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J. Biol. Chem., Vol. 275, Issue 24, 18462-18469, June 16, 2000

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Functional Analysis of Plp1 and Plp2, Two Homologues of Phosducin in Yeast^*

Paul L. Flanary^§, Paul R. DiBello, Paula Estrada^¶, and Henrik G. Dohlman^**

From the Departments of  Pharmacology and ^¶ Cell Biology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06536

Received for publication, March 14, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mammalian phosducins are known to bind G protein subunits in vitro, and are postulated to regulate their signaling function in vivo. Here we describe two homologues of phosducin in yeast, called PLP1 and PLP2. Both gene products were cloned, expressed, and purified as glutathione S-transferase fusions. Of the two isoforms, Plp1 bound most preferentially to G. Binding was enhanced by pheromone stimulation and by the addition of GTPS, conditions that favor dissociation of G from G. Gene disruption mutants and gene overexpression plasmids were prepared and analyzed for changes in signaling and nonsignaling phenotypes. Haploid spore products bearing the plp2 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 the plp1 mutant were viable and exhibited a 6-7% increase in pheromone-mediated gene induction. Cells overexpressing PLP1 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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). Generally speaking, receptor activation triggers a conformational change in the G protein  subunit, exchange of GDP for GTP, and dissociation of G from the G protein subunits. G is free to activate downstream effectors until GTP is hydrolyzed and the protein reverts to the inactive conformation. G does not undergo a conformational change and so activates its effector only until it can reassociate with G·GDP (2, 3).

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, 5). 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). Arrestins also bind to clathrin and promote receptor endocytosis (6, 7).

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, 9). Another accessory protein called phosducin appears to sequester G in the cytosol (10), thereby preventing it from reassociating with G and the receptor (11-15). Although phosducin-G 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, 17). Phosducin has also been reported to bind with low affinity to G (14), although this finding has not been reproduced (13, 15, 18). Phosducin could also act as a G effector, in the manner of phospholipase C and ion channels (19), or as a G 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, 20) and desensitization factors (receptor kinase, RGS protein) have been characterized in this manner (1, 21). 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 G 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Sequence Analysis-- 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.

Strains, Media, and Transformation-- Standard methods for the growth and maintenance of yeast and bacteria were used throughout (22). Escherichia coli strain DH10B was used for the maintenance and amplification of plasmids. S. cerevisiae stains used in this study were: YPH 500 (MAT ura3-52 lys2-801^am ade2-101^oc trp1-63 his3-200 leu2-1) (23), YDM400 (MATa ura3-52 lys2-801^am ade2-101^oc trp1-63 his3-200 leu2-1 sst2-2) (from Jeremy Thorner, University of California), Y270 (MATa/ ura3-52 lys2-801^am ade2-101^oc trp1-63 his3-200 (from Mike Synder, Yale University), and BJ2168 (MATa ura3-52 leu2-1 trp1-63 prb1-1122 prc-1-407 pep4-3) (24). YPH400/500 diploids were obtained by mating YDM400 with YPH500.

The plp1::TRP1 and plp2::URA3 mutants were constructed using polymerase chain reaction (PCR)-mediated gene disruption. Primers consisting of 35 nucleotides of PLP1 sequence or 50 nucleotides of PLP2 sequence, and 18 nucleotides of sequence homologous to plasmids pJJ248 (flanking the TRP1 selectable marker) and pJJ244 (flanking the URA3 selectable marker) were used to amplify each marker sequence (25). 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) 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 of plp1 or plp2 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 either PLP1 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 URA3 selectable 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.

Plasmid Construction-- 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 and BglII and subcloned directly into pRS315, pRS313, or pRS423 (23). 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 of GPA1-GST (glutathione S-transferase) and GST in pAD4M was described previously (26).

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 and PLP2-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 GAL1/10 promoter (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.

Northern Analysis-- PLP1, PLP2, and PGK1 probes were labeled with [-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 mM NaH₂PO₄, 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.

