An Essential Function of a Phosphoinositide-Specific Phospholipase C Is Relieved by Inhibition of a Cyclin-Dependent Protein

Home , Pho regulon, Restriction enzyme

An Essential Function of a Phosphoinositide-Speciﬁc Phospholipase C Is Relieved by Inhibition of a Cyclin-Dependent Protein Kinase in the Yeast Saccharomyces cerevisiae

Jeffrey S. Flick* and Jeremy Thorner† *Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 and †Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, California 94720-3202 Manuscript received July 24, 1997 Accepted for publication September 19, 1997

ABSTRACT The PLC1 gene product of Saccharomyces cerevisiae is a homolog of the ␦ isoform of mammalian phosphoinositide-speciﬁc phospholipase C (PI-PLC). We found that two genes (SPL1 and SPL2), when overexpressed, can bypass the temperature-sensitive growth defect of a plc1⌬ cell. SPL1 is identical to the PHO81 gene, which encodes an inhibitor of a cyclin (Pho80p)-dependent protein kinase (Pho85p) complex (Cdk). In addition to overproduction of Pho81p, two other conditions that inactivate this Cdk, a cyclin (pho80⌬) mutation and growth on low-phosphate medium, also permitted growth of plc1⌬ cells at the restrictive temperature. Suppression of the temperature sensitivity of plc1⌬ cells by pho80⌬ does not depend upon the Pho4p transcriptional regulator, the only known substrate of the Pho80p/Pho85p Cdk. The second suppressor, SPL2, encodes a small (17-kD) protein that bears similarity to the ankyrin repeat regions present in Pho81p and in other known Cdk inhibitors. Both pho81⌬ and spl2⌬ show a synthetic phenotype in combination with plc1⌬. Unlike single mutants, plc1⌬ pho81⌬ and plc1⌬ spl2⌬ double mutants were unable to grow on synthetic complete medium, but were able to grow on rich medium.

RANSMEMBRANE signaling in eukaryotes fre- Cellular responses after PIP2 hydrolysis and produc- quently involves activation of a phosphoinositide- T tion of IP3 and DAG depend on the cell type and in- specific phospholipase C (PI-PLC), which hydrolyzes clude proliferation, differentiation, and secretion (Ber- ridge Valius Kazlauskas Weiss phosphatidylinositol 4,5-bisphosphate (PIP2) and gener- 1993; and 1993; and Littman ates two second messengers, inositol 1,4,5-trisphosphate 1994). In mammalian cells, IP3 binds to intra- Lee Rhee (IP3) and 1,2-diacylglycerol (DAG; and 1995). cellular receptors, stimulating the release of sequestered Three classes of PI-PLC (␤, ␥, and ␦) have been charac- Ca2ϩ, thereby activating Ca2ϩ- and calmodulin-regu- terized at the molecular level; each contains a con- lated protein kinases and phosphoprotein phosphatases served catalytic domain comprised of two segments, (Clapham 1995). DAG remains in the membrane, designated X and Y, as well as nonconserved segments where it can activate members of the protein kinase C that confer distinct modes of regulation. Members of (PKC) family (Nishizuka 1992). Both second messen- the PI-PLC ␤ family can be stimulated by binding of the ger pathways can elicit changes in gene expression G␣ (Lee et al. 1992) and G␤␥ (Touhara et al. 1994; Wu (Clipstone and Crabtree 1992; Franz et al. 1994; et al. 1993) subunits released upon activation of G pro- Hill and Treisman 1995; O’Keefe et al. 1992). tein–coupled receptors. Stimulation of the PI-PLC ␥ In animal cells, the role of any given PI-PLC isoform family depends on SH2 domains, which mediate inter- is difficult to assess because of the multiplicity of PI- action with and phosphorylation by receptor-tyrosine PLC isotypes present. Moreover, the precise function of Koch kinases ( et al. 1991). In contrast, regulation of PIP2 turnover in any given cellular response after re- the PI-PLC ␦ family is less well characterized, although ceptor activation is complicated because of the simulta- it has been reported that a mammalian PI-PLC ␦ can be neous recruitment of additional distinct signaling sys- stimulated in vitro by a GTPase-activating protein (GAP) tems, for example, the Ras- and PI-3 kinase–dependent for the small G protein Rho (Homma and Emori 1995) pathways (Valius and Kazlauskas 1993). Genetic ap- Feng and by interaction with a novel G␣h GTPase ( et al. proaches have revealed cell type–specific requirements 1996). for particular PI-PLC isozymes. A Drosophila mutant (norpA) deficient in a PI-PLC ␤4 isoform lacks light- stimulated membrane potential in its photoreceptor cells and is blind (Bloomquist et al. 1988), defining a Corresponding author: Jeffrey S. Flick, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232-0146. role for PIP2 mobilization by this PI-PLC ␤ isoform in E-mail: fl[email protected] insect phototransduction. Disruption of the mouse PI-

Genetics 148: 33–47 (January, 1998)

34 J. S. Flick and J. Thorner

PLC ␥1 gene results in embryonic lethality, indicating growth at 35Њ. One of the genes, PHO81, is a critical an essential requirement for this isoform in multicellu- regulatory factor of the PHO regulon (Schneider et al. lar development (Ji et al. 1997). In contrast, loss of a PI- 1994). The second gene, SPL2, encodes a novel protein PLC ␦ homolog in Dictyostelium discoideum did not result that is regulated in part by the PHO regulon. In S. cerevi- in any apparent phenotype (Drayer et al. 1994); conse- siae, response to phosphate starvation involves the quently, the role of phosphoinositide turnover by PI- PHO81-dependent inhibition (Schneider et al. 1994) PLC ␦ in this organism remains unknown. of a cyclin-dependent protein kinase (Cdk) comprised As a means to elucidate cellular functions depen- of the PHO80 and PHO85 gene products (Kaffman et dent on the activity of a PI-PLC ␦, we have also under- al. 1994; Santos et al. 1995). Pho81p action reduces taken a genetic analysis using the budding yeast Saccha- the inhibitory phosphorylation of the Pho4p transcrip- romyces cerevisiae. We (Flick and Thorner 1993) and tion factor, thereby allowing high-level expression of others (Payne and Fitzgerald-Hayes 1993; Yoko-o et genes encoding secreted acid phosphatase (PHO5), al. 1993) isolated a yeast gene, PLC1, that encodes a ho- other phosphatases, phosphate transporters, and other molog of mammalian PI-PLC ␦ isoforms. Moreover, we products required for efficient phosphate assimilation (Flick and Thorner 1993) demonstrated that Plc1p is (Johnston and Carlson 1992). a PI-PLC, that its activity in vitro is Ca2ϩ-dependent and The findings presented here suggest, first, that Plc1p that, as observed for the mammalian enzymes, its sub- (or, more likely, the products of the reaction it cata- strate selectivity is influenced by the concentration of lyzes) acts to antagonize, directly or indirectly, either Ca2ϩ. Yeast cells lacking Plc1p are viable, but grow the Pho80p/Pho85p Cdk or one of the downstream slowly at 30Њ (and below), and display several other substrates of this protein kinase. Second, our results in- phenotypes, including temperature-sensitive lethality dicate that the target of Plc1p and Pho80p/Pho85p ac- (at 34Њ and above; Flick and Thorner 1993; Payne tion is a novel factor, not the Pho4p transactivator. Fi- and Fitzgerald-Hayes 1993), sensitivity to hyperos- nally, the genetic interactions we have uncovered motic conditions (Flick and Thorner 1993), missegre- among PLC1, SPL2, PHO81, and PHO80/PHO85 show gation of chromosomes during mitosis (Payne and that the products of these genes participate in overlap- Fitzgerald-Hayes 1993), and poor utilization of car- ping regulatory pathways necessary for adaptation to bon sources other than glucose (Flick and Thorner changing nutrient and temperature conditions. 1993; Payne and Fitzgerald-Hayes 1993). In at least one genetic background, a plc1 null allele is lethal (Yoko-o et al. 1993). MATERIALS AND METHODS As one approach for identifying the roles that Plc1p Strains, growth conditions, and general methods: The yeast plays in cellular physiology, we isolated and character- strains used in this study are listed in Table 1. Strain construc- ized two genes, which, when present in high-copy num- tion followed standard methods (Rose et al. 1990). Yeast was ber, bypass the requirement for Plc1p activity for grown on agar plates containing either YPGlc medium (1%

