Functional Analysis of Plp1 and Plp2, Two Homologues of Phosducin in Yeast
2000; Elsevier BV; Volume: 275; Issue: 24 Linguagem: Inglês
10.1074/jbc.m002163200
ISSN1083-351X
AutoresPaul L. Flanary, Paul R. DiBello, Paula Estrada, Henrik Dohlman,
Tópico(s)Carbohydrate Chemistry and Synthesis
ResumoMammalian phosducins are known to bind G protein βγ subunits in vitro, and are postulated to regulate their signaling function in vivo. Here we describe two homologues of phosducin in yeast, called PLP1 andPLP2. Both gene products were cloned, expressed, and purified as glutathione S-transferase fusions. Of the two isoforms, Plp1 bound most preferentially to Gβγ. Binding was enhanced by pheromone stimulation and by the addition of GTPγS, conditions that favor dissociation of Gβγ from Gα. Gene disruption mutants and gene overexpression plasmids were prepared and analyzed for changes in signaling and nonsignaling phenotypes. Haploid spore products bearing the plp2Δ mutant failed to grow, suggesting that PLP2 is an essential gene. Cell viability was not restored by a mutation in STE7 that blocks signaling downstream of the G protein. Haploid products bearing theplp1Δ mutant were viable and exhibited a 6–7% increase in pheromone-mediated gene induction. Cells overexpressingPLP1 or PLP2 exhibited a 70–80% decrease in gene induction but no change in pheromone-mediated growth arrest. These data indicate that phosducin can selectively regulate early signaling events following pheromone stimulation and has an essential role in cell growth independent of its regulatory role in cell signaling. Mammalian phosducins are known to bind G protein βγ subunits in vitro, and are postulated to regulate their signaling function in vivo. Here we describe two homologues of phosducin in yeast, called PLP1 andPLP2. Both gene products were cloned, expressed, and purified as glutathione S-transferase fusions. Of the two isoforms, Plp1 bound most preferentially to Gβγ. Binding was enhanced by pheromone stimulation and by the addition of GTPγS, conditions that favor dissociation of Gβγ from Gα. Gene disruption mutants and gene overexpression plasmids were prepared and analyzed for changes in signaling and nonsignaling phenotypes. Haploid spore products bearing the plp2Δ mutant failed to grow, suggesting that PLP2 is an essential gene. Cell viability was not restored by a mutation in STE7 that blocks signaling downstream of the G protein. Haploid products bearing theplp1Δ mutant were viable and exhibited a 6–7% increase in pheromone-mediated gene induction. Cells overexpressingPLP1 or PLP2 exhibited a 70–80% decrease in gene induction but no change in pheromone-mediated growth arrest. These data indicate that phosducin can selectively regulate early signaling events following pheromone stimulation and has an essential role in cell growth independent of its regulatory role in cell signaling. G protein-coupled receptor kinase glutathioneS-transferase guanosine 5′-O-(3-thiotriphosphate) polymerase chain reaction polyacrylamide gel electrophoresis phosducin-like protein regulator of G protein signaling 4-morpholinepropanesulfonic acid base pair(s) kilobase pair(s) G protein-coupled receptors are a large and diverse family of signaling proteins that can respond to chemosensory signals (hormones, neurotransmitters, odors) and light. In the yeast Saccharomyces cerevisiae, G protein-linked pheromone receptors mediate events needed for cell fusion and mating (1.Dohlman H.G. Song J. Apanovitch D.M. DiBello P.R. Gillen K.M. Semin. Cell Dev. Biol. 1998; 9: 135-141Crossref PubMed Scopus (42) Google Scholar). Generally speaking, receptor activation triggers a conformational change in the G protein α subunit, exchange of GDP for GTP, and dissociation of Gα from the G protein βγ subunits. Gα is free to activate downstream effectors until GTP is hydrolyzed and the protein reverts to the inactive conformation. Gβγ does not undergo a conformational change and so activates its effector only until it can reassociate with Gα·GDP (2.Bohm A. Gaudet R. Sigler P.B. Curr. Opin. Biotechnol. 