Phenotypic Assays-- Halo and reporter-transcription assays were performed as described (27), except that fluorescein di--D-galactopyranoside was used as the -galactosidase substrate. Mating assays were performed as described (28), with minor modifications. Briefly, MATa and MAT cells (spore products of Y270 PLP1/plp1::TRP1 diploids) were transformed with either pRS316 (URA3) or pRS313 (HIS3). Cells were crossed as described (28), 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), 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 1 M sorbitol. To assay for calcium sensitivity, cells were streaked onto YPD plates containing 0.1 M CaCl₂. 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.

G Binding-- Binding was performed as described earlier for Gpa1 binding to G, with minor modifications (26). 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 µM -factor. After 1 h, cells were shifted to ice and growth was stopped by the addition of NaN₃ to 10 mM. 30 A_{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 mM dithiothreitol, 1 mM 4-(2-aminoethyl)benzenesulfonylfluoride-HCl, 15 µg/ml leupeptin, 20 µg/ml pepstatin, 1 mM benzamidine, 10 µg/ml aprotinin, 100 µM -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 GTPS. 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.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Phosducin Homologues in Yeast-- Sequencing of the entire S. cerevisiae genome has been completed recently (29). 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) and PLP2 (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).

View larger version (64K): [in this window] [in a new window]   Fig. 1.   Alignment of bovine phosducin with yeast Plp1 and Plp2. Amino acid sequence alignments were performed with Clustal, using the PAM250 scoring matrix. Blocks indicate residues that are identical in Plp1 (YDR183w), Plp2 (YOR281c), or bovine phosducin (PHOS) (SWISSPROT accession no. P19632). Predicted molecular weights are 26,609 (Plp1), 32,767 (Plp2), and 28,185 (phosducin). Predicted pI values are 5.1, 4.5, and 4.5, respectively. Values were obtained from the YPD data base and from Ref. 37.

We then used Northern blot analysis to determine whether PLP1 and PLP2 are bona fide genes, which undergo transcription. Thus labeled nucleic acid probes were hybridized to RNA prepared from haploid (MATa, MAT) and diploid (MATa/) yeast cells, as well as from haploid (MATa) cells treated with -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 and PLP2 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.

View larger version (88K): [in this window] [in a new window]   Fig. 2.   PLP1 and PLP2 are transcribed genes. Total RNA was prepared from MATa, -factor treated MATa, MAT, and MATa/ cells, and resolved by Northern blotting (0.8% agarose gel). Identical blots were hybridized with PLP1 and PLP2 probes, yielding bands of approximately 0.8 and 1.0 kb, respectively (top panel). No band was observed in similar blots using RNA from plp1 cells (not shown). The same blots were subsequently rehybridized with a PGK1 probe to normalize loading of RNA (bottom panel).

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 PLP2 per 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). 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.

Plp1 and Plp2 Binding to G-- One of the defining characteristics of phosducin is the ability to bind G. Accordingly, we examined whether Plp1 or Plp2 can interact with the G 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 (11, 31-34). 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 G was then tracked by blotting with antibodies to G (Ste4). Equal loading of each lane was confirmed by blotting the same extracts with anti-GST antibodies.

As shown in Fig. 3, G bound specifically to Plp1-GST and to Gpa1-GST, but not to GST alone. G binding to Plp2-GST was evident, but only upon very long exposures of the autoradiograph (Fig. 3, inset). Significantly, G binding was enhanced by pretreating the cells with -factor pheromone, as well as by lysing the cells in the presence of GTPS. Both treatments should promote at least transient dissociation of the G protein subunits, thereby increasing the pool of G available to bind phosducin. Thus, Plp1 (and to a far less extent Plp2) can bind to G in yeast, in the manner of phosducin in mammals. Moreover, binding appears to be specific, stimulus-dependent, and subtype-selective.

