivity to ap-

proximately 2%ofwild-tme amfactorin the halo assay I1; Fig. 4).Truncation to remove the first isoleucine results in an additional orderof magnitude decrease in activity. The further removal of the Ile3 and Lys4 residues has no effect on activity. However, removal of Gly5results in an additional order of magnitude reduction of activity and subsequentremoval ofVal' and Phe7further lowersactivity resultingin a peptidewith 1/40,000 the activity of a-factor in the halo assay. The successive removal of amino acids from the NH, terminus of a-factor results ina gradient of activity loss until virtually allbioactivity appears lost with the tetrapeptide DPAC(Far)-OCH, peptide. This peptide does not cause growth arrest even when RESULTS tested a t 1 pg. Overall changes in bioactivity are not as dra(Table 11).Nevertheless, Biological Activity of Tkuncated a-Factor Peptides and Topol- matic in the mating restoration assay ogyAnalogs-We have previously studied the significance of the relative effect of sequential truncation of NH,-terminal COOH-terminal modifications to the biological activity of a- residues follows approximately the same trendsas in the halo factor and determined that the farnesyl and methyl groups assay. Thus, changes t o the peptide portionof the a-factor cause contribute approximately equally to, and are requiredfor, bio- only moderate effects on the activity of the pheromone until logical activity (14). However, the relative contribution to a- more than half of the residues areremoved. factor activity made by the individual amino acids present in As it is evident that structural determinants within pepthe the pheromone has not been addressed. A series of synthetic tidy1 portion of a-factor may contribute to recognition of this a-factor peptides containing serial truncations from the NH, pheromone by its receptor, the mannerby which this peptide is terminus of the pheromone was synthesized inorder to ascer- presented to Ste3p is clearly linked to its requirement for a tain the effect changes in peptide structure and length would hydrophobic COOH terminus. To investigate the putative efhave on a-factor bioactivity. It should be noted that allof these fects on bioactivity of altering the spatial orientation of the truncated peptides retained an intact COOH terminus cor- farnesyl and carboxyl methyl modifications on the COOH terrectly modified with both the C,, farnesyl andcarboxyl methyl minus of a-factor, we synthesized a-factor analogsin which the ester moieties. chirality andtopology of the cysteine thioether and the carboxyl We employed two different assays to measure the biological ester were varied.As seen inFig. 5, if the peptide portion of the activity of a-factor and its analogs. The halo, or growth arrest a-factor is bound in a specific orientation to the receptor the assay is an agar diffusion assay which serves to determine the spatial relationship (topology) of the farnesyl and methyl moiminimum quantityof peptide required to induce growth arrest eties of the Cys residueare very similarin a-factor and of a-cells. The mating restoration assay is a measure of the [~-Cys'~(CH,)OFarl-a-factor(XI). In contrast, the topological minimum quantityof exogenous peptide required to stimulate relationship of the large (farnesyl) and small (methyl)hydromating between a-cells and a-cells which do not have theabil- phobic group would be exactly the opposite in compounds (IX ity to produce their own a-factor. Mating is scored by the for- and X). Biological assays on these analogs indicated that an mation of colonies on plates that select for growth of only dip- exchange of the farnesylfor the methyl moiety in a-factor peploids in thepresence of exogenously added peptide. The results tides which contained the terminalCys residue in either a D- or of testing NH,-terminal truncations of a-factor in both of these L-configuration resulted in only a moderate change in bioactivI

19822

Activity

Biological

a-Factor

A

a-Factor

IX

R = TrlklkLysGlyValPkT~AspProA~

B

FIG. 4. Bioactivity of the truncated a-factor analog (IIKGVFWDPAC(Farnesy1bOCHJ. P a r d A, growth arrest assay. Growth arrest of a-strain R C i 5 i , which is supersensitiveto a-factor, is monitored as 5 pi of serial dilutionsof peptide are spotted onto a lawn ofthis strain (see "Materials andMethods"). Panel B , mating restoration assay. The mating restoration assay servesa means as for determining pheromone potency by judging the minimum quantity of exogenously added peptide requiredtoinducetheformation of diploid cellsbetween haploid a-strain RC757 and a-strain SM1229 (Amfal Amfa2).

ity when compared among themselves (none to 5-fold difference) or to wild-type a-factor (4-20-fold difference) (Table 11). These results indicate that the spatial orientation of the hydrophobic moieties at the carboxyl terminus of a-factor is not an importantfactor in the determination of pheromone potency. Identification of a-Factor Peptides Which Are Secreted but Not Biologically Active-To further investigate the functional significance of individual amino acids in the a-factor peptide, we used a genetic screen to identify residues that contribute to the bioactivity of the pheromone. Random oligonucleotide-targeted mutagenesis (35) was employed to generate a mutant pool of a-factors which contained base substitutions in the afactor gene, specifically covering all the residuesof the mature a-factor, with the exception of the COOH-terminal cysteine (Fig. 3). This mutagenesisscheme was designed to maintain an