TABLE 1 Yeast strains used in this study

Straina Genotypeb Source YPH499 MATa Sikorski and Hieter 1989 YPH500 MAT␣ Sikorski and Hieter 1989 YSS5 MAT␣ pho80⌬::HIS3 Salama et al. 1994 YJF32 MATa plc1⌬::HIS3 Flick and Thorner 1993 YJF132 MATa plc1⌬::LEU2 Flick and Thorner 1993 YJF133 MAT␣ plc1⌬::LEU2 Flick and Thorner 1993 YJF251 MATa plc⌬::LEU2 pho80⌬::HIS3 This work YJF252 MATa plc1⌬::LEU2 pho80⌬::HIS3 This work YJF277 MATa spl2⌬::HIS3 This work YJF306 MATa plc1⌬::LEU2 spl2⌬::HIS3 This work YJF386 MAT␣ plc1⌬::LEU2 pho80⌬::HIS3 spl2⌬::HIS3 This work YJF552 MATa pho81⌬::TRP1 This work YJF555 MAT␣ plc1⌬::HIS3 pho81⌬::TRP1 This work YJF567 MAT␣ plc1⌬::LEU2 pho80⌬::HIS3 pho4⌬::TRP1 This work BJ3501 MAT␣ pep4::HIS3 prb1-⌬1 can1 gal2 ADE2 LYS2 TRP1 LEU2 Jones 1991 a All strains are derived from YPH499 or YPH500, except for the protease-deficient strain BJ3501. b Except for strain BJ3501, all strains listed also carry the following mutations: ade2-101oc his3-⌬200 leu2-⌬1 lys2-801am trp1-⌬1 and ura3-52. Plc1p Function and the Pho80p/Pho85p Cdk in Yeast 35 yeast extract, 2% peptone, 2% glucose) or a synthetic com- BamHI and SalI to yield plasmid pJF181. The complete nucle- plete medium (SCGlc) that contained (per liter): 1.7 g Yeast otide sequence on both strands of this 1.8-kb genomic seg- Nitrogen Base without amino acids or ammonium sulfate ment was determined using a Sequenase kit (United States (Difco, Detroit, MI); 5 g (NH4)2SO4, 20 g glucose, 20 mg each Biochemical, Cleveland, OH) and conditions recommended uracil and l-Arg, 30 mg each l-Tyr and l-Ile, 40 mg adenine by the supplier from a series of nested deletions that were sulfate, 50 mg l-Phe, 60 mg l-His, 100 mg each L-Glu and generated by a procedure described previously (Henikoff l-Asp, 150 mg each l-Met and l-Val, 200 mg of l-Thr, 260 mg 1984). DNA sequences were compiled and analyzed using l-Leu, and 400 mg l-Ser. Either uracil or leucine was omitted Lasergene software (DNASTAR Inc., Madison, WI). Databases from SCGlc plates to provide selection for the maintenance of were searched using the BLAST algorithm (Altschul et al. plasmids. Phosphate-depleted YPGlc and SCGlc-Ura were pre- 1990). pared as described elsewhere (Bisson and Thorner 1982). To express the SPL2 coding sequence from the ADH1 pro- Plasmids were introduced into yeast using the lithium acetate moter, a 0.5-kb NcoI-NdeI fragment from pJF181 (containing transformation procedure in the presence of carrier DNA nucleotides Ϫ1 to ϩ499 relative to the first base of the ATG of (Gietz and Schiestl 1991). Conventional methods were used the SPL2 open reading frame) was converted to blunt ends by for the construction and manipulation of all plasmids (Ausubel treatment with the Klenow fragment of Escherichia coli DNA et al. 1994); Escherichia coli DH5␣FЈ (GIBCO BRL, Bethesda, polymerase I and all four deoxyribonucleotide triphosphates, MD) was used for the propagation of all plasmids. and it was then inserted into the SmaI site of vector pAD4M Selection of high-copy suppressors: Strain YJF32 (plc1⌬1::HIS3) (Martin et al. 1990). To create an in-frame fusion containing was transformed with an S. cerevisiae genomic DNA library a 17-residue c-myc epitope (-IEEQKLISEEDLLRKRD-cooh; (Nasmyth and Tatchell 1980), carried in a 2-␮m DNA vec- Evan et al. 1985) attached to the C-terminal residue (codon tor (YEp13), and plated on SCGlc-Leu plates. After incuba- 148) of Spl2, a 1.2-kb EcoRV-BspHI fragment was excised from tion at 23Њ for 12 hr, the plates were transferred to 35Њ for 5 plasmid pJF181, converted to blunt ends, and ligated into a -transformants, two colonies were ob- derivative of pBluescript containing the Myc tag coding se 20,000ف days. From tained. Plasmids were rescued from these two yeast clones in quence (pBS-MycTag; constructed by D. Ma) that had been bacteria (Robzyk and Kassir 1992) and were retested for digested with EcoRI and treated with mung bean nuclease. A their suppressor activity by transformation of YJF32 and exam- 1.3-kb BamHI-SalI fragment (nucleotides Ϫ725 to ϩ445 of ination of the resulting transformants for their ability to grow the SPL2 coding region) containing the resulting SPL2-myc fu- as isolated single colonies at 35Њ. One plasmid, pJF115, was sion was subcloned into BamHI- and SalI-digested YEp352 able to suppress the temperature-sensitive phenotype of the (Hill et al. 1986), yielding plasmid pJF264. To create plas- plc1⌬ cells and did not contain the PLC1 gene itself, as deter- mid pJF184, a 3.4-kb BamHI fragment containing the entire mined by restriction endonuclease cleavage site analysis. The PLC1 gene (Flick and Thorner 1993) was ligated into insert DNA in pJF115 was designated SPL1. The selection for BamHI-digested YEp351. dosage suppressors was repeated with a different recipient Physical mapping: The SPL1 and SPL2 loci were assigned strain, YJF132 (plc1⌬2::LEU2), and by using another S. cerevi- to their respective chromosomes by hybridization of appropri- siae genomic library (Carlson and Botstein 1982) carried ate 32P-labeled internal probes corresponding to each gene -transfor- (3-kb BamHI-BamHI fragment of pJF115 and 3.5-kb BamHI 18,000ف on a different 2-␮m vector (YEp24). From mants, three colonies grew at 35Њ. After recovery in bacteria BamHI fragment of pJF116, respectively) to whole yeast chro- and retesting, all three plasmids were found to reproducibly mosomes that had been separated by pulsed-field gel electro- rescue the temperature-sensitive growth of YJF132 at 35Њ. As phoresis (Chu et al. 1986) and transferred to a nylon membrane judged by restriction enzyme analysis, one plasmid carried the (a gift from G. Anderson). The positions of SPL1 and SPL2 PLC1 gene. The other two plasmids, pJF116 and pJF117, con- were located more precisely by hybridization of the same probes tained overlapping fragments of genomic DNA that were not to nitrocellulose filters (gift from L. Riles and M. Olson) con- the same as either PLC1 or SPL1. The insert DNA in pJF116 taining an ordered set of yeast genomic DNA segments in- and pJF117 was designated SPL2. Both SPL1- and SPL2-con- serted into a bacteriophage ␭ vector (Riles et al. 1993). taining plasmids were able to suppress the temperature-sensi- Construction of null mutations: The one-step gene replace- tive phenotype of yeast containing either the plc1⌬1::HIS3 or ment method (Rothstein 1983) was used to introduce all plc1⌬2::LEU2 null allele. mutant alleles into their corresponding chromosomal loci. To Plasmid constructions: The SPL1-containing insert in pJF115 inactivate the PHO81 gene, plasmid pPHO81⌬::TRP1B (gift kb. Initial attempts to retrieve conveniently a func- from E. O’Neil and E. O’Shea) was digested with NcoI and 10ف was tional subclone were unsuccessful. Therefore, restriction en- ApaI, and the resulting fragment (in which codons 35–421 of zyme digestion and deletion analysis (Sambrook et al. 1989) the PHO81 coding sequence have been removed and substi- were used to localize the complementing region within the in- tuted with the TRP1 gene) was used to transform YPH499 to sert. The DNA sequence adjacent to a BamHI restriction site tryptophan prototrophy. To disrupt the SPL2 gene, the 149- that fell within the region responsible for suppressor activity bp NcoI-BstEII fragment in plasmid pJF181 (codons 1–49 of was determined by standard methods (Biggin et al. 1983). the SPL2 open reading frame) was excised and replaced with The nucleotide sequence obtained was identical to that re- a 2.7-kb fragment containing the HIS3 gene derived from ported for the PHO81 gene (EMBL/GenBank accession num- plasmid pJJ217 (Jones and Prakash 1990) to create plasmid ber D13228; Ogawa et al. 1993). On this basis, a 4.7-kb DNA pJF230. Plasmid pJF230 was digested with EcoRV and HpaI, fragment containing the entire PHO81 gene and its promoter and the released fragment was used to transform a diploid (from the HindIII site at position Ϫ1011 to the AvaII site at strain, YPH501 (Sikorski and Hieter 1989), to histidine pro- position ϩ3726, relative to the first base of the ATG initiator totrophy; sporulation and tetrad dissection of a resulting dip- codon of the PHO81 open reading frame) was inserted into loid transformant yielded haploid segregants containing the the HindIII and SmaI sites of the vector YEp351 (Hill et al. spl2⌬::HIS3 allele. To eliminate the PHO4 gene, plasmid 1986). The resulting plasmid was found to contain full SPL1 pPHO4dv (gift from E. O’Shea) was digested with SacI and suppressor activity and was designated pJF243. XhoI, and the resulting fragment (in which codons 1–309 of A 1.8-kb BamHI-SalI fragment containing SPL2 suppressor the PHO4 coding sequence have been removed and substi- activity was subcloned from the insert in plasmid pJF117 and tuted with the TRP1 gene) was used to transform YPH499 to ligated into the vector YEp351, which had been cleaved with tryptophan prototrophy. Proper integration of all gene trans- 36 J. S. Flick and J. Thorner placements was confirmed either by restriction enzyme digestion and Southern blot hybridization analysis with appropriate DNA probes or by the polymerase chain reaction using the appropriate sets of flanking synthetic oligonucleotide primers. RNA and protein analysis: Total and poly(A)ϩ RNA were prepared as described (Ausubel et al. 1994; Elder et al. 1983) from yeast grown in YPGlc medium to early exponential phase and from a separate sample of the same culture 2.5 hr after its transfer to low-Pi YPGlc medium. Electrophoresis, membrane blotting, and hybridization of RNA were con- ducted as described (Flick and Johnston 1990). A 32P-labeled antisense SPL2 RNA probe was prepared by in vitro transcription with T7 RNA polymerase (Ausubel et al. 1994) using plasmid pJF242 linearized with NcoI as the template; the resulting RNA product is complementary to nucleotides ϩ1 to ϩ440 of the SPL2 sequence. To construct plasmid pJF242, the 1.2-kb EcoRV-BspHI fragment from pJF181 was converted to blunt ends and inserted into pBluescript II KS that had been cleaved with ClaI and converted to blunt ends. An antisense probe to detect the CMD1 gene transcript was generated by in vitro transcription with T3 RNA polymerase using plasmid pJF156 linearized with EcoRI as the template; plasmid pJF156 consists of the 229-bp EcoRI-HindIII fragment of CMD1 (Davis et al. 1986) cloned into pRS316 (Sikorski and Hieter 1989). Quantitative estimates of the relative levels of the SPL2 and CMD1 mRNAs were obtained by analyzing the intensity of the corresponding bands using a Phosphorimager (Molecular Dynamics, San Diego, CA). Spl2-myc protein was analyzed in extracts prepared from a protease-deficient strain (BJ3501; Jones 1991) carrying plasmid pJF264 that had been grown either in SCGlc-Ura medium or in low-Pi SCGlc-Ura medium. Cell lysis, fractionation, gel electrophoresis, and immunoblotting using anti-Myc mAb 9E10 were carried out as described previously (Flick and Thorner 1993).