1997; 8: 480-487Crossref PubMed Scopus (77) Google Scholar, 3.Sprang S.R. Annu. Rev. Biochem. 1997; 66: 639-678Crossref PubMed Scopus (896) Google Scholar). G protein signaling can be modulated at various steps throughout the pathway. Receptors become desensitized following phosphorylation by second messenger-dependent and activation-dependent protein kinases (G protein-coupled receptor kinases ( GRKs)1) (4.Premont R.T. Inglese J. Lefkowitz R.J. FASEB J. 1995; 9: 175-182Crossref PubMed Scopus (475) Google Scholar, 5.Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (862) Google Scholar). In some cases, accessory proteins contribute to receptor desensitization. For instance, arrestins bind to phosphorylated receptors and prevent further coupling to the G protein (5.Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (862) Google Scholar). Arrestins also bind to clathrin and promote receptor endocytosis (6.Goodman Jr., O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1190) Google Scholar, 7.Ferguson S.S. Downey 3rd, W.E. Colapietro A.M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-366Crossref PubMed Scopus (856) Google Scholar). More recently, it has become evident that G proteins are also subject to desensitization. Members of the RGS (regulators of G protein signaling) family accelerate G protein GTP hydrolysis, thereby shortening the lifetime of the active species and dampening the signal (8.Dohlman H.G. Thorner J. J. Biol. Chem. 1997; 272: 3871-3874Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar, 9.Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). Another accessory protein called phosducin appears to sequester Gβγ in the cytosol (10.Lee R.H. Whelan J.P. Lolley R.N. McGinnis J.F. Exp. Eye Res. 1988; 46: 829-840Crossref PubMed Scopus (64) Google Scholar), thereby preventing it from reassociating with Gα and the receptor (11.Schroder S. Bluml K. Dees C. Lohse M.J. FEBS Lett. 1997; 401: 243-246Crossref PubMed Scopus (19) Google Scholar, 12.Bauer P.H. Muller S. Puzicha M. Pippig S. Obermaier B. Helmreich E.J. Lohse M.J. Nature. 1992; 358: 73-76Crossref PubMed Scopus (167) Google Scholar, 13.Yoshida T. Willardson B.M. Wilkins J.F. Jensen G.J. Thornton B.D. Bitensky M.W. J. Biol. Chem. 1994; 269: 24050-24057Abstract Full Text PDF PubMed Google Scholar, 14.Bauer P.H. Bluml K. Schroder S. Hegler J. Dees C. Lohse M.J. J. Biol. Chem. 1998; 273: 9465-9471Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 15.Bauer P.H. Lohse M.J. Naunyn-Schmiedebergs Arch. Pharmakol. 1998; 357: 371-377Crossref Scopus (12) Google Scholar). Although phosducin-Gβγ interaction has been convincingly documented through in vitro studies, it is not clear how this activity should affect signaling in vivo. Indeed there is growing evidence that phosducin may have other functions in the cell. Phosducin and the phosducin-like protein PhLP were recently shown to bind p45 SUG1, the regulatory subunit of the 26 S proteasome (16.Zhu X. Craft C.M. Mol. Vis. 1998; 4: 13PubMed Google Scholar, 17.Barhite S. Thibault C. Miles M.F. Biochim. Biophys. Acta. 1998; 1402: 95-101Crossref PubMed Scopus (18) Google Scholar). Phosducin has also been reported to bind with low affinity to Gα (14.Bauer P.H. Bluml K. Schroder S. Hegler J. Dees C. Lohse M.J. J. Biol. Chem. 1998; 273: 9465-9471Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), although this finding has not been reproduced (13.Yoshida T. Willardson B.M. Wilkins J.F. Jensen G.J. Thornton B.D. Bitensky M.W. J. Biol. Chem. 1994; 269: 24050-24057Abstract Full Text PDF PubMed Google Scholar, 15.Bauer P.H. Lohse M.J. Naunyn-Schmiedebergs Arch. Pharmakol. 