FIG.5. Topology analogs of the S. cereuisiae a-factor. IX, [o-CyslzI-a-factor; X, ICys12(CH,)OFarl-a-factor; XI, [n-Cys"(CH,)OFarl-a-factor.

intact target for isoprenylation, the -CAAX box, which is required for export of the a-factor (16-18), while, theoretically, altering all other residues in the mature pheromone. Prior to pooling the putative mutants,individual E. coli transformants were sequenced to verify evidence of mutagenesis. The results of this analysis indicated that mutations were present a t a rate of 1.4 mutants pergene and exhibited a relatively evenspread over the bases encoding a-factor (data not shown). Following the subcloning of the altered gene pool into a yeast expression vector, expression of the mutatedM F a l genes ina yeast strain deleted in the a-factor genes (strain SM1229 Amfal Amfa2) was controlled by a galactose-inducible promoter. We have previously employed this methodology in the characterization of site-directed mutations in the M F a l gene that affect the biosynthesis of a-factor (16). Transformant colonies were isolated on selective medium and screened for evidence of loss of afactor bioactivityin thepresence of galactose, on the basisof an inability to inducegrowth arrest of a-cells. About 1000 colonies were screened and approximately 300 were identified a s putative mutants. DNA sequence analysis of the MFal gene from colonies exhibiting the mutant phenotype (40) led to identification of altered codons and predicted amino acid substitutions within the pheromone. An observed loss of a-factor bioactivity, as demonstrated by these mutants,could be attributed to either an interruption of proper biosynthesis, export, or ligand-receptor interaction. As the goal of this study focused on the identification of a-factor peptides which were successfully exported yet not recognized by the a-factor receptor, we proceeded to analyze a representative set of biologically inactive mutants for the presence of radiolabeled a-factor in the culture supernatant of cells labeled in vivo with [35S]cysteine. Immunoprecipitation of labeled afactor was performed using purified IgG from polyclonal antiserum generated specifically against the peptide backbone of a-factor (YIIKGVFWDPAC).Comparison of the counts immunopreciptiated from strain SM1229, deleted in the a-factor structural genes, and this same strain harboring a wild-type MFal-containing plasmid suggested our antibodies successfully recognize a-factor (Table 111). Similarly, a-factor peptides were also immunoprecipitated from transformed cultures of SM1229 expressingseveral of themutantMFal genes thereby indicating that these strains produce altered a-factor

a-Factor Biological Activity TABLEI11 Percent wild-type immunoprecipitation of a-factor peptides from phenotypically selected mutants Peptide'.b

19823

A

B

Wild-type cpm immunoprecipitated' Galactose

Glucosed %

YIIKGVFWDPAC 1.2 0.7 YI IKSFMPAC 0.7 YIIKGIFKDPAC YIIKSFWDPTC YIIKSVFWDPTC 1.3 YIIKGZFGDPAC Y 1.0 IIKSFWDPAC Amfal Amfa2

100.0 15.4 34.7

15.5 23.0 37.0 59.4 2.1

0.5 0.8

0.5

Altered residues within a-factor indicated by bold and underlined letters. a-Factor peptide sequences listedare as predicted by translation of DNA sequence obtained from each phenotypically selected mutant above. Percent immunoprecipitation based on average of three separate trials. Percent immunoprecipitation under growth in glucose (repressed) expressed as percentage of wild-type immunoprecipitated under galactose (induced).

'