RESULTS Selection of dosage suppressors: Inability of plc1⌬ cells to grow at 35Њ provided a stringent genetic selection for the isolation of genes, which, when overexpressed, could bypass the requirement for PLC1 for growth at elevated temperature. Two yeast genomic DNA libraries carried in different high–copy number vectors (see materials and methods) were surveyed for plasmids able to restore growth to plc1⌬ cells at 35Њ. This way, two separate genes, designated SPL1 and SPL2 (for suppressor of plc1⌬), that were distinct from PLC1 were identified (Figure 1). At 35Њ, the growth of plc1⌬ cells Figure 1.—Dosage suppressors of the temperature-sensi- supported by high-copy SPL1 and SPL2 was less vigor- tive phenotype of plc1⌬ cells. Strain YJF32 (plc1⌬), trans- ous than that sustained by the authentic PLC1 gene formed with vector alone (YEp351), or the same vector carrying PLC1 (pJF184), or carrying the suppressors SPL1 (Figure 1). At 37Њ, neither SPL1 nor SPL2 could rescue (pJF243) or SPL2 (pJF181), as indicated, were streaked on se- the lethality of plc1⌬ mutants. Another phenotype of lective medium (SCGlc-Leu) and incubated at either 25Њ for 4 plc1⌬ cells is an inability to grow under conditions of days (top plate) or at 35Њ for 5 days (middle plate) or SCGlc- osmotic stress (Flick and Thorner 1993). High-copy Leu medium containing 1.2 m sorbitol and incubated for 5 SPL2, but not SPL1, was able to restore growth to the days at 25Њ (bottom plate). plc1⌬ mutant on a hypertonic medium (Figure 1, bottom). Physical mapping (see materials and methods) from SPL1 plasmid insert corresponded to the pub- and nucleotide sequence analysis (see below) confirmed lished sequence of the PHO81 gene, a previously char- that SPL1 and SPL2 were distinct loci from PLC1. acterized locus encoding a positive regulator of PHO5 Identification of SPL1 as the PHO81 gene: The nu- (repressible acid phosphatase) gene expression (Toh-e cleotide sequence of a restriction fragment derived et al. 1973). Using the known PHO81 gene sequence Plc1p Function and the Pho80p/Pho85p Cdk in Yeast 37