1998; 357: 371-377Crossref Scopus (12) Google Scholar, 18.Lee R.H. Ting T.D. Lieberman B.S. Tobias D.E. Lolley R.N. Ho Y.K. J. Biol. Chem. 1992; 267: 25104-25112Abstract Full Text PDF PubMed Google Scholar). Phosducin could also act as a Gβγ effector, in the manner of phospholipase Cβ and ion channels (19.Clapham D.E. Neer E.J. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 167-203Crossref PubMed Scopus (708) Google Scholar), or as a Gβγ adaptor protein, in the manner of arrestin. Are phosducins regulators, effectors, or do they have some other function in the cell? To test these possibilities, it would be useful to obtain a cell or organism in which phosducin expression is disrupted. S. cerevisiae is an appropriate system to carry out such a genetic analysis, because gene disruption mutations are easily obtained through homologous recombination. Indeed, yeast is the only system in which all of the known signaling components (receptor, G protein, effector) (1.Dohlman H.G. Song J. Apanovitch D.M. DiBello P.R. Gillen K.M. Semin. Cell Dev. Biol. 1998; 9: 135-141Crossref PubMed Scopus (42) Google Scholar, 20.Sprague Jr., G.F. Thorner J. Broach J.R. Pringle J.R. Jones E.W. The Molecular and Cellular Biology of the Yeast Saccharomyces. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1992: 657-744Google Scholar) and desensitization factors (receptor kinase, RGS protein) have been characterized in this manner (1.Dohlman H.G. Song J. Apanovitch D.M. DiBello P.R. Gillen K.M. Semin. Cell Dev. Biol. 1998; 9: 135-141Crossref PubMed Scopus (42) Google Scholar, 21.Hicke L. Zanolari B. Riezman H. J. Cell Biol. 1998; 141: 349-358Crossref PubMed Scopus (247) Google Scholar). Now, with the completion of the yeast genome, we have identified two candidate phosducins that we named PLP1 and PLP2(phosducin-like proteins 1 and 2). Our analysis reveals that these proteins can bind and regulate Gβγ in vivo. In addition, one of the PLP isoforms has an essential role in maintaining cell viability, independent of its role in G protein signal transduction or desensitization. Bovine phosducin sequence (Swiss-Prot accession no. P19632) was used to screen on-line data bases with the BLAST, BLAST2, and FASTA programs. Multiple alignments were performed using the Clustal algorithm of MegAlign (Lasergene), using the PAM250 matrix. Standard methods for the growth and maintenance of yeast and bacteria were used throughout (22.$$$$$$ ref data missingGoogle Scholar). Escherichia coli strain DH10B was used for the maintenance and amplification of plasmids. S. cerevisiaestains used in this study were: YPH 500 (MATα ura3-52 lys2-801am ade2-101oc trp1-Δ63 his3-Δ200 leu2-Δ1) (23.Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar), YDM400 (MATa ura3-52 lys2-801am ade2-101oc trp1-Δ63 his3-Δ200 leu2-Δ1 sst2-Δ2) (from Jeremy Thorner, University of California), Y270 (MATa/α ura3-52 lys2-801am ade2-101oc trp1-Δ63 his3-Δ200 (from Mike Synder, Yale University), and BJ2168 (MATa ura3-52 leu2-Δ1 trp1-Δ63 prb1-1122 prc-1-407 pep4-3) (24.Jones E.W. Methods Enzymol. 1991; 194: 428-453Crossref PubMed Scopus (374) Google Scholar). YPH400/500 diploids were obtained by mating YDM400 with YPH500. The plp1::TRP1 and plp2::URA3mutants were constructed using polymerase chain reaction (PCR)-mediated gene disruption. Primers consisting of 35 nucleotides ofPLP1 sequence or 50 nucleotides of PLP2 sequence, and 18 nucleotides of sequence homologous to plasmids pJJ248 (flanking the TRP1 selectable marker) and pJJ244 (flanking theURA3 selectable marker) were used to amplify each marker sequence (25.Jones J.S. Prakash L. Yeast. 