peptides, however, these peptides are not recognized by Ste3p. Furthermore, the expression of both wild-type and mutant genes in these constructs wasdesigned to be controlled by the galactose-inducible GAL1 promoter thereby enabling us to directly compare the immunoprecipitable counts from the same strain in the presence or absence of inducer. The same mutants from which inactive extracellular a-factor was detected following growth in galactose exhibited only background levels of immunoprecipitation following i n vivo labeling and growth in glucose. This result confirms that the radioactivity immunoprecipitated by our a-factor antibodies was specifically representative of a-factor-like peptides. Although several of the mutants identified by this method clearly exported detectable quantities of pheromone, none appeared to immunoprecipitate at the same level as wild-type pheromone (Table 111). This may be attributed to altered antigenic determinants and a subsequent loss of optimal antibody recognition which resulted from the amino acid substitutions. Effects of this type would naturally be amplified in a small peptide the size of a-factor, especially for mutant peptides containingmultiple amino acid substitutions. An alternative method to determine whether a-factor-like peptides are secreted by cells harboring mutated M F a l genes is thin-layer chromatography (43). TLC was used to separate the smallhydrophobic a-factor secreted by in vivo labeled cells from all other labeled proteins priorto immunoprecipitation. Autoradiographs of silica thin layersonto which equal amountsof counts per min were spotted from each sample indicated that the phenotypically selected mutants yielded several spots characteristic of the wild-type a-factor species (Fig. 6A).In contrast, the deletion strain alone did not exhibit this pattern of compounds, nor did identical mutants grown and labeled under conditions (glucose) which repress theexpression of the M F a l gene (Fig. 6B). Therefore it appeared that these spots were a-factor related peptides. The existence of multiple a-factor related species of varied mobility is consistent with previous observations on partially purified a-factor in both TLC and HPLC systems and isbelieved to represent an artifactof oxidation (44,451. Oxidation during culturing has also been shown to result in the observation of several a-factorpeptides following TLC separation of that pheromone from supernatants (46). To further substantiate the biological effects of altering the specific residues within a-factor, we undertook the chemical synthesis of two a-factor peptides that were identified by our

Mutant Amfal Amfa2

W.T.

Galactose Glucose

FIG. 6. Thin layer chromatography o f in vivo labeled a-factor. Panel A, comparison of TLC separated samples from mutant (YIIKGk FLDPAC; Table HI), a-factor gene deletion (Arnful Amfu2), and wildtype (W.T ) strains. Panel B,comparison of TLC separated samples from the same a-factor mutant labeled in vivo under conditions that specifically either induce (galactose) or repress (glucose) MFul gene expression. Equal cpm were spotted in each sample in a volume of 2-7 pl. biological screen as being secretedby a-cells but not biologically active. These peptides (XI1 and XIII; Table I) corresponded to mutants whose predicted structure would be YIIKGIFLDPAC(Far)-OCH, and YIIKGIFmPAC(Far)-OCH,, respectively (Table 111).The substitution of these residues for the natural Val6 and Trp' resulted ina 16-25-fold drop in biological activity compared to wild-type as detected by the growth arrest assay. These results highlight the sensitivity of our selection scheme in successfully identifying substitutions at specific residues that result inphysiologically inactive peptides in vivo. It is difficult to compare the in vitro activities of the truncated peptides (Table 11)with the lack of in vivo activity of the biosynthetic products (Table 111)because these two sets of compounds contain no common members. It is clear, however, that for compounds XI1 and XI11 the transformed cells secrete insufficient quantities of the biosynthetically modified peptide to generate a response in thehalo assay. For example, in thecase of peptide XI11 (YIIKGIFaPAC(Far)-OCH,) a minimum of 0.625 ng of this mutant pheromone would be necessary to induce a response in the growth arrest assay. Although, as discussed above, the immunoprecipitation and TLC results do not give a precise indication of the amountof pheromone secreted, thetransformed cells never produce quantities of mutant pheromones higherthan those produced by the wild-type (Table I11 and Fig. 6). Since wild-type a-factor has an end point of 0.025 ng in the growth arrest assay, it is likely that the mutant biosynthesizing YIIKGIFmPAC(Far)-OCH, isnot secreting enough pheromone to elicit growth arrest. Thus, the selection assay that we used is sensitive enough to identify substitutions in the peptide backbone which result in physiologically inactive mutants and also allows us to discriminate between secretion and activity. Finally, it is probable that biosynthetic mutants producing truncated versions of a-factor similar tothose tested inTable I1 might also exhibit atotal loss of bioactivity as determined using our i n vivo selection scheme. However, as little is known concerning the processing at the amino terminus of the pheromone it isnot possible to rationally design such mutated pheromones at thistime.