(Ogawa et al. 1993), the portion of the SPL1-contain- plc1⌬ mutants carrying SPL1 plasmids secreted elevated ing plasmid insert that contained only the PHO81 cod- levels of acid phosphatase, even when grown in high ing region and its promoter was subcloned (Figure 2). phosphate medium, as assessed by a colorimeteric col- This DNA fragment carried on a high-copy vector ony overlay assay (data not shown). Domains of Pho81p (pJF243; Figure 2) permitted the growth of plc1⌬ cells required for effective inhibition of Pho80p/Pho85p in at 35Њ just as well as the original plasmid isolate. To con- vivo, as judged by the degree of Pho5p derepression, firm unequivocally that the SPL1 suppressor activity have been mapped previously (Ogawa et al. 1993). The was provided solely by the PHO81 gene product, three central region of Pho81p contains six tandem ankyrin mutant alleles were constructed. Two truncations of repeats, flanked on their amino-terminal side by a neg- the PHO81 open reading frame (plasmids pJF202 and atively acting domain and on their C-terminal side by a pJF237), even one that removed Ͻ20% of the predicted positively acting domain. Tandem ankyrin repeats are protein (codons 957–1179), eliminated SPL1 suppres- also found in p16INK4 and in other low molecular weight sor activity (Figure 2). Likewise, an allele in which we inhibitors of mammalian Cdk4 and Cdk6 (Serrano et introduced a frame shift mutation at codon 557 of the al. 1993). In addition to its ankyrin repeats, a C-termi- PHO81 open reading frame (plasmid pJF251) also to- nal segment of Pho81p (residues 772–1179) is required tally destroyed its SPL1 suppressor activity (Figure 2). for derepression of PHO5 expression (Ogawa et al. 1995). PHO81 is a phosphate-regulated inhibitor of a Cdk A plasmid, pJF237, which expressed a form of Pho81p comprised of a cyclin protein encoded by the PHO80 that retained its ankyrin repeats, but truncated the gene and a protein kinase catalytic subunit encoded C-terminal domain (removing amino acids 957–1179), by the PHO85 gene (Schneider et al. 1994). During failed to suppress the temperature sensitivity of plc1⌬ growth in normal media (i.e., high phosphate), the cells (Figure 2) or to derepress acid phosphatase activ- PHO4-encoded transcriptional activator is hyperphos- ity (data not shown). Thus, a region of Pho81p previ- phorylated by the Pho80p/Pho85p complex, which ously defined as critical for its ability to inhibit the leads to exclusion of Pho4p from the nucleus (O’Neil Pho80p/Pho85p enzyme and to cause derepression of et al. 1996). Starvation for phosphate stimulates Pho81 PHO5 was also required for its SPL1 activity. protein to inhibit Pho80p/Pho85p Cdk activity, thus al- Other conditions that inhibit Pho80p/Pho85p and dere- lowing underphosphorylated Pho4p to accumulate in press the PHO regulon suppress the temperature-sensitive the nucleus and activate transcription of genes in the growth of plc1⌬ mutants: To determine if PHO81 overex- PHO regulon, including PHO5 (Schneider et al. 1994). pression rescued the temperature-sensitive lethality of It has been shown previously that increased dosage of plc1⌬ cells solely because of its inhibition of Pho80p/ the PHO81 gene leads to partially constitutive expres- Pho85p and the consequent derepression of genes un- sion of PHO5, even in media containing abundant phos- der its control, we examined other conditions that phate (Yoshida et al. 1989). cause inhibition of this Cdk. First, we simply altered the

SPL1 activity of PHO81 correlates with its ability to in- Pi concentration in the growth medium. As shown pre- hibit the Pho80/Pho85 Cdk: Our results suggested that viously (Flick and Thorner 1993), on a medium con- high-copy PHO81 might suppress temperature-sensitive taining high Pi (where Pho80p/Pho85p activity is high growth of plc1⌬ cells by inhibiting Pho80p/Pho85p, or and PHO5 expression is prevented), plc1⌬ cells failed to via subsequent induction of a gene of the PHO regulon grow at 35Њ (Figure 3A, left side). In contrast, on a me-

(or both). Multicopy PHO81 was indeed capable of dium containing low Pi (where Pho80p/Pho85p activity causing derepression of the genes of the PHO regulon is low and PHO5 is expressed), plc1⌬ cells were able to in plc1⌬ mutants because, unlike plc1⌬ cells alone, propagate at high temperature (Figure 3A, right side).

Figure 2.—Demonstration that PHO81 encodes SPL1 function. Top line shows a restriction endonuclease cleavage site map of the PHO81-containing region [adapted from (Ogawa et al. 1993)] of the DNA insert in the original SPL1 isolate (pJF115). The PHO81 open reading frame (shaded bar) contains six ankyrin repeats (solid arrowheads). The indicated restriction fragments were subcloned into vector YEp351 and retested for their ability (ϩ) or inability (Ϫ) to transform strain YJF32 (plc1⌬) to leucine prototrophy at 35Њ. The insert in plasmid pJF251 contains a frameshift mutation (asterisk) introduced by cleavage with BamHI, ﬁll- ing in with the Klenow fragment of E. coli DNA polymerase I, and religation. Restriction sites: A, AvaII; B, BamHI; E, EcoRI; H, HindIII. 38 J. S. Flick and J. Thorner

Figure 3.—Nutritional and mutational inactivation of the Pho80/Pho85 Cdk suppresses the temperature-sensitive phenotype of plc1⌬ cells. (A) Normal PLC1ϩ cells (strains YPH499, left top, and YPH500, right top; WT) and their otherwise isogenic plc1⌬ derivatives (strains YJF132, left bottom, and YJF133, right bottom; plc1⌬) were streaked on a rich medium (YPGlc) containing excess inorganic phosphate (left plate; high [Pi]) and on the same medium that had been depleted of inorganic phosphate, as de- materials and methods scribed in (right plate; low [Pi]), and incubated for 3 days at 36Њ. Using a colorimetric colony staining assay (Schurr and Yagil 1971), all four strains grown on the phosphate-depleted plate were positive for acid phosphatase expression (data not shown). (B) Strains YJF32 (plc1⌬; top left), YSS5 (pho80⌬; top right), YJF251 (plc1⌬ pho80⌬; bottom left), and YJF252 (plc1⌬ pho80⌬; bottom right), all otherwise isogenic to either YPH499 or YPH500 (Table 1), were streaked on synthetic medium (SCGlc) and incubated at 25Њ for 5 days (left plate) or at 35Њ for 5 days (right plate).