View larger version (71K): [in this window] [in a new window]   Fig. 3.   G binds to Plp1. Cells expressing Plp1-GST, Plp2-GST, Gpa1-GST, or GST alone (in plasmid pAD4M), were grown to mid-log phase either in the presence (+) or absence () of -factor pheromone (MF), and then lysed either in the presence (+) or absence () of GTPS, as indicated. Solubilized lysates were immobilized on glutathione-Sepharose, washed, and eluted with SDS-PAGE sample buffer. Retained protein (Bound) was detected by immunoblotting with antibodies against Ste4 (G subunit; top panels) or GST (middle panels). Samples of the soluble cell lysate (Applied) were similarly probed with Ste4 antibody (bottom panels) to confirm equivalent levels of G expression in each case. The arrows indicate the band(s) specifically recognized by the indicated antibody. Nonspecific bands are present in ste4 mutant or vector-transformed cells. Ste4 is phosphorylated in -factor-treated cells (unless Gpa1 is overexpressed) and therefore migrates as a doublet (62). Inset at bottom, long exposure of the top left panel, showing weak binding to Plp2.

Function of PLP1 and PLP2-- To determine the in vivo function 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, plp1) or URA3 (plp2::URA3, plp2). 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/plp1 diploids yielded four viable spore products, whereas PLP2/plp2 cells segregated 2:2 for viability. All of the viable spore products from PLP2/plp2 cells were unable to grow in medium lacking uracil, confirming that the plp2::URA3 mutation 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 that PLP1 is not.

View larger version (86K): [in this window] [in a new window]   Fig. 4.   PLP2 is an essential gene. Heterozygous PLP2/plp2 cells (derived from strain YPH501) were sporulated, and each tetrad was dissected onto YPD plates. After 2 days, the resultant colonies were photographed and then tested for the presence of the URA3 marker. Only PLP2+ colonies (lacking the URA3 disruption marker) were viable.

To determine if plp1 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 (Table I). In no instance could we detect any difference in growth between the plp1 and wild-type cells.

View this table: [in this window] [in a new window]   Table I Relative growth of wild-type and mutant cells 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). Thus we examined whether the plp1 mutant, or overexpression of either PLP1 or PLP2, has any effect on pheromone sensitivity. For the overexpression studies, we placed both genes under the control of the galactose-inducible GAL1/10 promoter. First, we used a plate bioassay to measure pheromone-dependent growth arrest (halo assay). Second, we measured pheromone-induced gene transcription, using a -galactosidase reporter construct (FUS1 promoter, lacZ gene). Finally, we measured the efficiency with which plp1 cells mate, either with wild-type or a plp1 partner. As shown in Fig. 5, there was no difference in the growth (Fig. 5A) or mating (Fig. 5C) response, whether or not PLP1 was expressed. Overexpression of PLP1 or PLP2 also had no effect on the growth arrest assay (Fig. 5A). The plp1 mutants did have a modest effect on transcriptional induction, however. Compared with wild-type, the mutant strain exhibited an approximate 6-7% increase in the maximum response, with a change of <2-fold in EC₅₀ (Fig. 5B, left panels). Conversely, overexpression of either PLP1 or PLP2 reduced the maximum response by 16-20% in the plp1 strain and ~73-80% in the wild-type strain. No such differences were seen in dextrose media, which should repress PLP1/2 expression (Fig. 5B, DEX versus GAL). Thus, Plp1 and Plp2 share the ability to inhibit short term transcriptional changes, with no change in long term growth arrest, following stimulation with pheromone. This pattern of selective regulation is, to our knowledge, completely unprecedented in yeast.

View larger version (37K): [in this window] [in a new window]   Fig. 5.   Pheromone response assays. A, MATa wild-type (WT) or mutant (plp1) strains were transformed with pRS315-GAL (CEN vector, galactose-inducible GAL1/10 promoter) containing no insert, PLP1, or PLP2, as indicated. Cells were plated on galactose medium and exposed to filter disks containing 5, 15, or 45 µg of -factor for 48 h and then photographed. B, left, the mutant strain (plp1) was transformed with pRS315-GAL containing no insert (), PLP1 (), or PLP2 (). The isogenic wild-type strain containing the empty vector was also tested for comparison (). Right, the wild-type strain (WT) was transformed with the pRS315-GAL containing no insert (), PLP1 (), or PLP2 (). Cells were grown in the presence of galactose (GAL) or dextrose (DEX), and treated with the indicated concentrations of -factor. -Galactosidase activity was determined using a pheromone-responsive FUS1 promoter-lacZ reporter construct (27). Data shown are typical of two to five independent experiments performed in triplicate. Error bars, mean ± S.E. For clarity, larger versions of the symbols are provided to the right of each graph. C, MATa and MAT cells (wild-type or plp1, as indicated) were transformed with either pRS316 (URA3) or pRS313 (HIS3), crossed, serially diluted, and plated on complete synthetic media lacking histidine and uracil. Mating frequencies were calculated based on the total number of colonies resulting when mating mixtures were plated on nonselective (YPD) medium as a reference, as described in Ref. 28. Numbers shown are the average of three experiments.