19824

a-Factor BiologicalActivity DISCUSSION

Various models have been proposed for the mechanism of targeting isoprene-modified proteins (19,20,26).These include the possibility that prenylated proteins might be directed to membranes by interaction with specific membrane-bound receptors, by lipid-lipid interactions in membranes, or by secondary modifications subsequent to prenylation (20). The ease with which a-factor biological activity is assayed, along with our established methodologies for chemical synthesis of its analogs (30-33) and mutagenesis of the gene encoding the pheromone (161, provide an attractive systemby which structure-function relationships inlipopeptide-protein interaction may be studied. The results of our truncation analysis of a-factor indicate that only extreme changes in the peptidyl structure of the pheromone have as great aneffect on biological activity as does removal of the COOH-terminal modifications. For example, removal of residues 1-3 results inonly an 87%drop in activity as judged in the mating restoration assay (Table 11)in comparison to a 99% dropon removal of either themethyl ester or farnesyl moiety. Over two-thirds of the peptide must be removed from the NH, terminus t o lose nearly total activity. In contrast, an equivalent loss of bioactivity is found with thecomplete peptide missingboth thefarnesylandmethylester moieties. This strongly suggests that these post-translational modifications play a directrole in either the interaction with, or presentation of, the a-factor to its receptor, It is also clear that different amino acids contributein varyingdegrees to theoverall activity 50% of the of the a-factor. It issomewhat surprising that nearly peptidyl portion of the pheromone can be removed with retention of measurable biological activity. Despite these observations, the synthetic peptides do not provide insights into the efficiency of biosynthesis, the intracellular stability, and the export of such molecules from yeast. Nevertheless, it seems clear that the Ste3p receptor can be triggered by lipopeptides containing very small portions of the peptide sequence of mature a-factor. We have previously demonstrated by two-dimensional NMR methods that the structureof the peptide portion of synthetic a-factor is apparentlynot affected by the presence of a farnesyl on the terminal cysteine in solution (47). However, it remains possible that the farnesyl of a-factor interacts specifically with the Ste3p. The biological activities of the a-factor topology analogs used in this study suggest that such an interaction is unlikely (Table 11). Peptides IX, X, XI, and a-factor represent very differenttopologies for the carboxyl terminus (Table I; Fig. 5 ) .If the farnesylgroup on each of these pheromones interacted specifically with the receptor, the peptide portion of each of these a-factor molecules could not fit into a binding site in the same manner. Therefore, we suggest that therole of the farnesyl and methyl ester moieties on a-factor is totraffic the pheromone to the hydrophobic membrane and, perhaps, t o present the peptide portion of the molecule to Ste3p. Recent studies on a-factor in the presence of membrane vesicles in vitro suggest that the farnesylgroup bestows a particular physical interaction of a-factor with membranes (48). Specifically, studies on the intrinsicfluorescence of the tryptophan residueat position 8 of the a-factor indicated that isoprenylation causes this residue to be more deeply imbedded into bilayers. Furthermore, truncation of the NH,-terminal amino acidsof a-factor reduced the penetrationof the peptide into membranesas measured by tryptophan fluorescence. This reduced penetration correlates with the lower bioactivity of NH,-terminal truncations of afactor. It is interesting note to the differences that exist betweenthe end points of biological activity in the growth arrest assay compared to those exhibited in the mating restoration assay

(Table 11).Although sequential truncationof residues from the a-factor NH, terminus results ina similar gradientof activity loss when comparing individual analogs within either assay, the removal of specific amino acids from the NH, terminus clearly exhibits a more dramatic effect on pheromone potency in the growth arrest assay than in mating restoration. If the initial event of ligand binding to its receptor triggers a single cellular response leading to diverse physiological changes in the cell, then all measurements of analog potency should yield approximately equivalent values as end points of pheromone activity. It hasbeen previously observed that different ratios of pheromone potency are exhibited with &-factoranalogs in comparative analyses of morphogenesis versus agglutination, two physiological responses tothis pheromone in a-cells (49). Marsh (50) has isolated an &-factor receptor mutant that appears toexhibit acharacteristic ability to activateonly a subset of specific responses following a-factor treatment. This mutant receptor is >1000-fold less sensitive than wild-type in growth arrest assays, but only 2-fold less sensitive in its ability to mediate pheromone-dependent reporter gene expression. These previous observations, in thecontext of our data, suggest that structural changes within the ligands or receptors involved in yeast mating may serve to uncouple biologically related, yet distinctresponses to pheromone binding. Additional structure-function studies on a-factor may facilitate the elucidation of such differential signal-response relationships. The Ste3p receptor and Ste6p export protein represent two specific polypeptides which recognize the a-factor as an isoprenylated ligand in vivo. We have previously shown that altering the isoprenylation targeting motif on a-factor to encode for a geranylgeranylated pheromone does not functionally affect afactor export or bioactivity (15). The identification of mutant a-factor peptides which are successfully exported yet not biologically active (Table 111) indicates that a distinction exists in the mechanisms by which a-factor is recognized by either Ste3p or Ste6p. The fact that a-factor has been previously shown to induce growth arrest at picogram levels (14, 30) suggests that secretion of even small quantitiesof biologically active peptides would be detected physiologically. Therefore, it is reasonable to conclude that the mutant a-factors which were immunoprecipitated are not well recognized by the Ste3p receptor. The decrease in bioactivity exhibited by synthetic analogs (XI1 and XIII; Table I) designed to correspond to the putativepheromone products produced by two specific a-factor mutants of this type further implies a lack of optimal ligand-receptor interaction existed with these mutants and indicates the sensitivity of this approach. The use of a selection scheme to identify colonies lacking a-factor activity, coupled with inducible and specific control of gene expression, and biochemical verification of peptide export allows us to unequivocally identify candidate peptides for subsequent chemical synthesis and expand these studies on pheromone potency and structure. We are currently in the process of extending thismethodology t o find and synthesize antagonists of a-factor activity as well. The cumulative results from synthetic peptide analysisand molecular mutagenesis experiments suggest that removal or substitution of specific residues within thepheromone exhibits deleterious effects on bioactivity. Goldstein and Brown (51) have suggested that prenylated proteins may directlyinteract withspecific membrane proteins a regulatory role in the pathway for cholesterol and might serve is the biosynthesis. The key regulatory protein in this pathway membrane-bound enzyme HMG-CoA reductase. Cellulardepletion of isoprenoid containing proteins results in an elevated level of HMG-CoA expression, thereby suggesting a prenyldependent feedback inhibition mechanism may be involved in