1990; 6: 363-366Crossref PubMed Scopus (334) Google Scholar). A PLP1 gene disruption fragment was constructed by amplification of pJJ248 with primers 1 (5′-GGCGAGGAAAATTTAGATGAACTACTTAATGAATTGGATAGAGAATTAGACGAGCACAGGAAACAGCTATGACC-3′) and 2 (5′-GAAGCGAAGCGTTCCGTATTCACAGATGAATGCTTTCTTATTTCGAATGTGTCTTCCGACGTTGTAAAACGACGG-3′). A PLP2 gene disruption fragment was amplified from pJJ244 with primers 3 (5′-GAAGCAATTGCCAAGCAGCATGAAAATAGACTAGAAGATAAAGACTTGTCGGATTTGGAACACGACGTTGTAAAACGACG-3′) and 4 (5′-TTTTCCTCTAATACCTGACCTGATCGATTTTTTTTCACCGTAATGCAATTTTCTCTCTTCCAGCTATGACCATGATTACG-3′). The amplified product was transformed directly into either YPH400/500 or Y270. To confirm each gene disruption, genomic DNA was prepared (22.$$$$$$ ref data missingGoogle Scholar) and PCR amplified using primers flanking, as well as internal to, the disrupting auxotrophic markers. For PLP1, primers 5 (5′-ACGCGTCGACCTCCATTCTCTTAACAACTC-3′) and 6 (5′-ACGCGAGCTCTTTTAGTAGGGAGGTAATGG-3′) were used in combination with primer 248 (5′-CTCTCTTGCCTTCCAACCCAGTC-3′). For PLP2, primers 7 (5′-ACGCGTCGACCCAATTTAGTGGCTTGTTCTTC-3′) and 8 (5′-ACGCGAGCTCTGGCTGAATCCAATGACACCTC-3′) were used in combination with primer 244 (5′-GAACGTTACAGAAAAGGAGGC-3′). In some cases, additional mutant strains were made by transformation of a fragment derived from PCR amplification of the disrupted gene from genomic DNA ofplp1Δ or plp2Δ cells (using primers 5 and 6, or 7 and 8). These new strains were tested by PCR amplification of mutant genomic DNA, with one primer complementary to sequence distal to the amplified product and again with primers complementary to the auxotrophic marker (9: 5′-CACGACCTGATGGTAACACCTCAG-3′or 10: 5′-AAGGGTTATACTGGCAAGGCATC-3′, 11: 5′-TTCTGCTACAACTTCATCTAACTCC-3′). PCR amplification, using primers completely flanking eitherPLP1 or PLP2, was also used to test if diploid yeast containing a gene disruption were homozygous (one amplified product) or heterozygous (two amplified products) for the gene disruption. The presence of the TRP1 or URA3selectable marker was used to follow the mutated genes after sporulation and tetrad dissection. Taq DNA polymerase (Roche Molecular Biochemicals) was used according to the manufacturer's recommendations. Primers were synthesized by the Keck Biotechnology Facility, Yale University. All DNA-modifying enzymes were purchased from New England BioLabs. PLP1 single copy plasmids were made by PCR amplification of genomic DNA with primers 9 and 10. The amplified product was digested with XbaI andBglII and subcloned directly into pRS315, pRS313, or pRS423 (23.Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). A single copy PLP2 plasmid was constructed by PCR amplification of wild-type genomic DNA, using primers 7 and 8. The amplified PLP2 gene was then digested with SalI and SacI and subcloned directly into pRS313. Construction ofGPA1-GST (glutathione S-transferase) and GST in pAD4M was described previously (26.Song J. Hirschman J. Gunn K. Dohlman H.G. J. Biol. Chem. 1996; 271: 20273-20283Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). PLP1-GST and PLP2-GST were constructed by PCR amplification of PLP1 and PLP2 from wild-type genomic DNA with primers 13 (5′-ACGCGTCGACCTCCATTCTCTTAACAACTC-3′) and 14 (5′-CGCGGATCCTATATCTAAATCACTATC-3′), or 15 (5′-ACGCGTCGACCCAATTTAGTGGCTTGTTCTTC-3′) and 16 (5′-CGCGGATCCGTCAAAAAATCCATCATCATC-3′). The 3′ primer for both genes allows for an in-frame fusion to GST upon subcloning into a GST fusion cassette. The GST cassette was constructed by