Bisson Thus, like PHO81 overexpression, a natural nutritional PHO5 in yeast grown in high Pi media ( and signal that leads to inhibition of Pho80p/Pho85p and Thorner 1982), and we observed constitutive expres- PHO5 derepression also permitted the growth of plc1⌬ sion of acid phosphatase in plc1⌬ pho80⌬ double mu- cells at elevated temperature. tants (data not shown). Like plc1⌬ cells on low Pi me- Second, we used another genetic manipulation to dium, plc1⌬ pho80⌬ double mutants were able to grow inhibit the Pho80p/Pho85p enzyme. The PHO80 gene at 35Њ, even on a high Pi medium (Figure 3B). Thus, encodes the cyclin component of this Cdk. Because it is three different conditions that lead to inactivation of required for activity of the kinase, Pho80p acts as a neg- the Pho80p/Pho85p Cdk and concomittant derepres- ative regulator of PHO5 expression (Toh-e and Shi- sion of the genes under its control allowed plc1⌬ cells mauchi 1986; Yoshida et al. 1989). Indeed, pho80 mu- to grow at a temperature that would otherwise be non- tations cause high-level constitutive derepression of permissive for their growth. Plc1p Function and the Pho80p/Pho85p Cdk in Yeast 39

Disruption of the genes for several other Pho85-asso- (30%), and Pho81 (27%). By comparison, Far1p, a ciated cyclins does not suppress plc1⌬: Pho85p has re- demonstrated inhibitor of the Cdc28/Cln2 Cdk (Peter cently been shown to associate with nine other putative and Herskowitz 1994), does not bear detectable ho- cyclin proteins, in addition to Pho80p, including: mology to any mammalian Cdk inhibitor yet identified. Pcl1p, Pcl2p, Pcl5p, Pcl6p, Pcl7p, Pcl8p, Pcl9p, Pcl10p, We noted that the N-terminal 20 residues of Spl2p are and Clg1p (Measday et al. 1997). To determine if loss comprised almost exclusively of hydrophobic and un- of other Pho85 Cdk complexes can bypass the tempera- charged amino acids, and that Gly2 is a potential target ture sensitivity caused by the plc1⌬ mutation, we gener- for N-myristoylation, although Spl2 lacks other consen- ated double mutants of plc1⌬ with pcl1⌬, pcl2⌬, pcl5⌬, sus residues found in efficiently myristoylated proteins and clg1⌬. Unlike plc1⌬ pho80⌬ cells, none of these (Towler et al. 1988). The remainder of the protein can double mutants was able to grow at a restrictive temper- be divided into two regions: residues 32–97 constitute a ature, indicating that suppression was not a general re- highly basic segment (net charge ϩ9), and residues 98– sult of perturbed Pho85p activity or altered cyclin ra- 148 comprise a highly acidic segment (net charge Ϫ11; tios. A plc1⌬ pho85⌬ double mutant also failed to grow Figure 4B). Potential phosphorylation sites for several at restrictive temperature (data not shown). This find- classes of protein kinase are also present in Spl2, in- ing might mean that a Pho85p-independent activity of cluding cAMP-dependent protein kinase (Ser59 and overproduced Pho81p (or of loss of Pho80p) is respon- Ser86), proline-directed protein kinases (Thr5, Ser30 sible for suppression of the plc1⌬ mutation. It is more and Thr81), and casein kinase II (Ser142 and Ser143). likely, however, that alteration or reduction of Pho85p The functional significance of these sites has not been activity is able to suppress the plc1⌬ temperature-sensi- explored. tive defect, whereas total elimination of all Pho85p- SPL2 gene expression and Spl2 protein level are reg- dependent Cdk activity has such a deleterious effect on ulated by phosphate: The SPL2 promoter region con- cells as to preclude growth of a plc1⌬ pho85⌬ double tains three matches to the consensus (5Ј-CACGTG-3Ј) mutant under any condition. In support of this view, for binding of the Pho4p transactivator located at posi- pho85⌬ mutants, which lack 10 different Cdk complexes, tions Ϫ147, Ϫ90, and Ϫ31 (where ϩ1 is the ATG; Fig- Gilliquet Berben grow quite poorly even at 30Њ ( and ure 4B). When Pi is limiting, Pho4p is in the nucleus, 1993), show defects in glycogen metabolism (Timblin binds to such sites in the upstream regions of PHO5 et al. 1996), and display abnormal cellular morphology and the other genes of the PHO regulon, and activates (Measday et al. 1997). transcription (Fisher et al. 1991). To determine if its Molecular characterization of the SPL2 gene: The SPL2 expression is regulated by phosphate, a 32P-labeled suppressor locus was delimited to a 1.8-kb BamHI-SalI SPL2 probe was hybridized to size-fractionated RNA fragment by subcloning restriction fragments from one prepared from yeast grown in either a high or a low Pi of the original plasmid isolates and retesting for sup- medium. The internal control for loading was a probe pressor activity (Figure 4A). Within this DNA fragment, for CMD1 mRNA, that encodes calmodulin. The SPL2 0.45ف) nucleotide sequence analysis (EMBL/GenBank acces- transcript was polyadenylated and long enough sion number P38839) delineated a 148-codon open kb) to encode the SPL2 open reading frame (Figure reading frame (YHR136c) that originated from the 5A). In cells grown in high Pi medium, SPL2 mRNA was -of the level of the CMD1 mRNA. Af %30ف right arm of chromosome 8 (Johnston et al. 1994). expressed at

Two different approaches were taken to confirm that ter shift to low Pi medium for 2.5 hr, the steady-state the 148-codon open reading frame encoded the SPL2 level of SPL2 mRNA increased 2.7-fold (when normal- suppressor activity. First, a frameshift mutation intro- ized to CMD1 RNA), indicating that SPL2 expression is duced at codon 49 abolished the suppressor activity of induced and suggesting that its potential Pho4-binding the subcloned BamHI-SalI fragment (plasmid pJF217; sites have a functional role. In further support that in- Figure 4A). Second, expression of just the 148-codon duction occurs at the transcriptional level, expression sequence from a different promoter (ADH 1) on a high of an SPL2-lacZ fusion (containing SPL2 sequences copy plasmid vector was able to suppress the tempera- from Ϫ724 to ϩ3) in wild-type cells increased 4.4-fold ture-sensitive growth defect of plc1⌬ cells (plasmid after transfer to a low Pi medium, whereas in pho80⌬ pJF235; Figure 4A). Furthermore, our subsequent char- cells, expression was constitutively high and did not in- acterization (see below) confirmed that SPL2 encodes crease significantly upon transfer to low Pi medium, as the predicted protein. expected if SPL2 is regulated by Pho4p (data not The deduced SPL2 gene product (Figure 4B) has a shown). calculated molecular mass of 17 kD and, allowing for For detection, sequences encoding a c-Myc epitope conservative amino acid replacements, is similar to the were fused in-frame to the C terminus of the SPL2 cod- sequences of mammalian low molecular weight Cdk in- ing region. The epitope used is recognized by the mono- hibitors and also to the ankyrin repeat region in clonal antibody 9E10. The epitope-tagged Spl2 (Spl2myc) Pho81p (Figure 4C). Similarity between Spl2p and the expressed from the SPL2 promoter suppressed the tem- other proteins is as follows: p16 (29%), p18 (33%), p19 perature sensitivity of plc1⌬ cells as efficiently as untagged 40 J. S. Flick and J. Thorner