Besides PLP2, there is at least one other component of the pheromone response pathway that is essential for cell viability. Mutants in the G subunit gene GPA1 result in constitutive G1 arrest, apparently due to unregulated expression of free G and sustained activation of the mating pathway. This constitutively active phenotype can be rescued by a ste7 mutant. Thus we examined whether plp2 could be rescued by ste7, in the manner of gpa1. A diploid strain bearing single gene mutations in STE7 and PLP2 was sporulated, and the resulting tetrads were dissected. Whereas gpa1 mutants are rescued by a ste7 mutation, all of the plp2 spore products failed to grow, whether or not STE7 was expressed (Fig. 4, and data not shown). We conclude that the growth arrest phenotype of the plp2 mutation is unrelated to the growth arrest typically seen in response to G protein activation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Phosducin was originally discovered in the retina by virtue of its association with purified transducin G (35). Although phosducin is best known for its role in the visual system, it has been proposed to regulate other signaling systems as well. Phosducin (12, 36) and several phosducin-like proteins (37-41) are now known to be expressed in a wide variety of tissues, including brain, liver, pineal, and olfactory epithelium (12, 36). Moreover, binding is not selective for the retinal-specific isoforms of G and G (36, 42). One member of the family, called PhLP (phosducin-like protein) (41) shares only limited sequence similarity with phosducin but is still able to bind to G (34, 43). Two additional homologues, PhLOP1 (an N-terminally truncated form of phosducin) and PhLOP2, do not appear to bind to G at all (44).

Although past studies have demonstrated that some phosducin family members can regulate G protein signaling in vitro (12, 18, 45), we sought to determine whether they regulate G protein function in vivo. We have shown that phosducins in yeast, like phosducin and PhLP in mammals, have the ability to bind to G. As expected, overexpression of either PLP1 or PLP2 can greatly reduce pheromone-mediated gene transcription. Surprisingly, overexpression of the same genes had no effect on pheromone-mediated growth arrest. Finally, and perhaps most significantly, gene disruption mutants of PLP1 had only modest effects on signaling, whereas the PLP2 disruption was lethal.

Both PLP1 and PLP2 can inhibit short term but not long term pheromone responses. To our knowledge, this type of selective regulation has not been reported previously in yeast. This is understandable, considering that most genetic screens are designed to isolate mutants affecting growth, which is simpler to assay than is transcription. This raises the exciting possibility that new genetic screens based on transcriptional induction rather than growth arrest could yield additional novel regulators of short term desensitization.

Compared with Gpa1, Plp1 and Plp2 bind poorly to Ste4. In mammalian cells the binding of phosducin (and PhLP) to G is also quite weak, with published affinity constants of 17-110 nM (12, 13, 15, 31, 32, 42, 43, 46). By comparison, flow cytometry measurements of G binding to G yield considerably higher affinities of 1-3 nM in the presence of GDP and >100 nM in the presence of GTPS (47, 48). Unfortunately, similar quantitative methods are not yet available for the yeast proteins.

Of the two yeast phosducins, Plp2 is most similar to other members of the family, yet Plp1 is best able to bind to . An explanation may be found in the recently solved crystal structures of the retinal phosducin-G complex (49-51). These studies reveal extensive contacts between the N-terminal domain of phosducin with the top of the G "propeller," a region that also binds to G (52). The C-terminal domain forms less extensive contacts with the side of the G propeller, a region that is believed to face the lipid bilayer (52). A structurally based alignment of Plp2 with phosducin reveals several amino acid deletions and charge substitutions in helix I (residues 20-34 in phosducin), which caps the central hole formed by the G propeller. These changes could impair binding of Plp2 to G. There may also be other subtle differences that are not obvious from broad comparisons of sequence or structure. For instance, phosducin binding is reduced ~5-fold when it is phosphorylated at Ser-73 (13, 53), even though this residue is oriented away from G (49) and has only modest effects on the order of the loop that contains the site of phosphorylation (50). Apparently, even small differences in phosducin structure, at sites removed from the binding interface, can have significant effects on phosducin function. The structural and functional differences between Plp1 and Plp2 could be just as subtle.