PCR amplification of GST from GPA1-GST using the primers 17 (5′-GCGGGATCCATCGAAGGTCGTGGGATGTCC-3′) and 18 (5′-ACGCGAGCTCTATTTTGGAGGATGGTCGCC-3′) and subcloning into Bluescript (Stratagene) as a BamHI-SacI fragment. High expression of PLP1-GST and PLP2-GST in yeast was accomplished by subcloning PLP1-GST andPLP2-GST from the Bluescript GST fusion cassette into pAD4M (as SalI-SacI fragments) and transforming into yeast. All fusions were confirmed by DNA sequencing (Keck Biotechnology Facility, Yale University) and immunoblot detection using anti-GST antibodies (from J. Steitz, Yale University). Overexpression of PLP1 and PLP2 was attained using pRS315-GAL, containing the galactose-inducible GAL1/10promoter (EcoRI-BamHI fragment). Each gene was PCR-amplified from genomic DNA using PLP1 primers 5 and 6, or PLP2 primers 19 (5′-GCGGTCTAGAAGGCATATTCAGCAGACATA-3′) and 20 (5′-GCGGGAGCTCTGGGGATAGTGACACCACTT-3′). The amplified products were digested with XbaI and SacI (PLP2, 930 base pairs (bp)) or SalI (blunt-ended with T4 polymerase) and SacI (PLP1, 766 bp) and subcloned into the XbaI and SacI sites of pRS315-GAL. PLP1, PLP2, andPGK1 probes were labeled with [α-32P]CTP, using the Prime-a-Gene system (Promega). Labeled fragments were separated from unincorporated label by desalting on a NICK spin column (Amersham Pharmacia Biotech). PLP1 and PLP2 DNA fragments were prepared as gel-purified restriction fragments of subcloned genes using Qia-quick spin columns (Qiagen).PGK1 DNA was PCR-amplified from wild-type genomic DNA (primers: 5′-AATCGTGTGACAACAACAGCCTG-3′; 5′-CGGATAAGAAAGCAACACCTGG-3′), gel-purified, and labeled directly. RNA was isolated using RNeasy Mini (Qiagen), separated on a 0.8% agarose/5% formaldehyde gel in 1× MOPS running buffer (5 mm NaOAc, 20 mm MOPS, 1 mm EDTA, pH 7.0). The separated RNA was transferred to positively charged nylon (Roche Molecular Biochemicals) and fixed by UV cross-linking (Stratalinker 1800). RNA-containing nylon membranes were incubated at 42 °C for 3 h in prehybridization buffer containing 5× SSPE (1× = 150 mm NaCl, 10 mmNaH2PO4, 1 mm EDTA, pH 7.4), 50% formamide, 100 μg/ml salmon sperm DNA, 5× Denhardt's (0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin, 0.2% sodium dodecyl sulfate) and overnight in prehybridization buffer containing 1 × 106 cpm of purified probe. Hybridized membranes were washed three times with 1× saline/sodium phosphate/EDTA, 1% SDS at 24 °C, and once with 0.2 × saline/sodium phosphate/EDTA, 0.1% SDS at 42 °C for 30 min. Membranes were then exposed to XAR x-ray film (Kodak) from overnight to 3 weeks at −80 °C. Halo and reporter-transcription assays were performed as described (27.Sprague Jr., G.F. Methods Enzymol. 1991; 194: 77-93Crossref PubMed Scopus (232) Google Scholar), except that fluorescein di-β-d-galactopyranoside was used as the β-galactosidase substrate. Mating assays were performed as described (28.Gehrung S. Snyder M. J. Cell Biol. 1990; 111: 1451-1464Crossref PubMed Scopus (114) Google Scholar), with minor modifications. Briefly, MAT a andMATα cells (spore products of Y270PLP1/plp1::TRP1 diploids) were transformed with either pRS316 (URA3) or pRS313 (HIS3). Cells were crossed as described (28.Gehrung S. Snyder M. J. Cell Biol. 1990; 111: 1451-1464Crossref PubMed Scopus (114) Google Scholar), serially diluted, and plated on complete synthetic media lacking histidine and uracil. Viable colonies were scored. No colonies resulted from mixing cells of the same mating type. Mating frequencies were calculated based on the total number of colonies resulting when mating mixtures were plated on nonselective (YPD) medium as a reference. Note that the mating frequencies are lower than reported earlier (28.Gehrung S. Snyder M. J. Cell Biol. 1990; 111: 1451-1464Crossref PubMed Scopus (114) Google Scholar), possibly due to the use of plasmid-borne selectable markers. To measure the ability of cells to grow on different media, cells were initially grown on YPD plates at 30 °C and then restreaked on plates containing various media and incubated overnight at 30 °C, unless otherwise noted. To assay for osmotic sensitivity, cells were streaked onto YPD plates containing 1.0 or 2.0 m NaCl, or 1m sorbitol. To assay for calcium sensitivity, cells were streaked onto YPD plates containing 0.1 mCaCl2. To assay for differences in carbon source utilization, cells were restreaked onto YP plates containing either 2% dextrose, 3% galactose, 3% ethanol, 2% raffinose, 2% maltose, 2% glycerol, or 0.1 m KAc. To assay for nitrogen sensitivity, cells were streaked onto normal synthetic plates, synthetic plates lacking (NH4)2SO4 (low nitrogen), and synthetic plates lacking (NH4)2SO4 and lacking all amino acids except those required for the growth of the strain (lysine, tryptophan, and histidine) (very low nitrogen). Cells were assayed for oxidative stress by streaking on YPD plates containing 2.0 mm H202 and for chemical stress by streaking onto YPD plates containing 3.0% formamide. Cells were assayed for their ability to grow after a heat shock by placing restreaked cells at 50 °C for 1 h and then 30 °C overnight. To assay for growth at different temperatures, cells were streaked onto YPD plates and were allowed to grow overnight at 18, 24, 34, and 37 °C. Binding was performed as described earlier for Gpa1 binding to Gβγ, with minor modifications (26.Song J. Hirschman J. Gunn K. Dohlman H.G. J. Biol. Chem. 1996; 271: 20273-20283Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Briefly, 50 ml of BJ2168 cells expressing pAD4M-PLP1-GST, pAD4M-GST, or pAD4M-GPA1-GST were grown to mid-log phase (A 600 nm ∼ 1.0) in SCD medium lacking leucine. Cells were split into two 25-ml cultures, and one was treated with 1 μmα-factor. After 1 h, cells were shifted to ice and growth was stopped by the addition of NaN3 to 10 mm. 30A 600 nm units of cells was harvested by centrifugation at 1000 × g for 10 min at 4 °C. Cells were washed once with 10 mm NaN3 and once with lysis buffer (40 mm triethanolamine, pH 7.2, 2 mm EDTA, 150 mm NaCl, 2 mmdithiothreitol, 1 mm4-(2-aminoethyl)benzenesulfonylfluoride-HCl, 15 μg/ml leupeptin, 20 μg/ml pepstatin, 1 mm benzamidine, 10 μg/ml aprotinin, 100 μm β-glycerol phosphate, 0.5 mm sodium orthovanadate). Cells were then split and resuspended in ice-cold lysis buffer containing either 3 mm MgCl2/10 μm GDP or 3 mm MgCl2/20 μm GTPγS. Cells were subjected to glass bead lysis by vortexing for 4 min at 4 °C. The resultant lysate was solubilized by the addition of Triton X-100 to 1% and rocking at 4 °C for 1 h. 100 μl of a 20% slurry of glutathione-Sepharose 4B in lysis buffer was used to bind GST fusion proteins at 4 °C for 4 h. The glutathione-Sepharose was washed three times with phosphate-buffered saline (pH 7.3) before the bound protein was eluted by heating to 100 °C in 1× SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer (60 mm Tris-HCl, pH 6.8, 10% glycerol, 144 mm 2-mercaptoethanol, 10 μg/ml bromphenol blue, 4% SDS). Samples were resolved on a 12% SDS-PAGE gel, transferred to nitrocellulose, and probed with antibodies to GST or Ste4 (from Duane Jenness, University of Massachusetts). Antibody detection was achieved using horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad) and the ECL chemiluminescence system (Amersham Pharmacia Biotech) according to the manufacturer's instructions. Sequencing of the entireS. cerevisiae genome has been completed recently (29.Goffeau A. Barrell B.G. Bussey H. Davis R.W. Dujon B. Feldmann H. Galibert F. Hoheisel J.D. Jacq C. Johnston M. Louis E.J. Mewes H.W. Murakami Y. Philippsen P. Tettelin H. Oliver S.G. Science. 