Figure 4.—Restriction map, deduced primary structure, and sequence comparison of the SPL2 gene. (A) Physical map of the genomic DNA (thin line) in the region containing the SPL2 gene (shaded bar), which is adjacent to the 5Ј end of the YCK1 gene (hatched box) and transcribed in the same direction (single-headed arrows). The indicated DNA fragments were subcloned into vector YEp351 and retested for their ability (ϩ) or inability (Ϫ) to transform YJF32 (plc1⌬) to leucine prototrophy at 35Њ. The insert in plasmid pJF217 contains a frameshift mutation (asterisk) introduced by cleavage with BstEII, filling in with the Klenow fragment of E. coli DNA polymerase I, and religation. In plasmid pJF235, the SPL2 open reading frame was expressed from the ADH1 promoter, as described in materials and methods, and was sufficient for complementation (#). Re- striction sites: B, BamHI; Bs, BspHI; Bt, BstEII; N, NcoI; Nd, NdeI; R, EcoRV; and, S, SalI. (B) Nucleotide sequence (where ϩ1 indicates the first base of the ATG initiator codon; EMBL/Gen- Bank accession number P38839) and predicted amino acid sequence (where 1 indicates the initiator methionine residue) of the SPL2 gene. Three consensus Pho4-binding sites in the promoter region (underlined), basic residues (K, R; boxed), and acidic residues (D, E; circled) are indicated. (C) Comparison of the primary sequence of Spl2 to those of known Cdk inhibitors, p16 (Serrano et al. 1993), p18 (Guan et al. 1994), p19 (Chan et al. 1995), and Pho81 (Ogawa et al. 1993). Identities and conventional conservative substitu- tions (Tanaka et al. 1990) (AϭGϭPϭS; LϭIϭVϭM; RϭKϭH; DϭE; NϭQ; FϭYϭW; SϭT; NϭD; QϭE) between Spl2p and any of the other proteins listed are given as white-on-black let- ters.

Spl2 (data not shown), demonstrating that Spl2myc was kD). However, anomalous migration has been observed functional. Extracts of yeast expressing Spl2myc from for other polypeptides that are also very highly charged the SPL2 promoter on a multicopy plasmid were ana- (Benton et al. 1994; Swartzman et al. 1996). As judged lyzed by SDS-PAGE and immunoblotting (Figure 5B). by densitometry, Spl2myc level increased about three-

Spl2myc migrated with a mobility corresponding to an fold in cells grown in a low Pi medium compared to its kD, which was some- level in an equivalent amount of extract from cells 23ف apparent molecular mass of what larger than its calculated molecular mass (19.2 grown on the same medium containing high Pi (Figure Plc1p Function and the Pho80p/Pho85p Cdk in Yeast 41

Figure 5.—SPL2 mRNA expression, Spl2 protein production and subcellular fractionation. (A) Total RNA (20 ␮g; lanes 1 and 2) or of poly(A)ϩ RNA (1 ␮g; lane 3) were prepared from strain YPH499 grown at 30Њ in either rich medium (YPGlc) containing excess inorganic phosphate (Hi; lanes 1 and 3) or the same medium depleted of inorganic phosphate (Lo; lane 2), resolved by electrophoresis in an agarose gel, transferred to a membrane filter, and hybridized to 32P-labeled antisense RNA probes corresponding to the SPL2 and CMD1 (Davis et al. 1986) genes, generated as described in materials and methods. After hybridization, the filter was washed at high stringency and used to expose X-ray film for 6 hr with an intensifying screen. Migration positions and sizes (in kilobases) of length standards (single-stranded RNAs) are indicated. (B) A protease-deficient strain (BJ3501) carrying plasmid pJF264 expressing SPL2myc was grown to mid-exponential phase in selective medium (SCGlc-Ura) containing excess inorganic phosphate (High [Pi]; lanes 1–6) or in the same medium depleted of inorganic phosphate (Low [Pi]; lanes 7–12), and was disrupted by vigorous vortex mixing with glass beads in a buffer containing 25 mm Tris-HCl, pH 7.5, 50 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, and 1 mm phenylmethylsulfonylfluoride. The resulting crude lysate was clarified by low speed (450 g) centrifugation to yield a supernatant fraction (S450; lanes 1 and 6), samples of which were subjected to centrifugation at either 10,000 g for 20 min to yield a supernatant (S104; lanes 2 and 7) and a pellet (P104; lanes 3 and 8) fraction, or at 100,000 g for 1 hr to yield a supernatant (S105; lanes 4 and 9) and a pellet (P105; lanes 5 and 10) fraction. Samples (each representing total protein derived from an equivalent number of cells) were solublized in SDS gel loading buffer, resolved on a 13% polyacrylamide gel, transferred to a membrane filter, incubated with monoclonal anti-c-myc antibody (9E10) followed by horseradish peroxidase-conjugated sheep anti-mouse antibody, and visualized by a commercial chemiluminescence detection system (ECL; Amersham, Arlington Heights, IL). Molecular mass markers: bovine carbonic anhydrase, soybean trypsin inhibitor, and chicken lysozyme.