a-Factor Biological Activity HMG-CoA regulation (51). It is interesting to note that the receptor for the a-factor and HMG-CoA reductase sharea similar structural organization, since both are seven transmembrane-domain containing proteins. In this sense, the interaction of a-factor with itsreceptor serves asa model for our future understanding of how prenylated proteins may interact with other cellular targets, perhaps in a regulatory role, as in the case of HMG-CoA reductase, or as previously described for US-adenylyl cyclase interaction (24). Acknowledgments-We thank Susan Michaelis for yeaststrain SM1229, Russ Chan for strain RC757, Jeremy Thorner and Karl Kuchler for the MFul gene, Phil Hieter and Bill Michaud for plasmid vectors, DavidMiller and Henry-York Steiner for their assistance in the generation and purification of antibodies, Wei Yang for technical assistance, and A n g u s Dawe for suggesting the random mutagenesis procedure and assistance with TLC. REFERENCES 1. Kurjan, J. (1992)Annu. Reu. Biochem. 61,1097-1129 2. Sprague, G. F., Jr., and Thorner,J. (1993) in The Molecular Biology of the Yeast Saccharomyces cereuisiae (Broach, J. R., Pringle, J. R., and Jones, E. W., eds) 2nd Ed., pp. 657-744, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 3. Dohlman, H. G., Thorner, J., Caron, M. G., and Lefiowitz, R. J. (1991)Annu. Rev. Biochem. 60,653-688 4. Burkholder,A. C., and Hartwell,L. H. (1985) Nucleic Acids Res. 13,8463-8475 5. Hagen, D. C., McCaffrey, G., and Sprague, G. F., Jr. (1986) Proc. Natl. Acad. Sci. U.S. A. 83, 1418-1422 6. Nakayama, N., Miyajima, A,, and Arai, K. (1987)EMBO J . 6, 249-254 7. Fuller, R. S., Sterne, R. E., and Thorner, J. (1988) Annu. Rev. Physiol. 50, 345-362 8. Anderegg, R. J., Betz, R., Carr, S. A., Crabb, J. W., and Duntze, W. (1988) J . B i d . Chem. 263, 18236-18240 9. Kuchler, K., Sterne, R. E., and Thorner, J. (1989) EMBO J. 8,3973-3984 10. McGrath, J. P., and Varshavsky, A. (1989) Nature 340, 400-404 11. Eriotou-Bargiota, E., Xue, C.-B., Naider, F., and Becker, J. M. (1992)Biochemistry 31, 551-557 12. Raths, S. K., Naider, E , and Becker, J . M. (1988)J. Biol. Chem. 263, 1733317341 13. Xue, C.-B., Eriotou-Bargiota, E., Miller, D., Becker, J. M., andNaider, F. (1989) J . Biol. Chem. 264, 19161-19168 14. Marcus, S., Caldwell, G.A., Miller, D., Xue, C.-B., Naider, F., and Becker, J. M. (1991) Mol. Cell. B i d . 11, 3603-3612 15. Caldwell, G. A., Wang, S.-H., Naider, F., and Becker, J. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1275-1279 16. Marcus, S., Caldwell, G. A,, Xue, C.-B., Naider, F., and Becker J. M. (1990) Biochem. Biophys. Res. Commun. 172, 1310-1316 17. Schafer, W. R., Kim, R., Sterne, R., Thorner, J., Kim, S.-H., and Rine, J. (1989) Science 245, 379-385

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