1996; 274 (563–547): 546Crossref PubMed Scopus (3347) Google Scholar). We used a variety of nucleic acid search tools to identify two open reading frames with significant similarity to mammalian phosducin. The putative genes were designated PLP1 (YDR183w) andPLP2 (YOR281c). An alignment of the conceptually translated gene products (Plp1, Plp2) is provided in Fig.1, along with the prototypic phosducin from bovine retina. All of the proteins exhibit significant sequence similarity. Moreover, the length and unusually acidic amino acid composition of Plp1 and Plp2 are highly characteristic of the known phosducins (Fig. 1). We then used Northern blot analysis to determine whetherPLP1 and PLP2 are bona fide genes, which undergo transcription. Thus labeled nucleic acid probes were hybridized to RNA prepared from haploid (MAT a,MATα) and diploid (MAT a/α) yeast cells, as well as from haploid (MAT a) cells treated with α-factor pheromone. These conditions were chosen, because many genes involved in mating are expressed only in haploids and are commonly up-regulated by pheromone stimulation. As shown in Fig. 2, the PLP1 andPLP2 probes hybridized to mRNA species of approximately 0.8 and 1.0 kbp, respectively. The size and abundance of these transcription products were identical in all three cell types tested and were not affected by pheromone treatment. Thus PLP1 and PLP2 are transcribed, and these transcripts are of sufficient size to encode the expected gene products. However, the abundance of both mRNA species (PLP1 in particular) is quite low, at least 100-fold lower than our positive control PGK1. Given that a single cell contains approximately 100 copies of PGK1 mRNA, there is (on average) less than one copy of PLP1 or PLP2per cell. This is not unusual in yeast, where genome-wide expression studies revealed that 69% of all mRNAs are present at one or fewer copies per cell (30.Wodicka L. Dong H. Mittmann M. Ho M.H. Lockhart D.J. Nat. Biotechnol. 1997; 15: 1359-1367Crossref PubMed Scopus (860) Google Scholar). Low abundance transcripts may be expressed at low levels, or they may be extremely stable or simply not induced by any of the conditions tested. One of the defining characteristics of phosducin is the ability to bind Gβγ. Accordingly, we examined whether Plp1 or Plp2 can interact with the Gβγ in yeast. Full length versions of both genes were fused in-frame to the coding sequence of GST. GST was placed at the C terminus, because this arrangement was shown not to interfere with the ability of mammalian phosducin to bind βγ (11.Schroder S. Bluml K. Dees C. Lohse M.J. FEBS Lett. 1997; 401: 243-246Crossref PubMed Scopus (19) Google Scholar, 31.Xu J. Wu D. Slepak V.Z. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2086-2090Crossref PubMed Scopus (66) Google Scholar, 32.Hawes B.E. Touhara K. Kurose H. Lefkowitz R.J. Inglese J. J. Biol. Chem. 1994; 269: 29825-29830Abstract Full Text PDF PubMed Google Scholar, 33.Bluml K. Schnepp W. Schroder S. Beyermann M. Macias M. Oschkinat H. Lohse M.J. EMBO J. 1997; 16: 4908-4915Crossref PubMed Scopus (35) Google Scholar, 34.Thibault C. Sganga M.W. Miles M.F. J. Biol. Chem. 1997; 272: 12253-12256Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The resulting Plp1-GST and Plp2-GST fusions were then expressed in haploid cells using a high copy plasmid and under the control of a strong constitutive promoter (from ADH1). Both fusion proteins were purified by glutathione-Sepharose affinity chromatography and resolved by SDS-PAGE and immunoblotting. A