5B), which is consistent with the increase observed in low-Pi medium, the amount in the particulate fraction the level of the SPL2 mRNA and SPL2-lacZ expression did not increase (Figure 5B). under the same conditions. Genetic interactions of plc1⌬, pho81⌬, and spl2⌬ mu- Despite its apparent homology to other small Cdk tations: One mechanism to explain how increased ex- inhibitors, the hydrophobic nature of the N terminus pression of PHO81 and SPL2 can compensate for the of Spl2 and its potential myristoylation site suggested loss of PLC1 to permit growth at 35Њ is that these gene that this segment of the protein might serve as a signal products might be components in a pathway required sequence or act as a membrane anchor. To determine for growth at elevated temperature that lies down- whether Spl2 was a secretory protein or localized to a stream of PLC1 and that requires PLC1 function for its membrane-bound compartment, total extracts of cells full activity. If so, then one might expect that null muta- expressing Spl2myc from its own promoter on a multi- tions in PHO81 and/or SPL2 might result in pheno- copy plasmid were fractionated by differential centrifu- types that resemble those of a plc1⌬ mutant. Unlike a gation into soluble and particulate material. As ex- plc1⌬ mutant, however, pho81⌬ and spl2⌬ single mu- pected for a soluble protein, the majority of Spl2myc tants and pho81⌬ spl2⌬ double mutants grew as well as remained in the supernatant fraction even after pro- wild-type cells on SCGlc at 35Њ (data not shown), on hy- longed centrifugation at 100,000 g (Figure 5B). A small perosmotic medium (data not shown), on synthetic amount of the Spl2myc was found in the pellet from medium at 25Њ (Figure 6A), and on glycerol-containing the 100,000 g centrifugation. When this material was re- medium (data not shown). The pho81⌬ mutant failed suspended in buffer containing 1% Triton X-100 and to derepress acid phosphatase activity and was unable subjected to recentrifugation, none of the Spl2myc was to grow on low-Pi medium (data not shown), as observed extracted, suggesting that the particulate Spl2myc was previously by others (Toh-e et al. 1973). By contrast, the not membrane-associated (data not shown). While the plc1⌬ mutant (Figure 3A) and the spl2⌬ mutant (data amount of soluble Spl2myc increased in cells grown in not shown) grew well on low-Pi medium and, when lim- 42 J. S. Flick and J. Thorner ited for phosphate, displayed levels of secreted acid pho81⌬ and plc1⌬ spl2⌬ double mutants grew (Figure phosphatase comparable to those seen in derepressed 6B) at a rate similar to that displayed by the plc1⌬ single wild-type cells (data not shown). The lack of similarity mutant (Figure 6A). Unlike the plc1⌬ single mutant in phenotypes between a plc1⌬ mutant and the pho81⌬ (Figure 6A), however, neither the plc1⌬ pho81⌬ double and spl2⌬ single and double mutants suggests that nei- mutant nor the plc1⌬ spl2⌬ double mutant was able to ther PHO81 nor SPL2 function in a strictly linear path- form visible colonies on the synthetic medium (SCGlc), way downstream of PLC1. even after incubation for 5 days at the same tempera- An alternative hypothesis to explain suppression of ture (Figure 6B). In contrast, as might have been antic- the temperature sensitivity of plc1⌬ cells by high-level ipated from the ability of a pho80⌬ mutation to sup- expression of PHO81 or SPL2 is that PHO81 and SPL2 press the temperature sensitivity of a plc1⌬ mutation act in an independent pathway(s) that is necessary for (Figure 3B), the plc1⌬ pho80⌬ double mutant grew on growth at elevated temperature and that overlaps (or is the synthetic medium at a rate similar to that of the partially redundant in function) with the PLC1-depen- plc1⌬ single mutant. Surprisingly, the plc1⌬ pho80⌬ dent pathway. If so, double mutants in which both path- double mutant was unable to form single colonies on ways are disrupted should display more severe defects rich medium after 5 days at 25Њ. Thus, with respect to than single mutants in which only one pathway is non- growth on synthetic medium, the plc1⌬ pho81⌬ and functional. To test this possibility, plc1⌬ pho81⌬ and plc1⌬ spl2⌬ double mutants did indeed display a much plc1⌬ spl2⌬ double mutants were constructed and ex- more severe phenotype than a plc1⌬ single mutant. amined, along with the plc1⌬ pho80⌬ double mutant If the loss of SPL2, like the loss of PHO81, leads to in- (Figure 3B). On rich medium (YPGlc) at 25Њ, the plc1⌬ creased activity of the PHO80/PHO85-encoded Cdk,

Figure 6.—Nutritional effects and genetic interactions among plc1⌬, spl2⌬, pho81⌬, and pho80⌬ mutations (A) Strains YPH499 (WT; upper left), YJF32 (plc1⌬; upper right), YJF277(spl2⌬; lower left), and YJF552 (pho81⌬; lower right), constructed as described in materials and methods, were streaked either on a rich medium (YPGlc; left plate) or on a synthetic complete medium (SCGlc; right plate) and incubated at 25Њ for 3 and 5 days, respectively. (B) Strains YJF306 (plc1⌬ spl2⌬; upper left), YJF251(plc1⌬ pho80⌬; upper right), YJF386 (plc1⌬ pho80⌬ spl2⌬; lower left), and YJF555 (plc1⌬ pho81⌬; lower right), constructed as described in materials and methods, were streaked as indicated in A and incubated at 25Њ for 4 and 5 days, respectively. Plc1p Function and the Pho80p/Pho85p Cdk in Yeast 43 then the inability of the plc1⌬ pho81⌬ and plc1⌬ spl2⌬ stimulation of SPL2 expression. By this model, suppres- double mutants to grow on SCGlc might be caused by sion of a plc1⌬ mutation by a pho80⌬ mutation (Figure hyperelevation of the Pho80/Pho85 Cdk. To test this 3) should also require SPL2 activity. To test this hypoth- possibility, a plc1⌬ spl2⌬ pho80⌬ triple mutant was con- esis, the phenotype of the plc1⌬ pho80⌬ spl2⌬ triple mu- structed. Indeed, this strain was able to grow on the syn- tant was compared to that of an otherwise isogenic thetic medium (Figure 6B) just as well as a plc1⌬ single plc1⌬ pho80⌬ double mutant. As observed before, the mutant (Figure 6A). Conversely, this triple mutant was plc1⌬ pho80⌬ double mutant was able to grow at 34Њ, a also able to grow well on rich medium (Figure 6B), sug- temperature that is nonpermissive for the plc1⌬ single gesting that the inability of the plc1⌬ pho80⌬ cells to mutant (Figure 7). Likewise, the plc1⌬ pho80⌬ spl2⌬ tri- grow on YPGlc might be caused, directly or indirectly, ple mutant was also able to grow at 34Њ, demonstrating by the derepression of SPL2. Taken all together, these that SPL2 is not essential for the ability of a pho80⌬ mu- results suggest that, in addition to their role in support- tation to bypass the plc1⌬ mutation. This result further ing growth at elevated temperature, the functions of suggests that when the Pho80p/Pho85p Cdk is inacti- PLC1, PHO81, SPL2, and the PHO80/PHO85-encoded vated, the activity of a factor(s) other than SPL2 is stim- Cdk all converge on a pathway required for proper re- ulated that allows the Plc1-deﬁcient cells to grow at an sponse to elevated temperature and changing nutrient elevated temperature. On the other hand, growth of levels. the plc1⌬ pho80⌬ spl2⌬ triple mutant at 34Њ and 35Њ was Epistasis relationships in plc1⌬ suppression: Raising discernibly weaker than that of the plc1⌬ pho80⌬ dou- the dosage of either PHO81 or SPL2 suppressed the ble mutant, indicating that elevated SPL2 expression, temperature-sensitive growth of a plc1⌬ mutant (Figure while not obligatory, contributes to suppression. 1). Because SPL2 is a phosphate-regulated gene (Fig- When the Pho80p/Pho85p Cdk is inactivated, there ures 4 and 5), and because overexpression of PHO81 is a dramatic increase in the transcription of PHO4- derepresses expression of other genes in the PHO regu- dependent genes. Because SPL2 was not essential for lon (Yoshida et al. 1989), it was possibile that high-copy suppression of the temperature-sensitive growth of a PHO81 suppressed the plc1⌬ mutation solely because of plc1⌬ mutant by a pho80⌬ mutation, it seemed reason-