Our finding that PLP2 is an essential gene was unexpected. If the only role of phosducin is to desensitize G signaling, a gene disruption should be viable. One explanation is that phosducin homologues have different functions in different organisms. We think this is highly unlikely, given the well documented structural and functional similarities between yeast and mammalian signaling components. A second possibility is that phosducins do not ordinarily modulate G protein signaling. Although they clearly have the capacity to sequester G and perturb signaling, this may not be their principal function in vivo. Some GRKs bind G to facilitate membrane localization as well as phosphorylation and desensitization of receptors (5, 19, 40, 54). Although GRKs also have the capacity to sequester G and perturb their function (55), it is not, however, considered to be their main function in the cell. A third scenario (which we favor) is that phosducin can regulate signaling but only in the visual system. Phosducin is expressed at much higher concentrations in the retina (~380 µM) than in other tissues (<1 µM), and the retina is the only tissue in which there is sufficient phosducin to bind more than about 10% of the available G (36, 56).

If phosducins do not act primarily as regulators of G protein function, what do they do? One possibility is that they act as chaperones, delivering G (and most likely other proteins) to or from the plasma membrane. A chaperone function would likely require that hydrophobic regions of G be masked so that it can be transported through the cytosol. In fact, phosducin normally resides within the cytoplasm of rod and cone photoreceptors, whether or not it is bound to G (10, 35, 57-60). Moreover, binding of phosducin to G promotes its dissociation from the membrane (60). In Cryphonectria parasitica, disruption of a phosducin-like protein produces a phenocopy of G null mutants, suggesting a role in facilitating G protein function (61). There is also evidence from two-hybrid screens that phosducin and PhLP bind to the mammalian 26 S proteasome regulatory subunit, p45 SUG1 (16, 17). Perhaps phosducin helps to deliver G and other proteins to the proteasome.

Many investigators have equated phosducin with G binding. This view may be too limited. Indeed, our analysis reveals an essential (and -independent) function for at least one phosducin homologue in yeast. These findings support an emerging model in which phosducin family members have functions other than to simply regulate G proteins. It remains to be determined if these proteins modulate some other aspect of G protein function, such as subunit assembly, stability, or subcellular localization. Our gene disruption mutants represent an essential first step to addressing these questions. Currently, we are attempting to identify downstream targets of Plp1 and Plp2, through genetic suppression, synthetic interaction, and two-hybrid interaction studies in yeast. Given the extensive similarity between yeast and human signaling pathways, these studies should help determine the in vivo function of phosducins in other organisms as well.

	ACKNOWLEDGEMENTS

We thank Rachelle Gaudet and members of the Dohlman laboratory for advice and discussion.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health (NIH) Grants GM55316 and GM59167 (to H. G. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ An NIH post-doctoral trainee (T32-NS07136).

An NIH pre-doctoral trainee (T32-GM07527).

^** An Established Investigator of the American Heart Association.

To whom correspondence should be addressed: Dept. of Pharmacology, Boyer Center for Molecular Medicine, Yale University School of Medicine, 295 Congress Ave., Rm. 436, P. O. Box 9812, New Haven, CT 06536-0812. Tel.: 203-737-2203; Fax: 203-737-2290; E-mail: henrik. dohlman{at}yale.edu.

Published, JBC Papers in Press, April 3, 2000, DOI 10.1074/jbc.M002163200

	ABBREVIATIONS

The abbreviations used are: GRK, G protein-coupled receptor kinase; GST, glutathione S-transferase; GTPS, guanosine 5'-O-(3-thiotriphosphate); PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; PhLP, phosducin-like protein; RGS, regulator of G protein signaling; MOPS, 4-morpholinepropanesulfonic acid; bp, base pair(s); kbp, kilobase pair(s).

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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