Gpa1-GST fusion was purified as a positive control. GST alone was purified as a negative control, to detect any nonspecific binding. Binding of Gβγ was then tracked by blotting with antibodies to Gβ (Ste4). Equal loading of each lane was confirmed by blotting the same extracts with anti-GST antibodies. As shown in Fig. 3, Gβγ bound specifically to Plp1-GST and to Gpa1-GST, but not to GST alone. Gβγ binding to Plp2-GST was evident, but only upon very long exposures of the autoradiograph (Fig. 3, inset). Significantly, Gβγ binding was enhanced by pretreating the cells with α-factor pheromone, as well as by lysing the cells in the presence of GTPγS. Both treatments should promote at least transient dissociation of the G protein subunits, thereby increasing the pool of Gβγ available to bind phosducin. Thus, Plp1 (and to a far less extent Plp2) can bind to Gβγ in yeast, in the manner of phosducin in mammals. Moreover, binding appears to be specific, stimulus-dependent, and subtype-selective. To determine the in vivofunction of the phosducins in yeast, we constructed disruption mutations of both PLP1 and PLP2. Diploid cells were transformed with a version of each gene in which most of the coding region had been replaced with a nutritional marker,TRP1 (plp1::TRP1, plp1Δ) or URA3 (plp2::URA3,plp2Δ). Each mutation was confirmed by PCR amplification of genomic DNA. Confirmed mutants were sporulated, and the resulting tetrads were dissected and allowed to grow on rich (YPD) medium.PLP1/plp1Δ diploids yielded four viable spore products, whereas PLP2/plp2Δ cells segregated 2:2 for viability. All of the viable spore products fromPLP2/plp2Δ cells were unable to grow in medium lacking uracil, confirming that the plp2::URA3mutation is lethal. Cell viability was restored by the presence of a cloned PLP2 gene on a single copy plasmid (Fig.4 and data not shown). These findings demonstrate that PLP2 is an essential gene and thatPLP1 is not. To determine if plp1Δ cells have any other growth-related phenotype, wild-type and mutant cells were compared in their ability to grow on solid medium containing different sources of carbon or different levels of nitrogen as well as under conditions of osmotic, thermal, oxidative, or chemical stress (TableI). In no instance could we detect any difference in growth between the plp1Δ and wild-type cells.Table IRelative growth of wild-type and mutant cellsWild-typeplp1∷TRP1YPD++++++YPD + 1 msorbitol++++++YPD + 2 m NaCl−−YPD + 1.0 m NaCl++++++YPD + 0.1m CaCl2++++++YP KAc−−YP galactose++++++YP ethanol++++++YP raffinose++++++YP maltose++YP glycerol++++YPD + heat shock++++++Low nitrogen++++Very low nitrogen++SCGaSCG, synthetic complete medium + galactose.++++++SCDbSCD, synthetic complete medium + dextrose.++++++YPD at 18 °C++++++YPD at 24 °C++++++YPD at 34 °C++++++YPD at 37 °C++++++YPD + 3% formamide++++YPD + 2 mmH2O2++++++Cells were scored (from none, −, to full growth, +++) after incubation in the indicated conditions, as detailed under “Experimental Procedures.”a SCG, synthetic complete medium + galactose.b SCD, synthetic complete medium + dextrose. Open table in a new tab Cells were scored (from none, −, to full growth, +++) after incubation in the indicated conditions, as detailed under “Experimental Procedures.” Most of the components of the mating response pathway are not essential, but can block or otherwise modulate the response to pheromone (20.Sprague Jr., G.F. Thorner J. Broach J.R. Pringle J.R. Jones E.W. The Molecular and Cellular Biology of the Yeast Saccharomyces. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1992: 657-744Google Scholar). Thus we examined whether the plp1Δ
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