Figure 7.—PHO4 or SPL2 are not essential for suppression of the temperature-sensitive phenotype plc1⌬ cells by a pho80⌬ mutation. Strains YJF32 (plc1⌬; upper left), YJF251 (plc1⌬ pho80⌬; upper right), YJF567 (plc1⌬ pho80⌬ pho4⌬; lower left), and YJF386 (plc1⌬ pho80⌬ spl2⌬; lower right), constructed as described in materials and methods, were streaked on synthetic medium (SCGlc) and incubated for 5 days at the indicated temperatures. 44 J. S. Flick and J. Thorner

able to assume that elevated expression of another low-Pi medium causes a PHO81-dependent reduction in PHO4-dependent gene(s) was responsible for the sup- Pho80p/Pho85p activity in vitro, and that a pho80⌬ mu- pression since, to date, Pho4p is the only known in vivo tation totally inactivates a PHO85-dependent kinase substrate of the Pho80p/Pho85p Cdk. To determine if activity (Kaffman et al. 1994; Schneider et al. 1994). any genes under Pho4p control were required for sup- Hence, the observed suppression of the temperature- pression, a plc1⌬ pho80⌬ pho4⌬ triple mutant was con- sensitive phenotype of plc1⌬ cells by high-copy PHO81 structed. As expected, this strain failed to grow on a (Figure 1), by growth on low-Pi medium (Figure 3A), low-Pi medium and did not express detectable acid and by a pho80⌬ mutation (Figure 3B) is most likely phosphatase activity (data not shown). In marked con- caused by inhibition of Pho80p/Pho85p activity. There- trast, the plc1⌬ pho80⌬ pho4⌬ triple mutant grew at 34Њ fore, in a formal genetic sense, Plc1p activity may either and at a rate comparable to that of the plc1⌬ pho80⌬ negatively regulate the Pho80p/Pho85p Cdk or pro- spl2⌬ triple mutant (Figure 7). Thus, PHO4-dependent duce a signal that opposes the effect of Pho80p/ transcription was not required for suppression of the Pho85p on a downstream target. The interaction of PI- plc1⌬ mutation when the Pho80p/Pho85p Cdk was in- PLC ␦ activity with a phosphate-regulated pathway ap- activated by a pho80⌬ mutation. Furthermore, the fact pears to be conserved. In Schizosaccharomyces pombe, a that plc1⌬ pho80⌬ pho4⌬ and plc1⌬ pho80⌬ spl2⌬ strains mutation in the plc1ϩ gene, encoding a PI-PLC ␦ ho- grew similarly and somewhat more weakly than the molog, is partially suppressed by growth on low-Pi me- plc1⌬ pho80⌬ double mutant (Figure 7) provides an ad- dia (Fankhauser et al. 1995). ditional demonstration that SPL2 is under PHO4 con- Pho4p is the only known in vivo target of the trol, and that SPL2 expression contributes to the ability Pho80p/Pho85p Cdk, and phosphorylation of Pho4p of cells to grow at elevated temperatures. Nonetheless, by Pho80p/Pho85p prevents it from stimulating tran- the fact that PHO4 is not required for suppression sug- scription by excluding Pho4p from the nucleus (O’Neil gests that loss of the Pho80p/Pho85p Cdk suppresses a et al. 1996). Hence, inactivation of Pho80p/Pho85p plc1⌬ mutation because the Pho80p/Pho85p normally could perhaps allow growth of plc1⌬ cells at elevated antagonizes the function of an as yet unidentiﬁed fac- temperature because it permits Pho4p to stimulate the tor that is required for the ability of cells to grow at ele- expression of a gene(s) that are required for growth at vated temperatures. high temperature. However, we found that a plc1⌬ pho80⌬ pho4⌬ triple mutant grew almost as well as the plc1⌬ pho80⌬ double mutant at a temperature (34Њ) that is DISCUSSION nonpermissive for a plc1⌬ single mutant (Figure 7). These On the basis of the pleiotropic phenotypes displayed results suggest that Pho80p/Pho85p-dependent inhibi- by yeast cells lacking a PLC1 gene, we proposed that tion (presumably by direct phosphorylation) of another Plc1p deﬁciency causes defects in nutritional and factor, different from Pho4p, is responsible for block- stress-related responses (Flick and Thorner 1993). As ing growth of plc1⌬ cells at elevated temperature. In shown here, two genes, PHO81 and SPL2, when overex- further support of this conclusion, high-copy PHO4 pressed, can bypass the need for PLC1 to permit growth causes robust constitutive expression of PHO5 in plc1⌬ Ͼ34Њ. Because elevated dosage of either gene sup- cells, as in wild-type cells (Yoshida et al. 1989), yet it is pressed a plc1 null allele, Pho81p and Spl2p might rep- only a very weak suppressor of the temperature sensitiv- resent components in a pathway directly downstream ity and does not suppress at all in a plc1⌬ pho81⌬ double of Plc1p. However, the genetic interactions of the mutant (J. Flick, unpublished results). These observa- plc1⌬, pho81⌬, and spl2⌬ mutants (Figure 6) suggest tions indicate that the weak suppression by multicopy that Plc1p, Pho81p, and Spl2p act independently, but PHO4 is caused by elevated expression of PHO81 and in a partially redundant or overlapping manner. Thus, possibly SPL2, which are both PHO4-dependent genes the functions of Plc1p, Pho81p, and Spl2p appear to (Yoshida et al. 1989; Creasy et al. 1993; Figure 5), and converge on a common target that is required for the resulting inhibition of the Pho80p/Pho85p Cdk growth at elevated temperature and proper response to (rather than caused by induction of other PHO genes). nutrient levels. The second plc1⌬ suppressor was the SPL2 gene

When Pi is limiting, Pho81p inhibits the Pho80p/ (Figure 4). SPL2 encodes a 17-kD soluble protein. Al- Pho85p Cdk (Schneider et al. 1994). Although the in- though both SPL2 mRNA (Figure 5A) and Spl2 protein hibitory action of Pho81p apparently is enhanced by (Figure 5B) appear to be expressed at a signiﬁcant low-Pi conditions, overexpression of PHO81 causes par- basal level, growth of cells in low-Pi medium caused a tially constitutive derepression of PHO5, even when readily detectable induction (approximately threefold). Yoshida cells are grown in high-Pi medium ( et al. However, suppression of the temperative-sensitive de- 1989). Thus, Pho81p must be capable, even in its basal fect of plc1⌬ cells by high-copy SPL2 did not require state, of inhibiting Pho80p/Pho85p. In fact, it has been stimulation by low-Pi conditions because suppression shown by others that overexpression of Pho81p does re- was observed on high-Pi medium (Figure 1). An spl2⌬ duce Pho80p/Pho85p activity in vitro, that growth in mutant had no readily discernible growth phenotype Plc1p Function and the Pho80p/Pho85p Cdk in Yeast 45

(Figure 6), and computer searches determined that no homolog or related gene exists in the S. cerevisiae ge- nome. Given that both PHO81 and SPL2 were isolated as dosage suppressors, that both are Pho4p-dependent genes, that the phenotypes of plc1⌬ pho81⌬ and plc1⌬ spl2⌬ mutants are very similar, and that SPL2 is not required for suppression by a pho80⌬ mutation, Spl2p may represent a novel inhibitor of the Pho80p/Pho85p Cdk. Indeed, based on its molecular weight and sequence, Spl2 (p17) resembles several known low molecular weight ankyrin-repeat–containing inhibitors (p16, p18, and p19) of mammalian Cdks (Sherr and Rob- erts 1995), as well as a portion of the ankyrin-repeat region of Pho81p (Figure 4C). However, neither an spl2⌬ mutation nor high-copy SPL2 (unlike high-copy PHO81) perturb the regulation of PHO5 expression in plc1⌬ yeast (J. Flick, unpublished results). Thus, Spl2p may block the ability of Pho80p/Pho85p to phosphory- Figure 8.—Model for genetic interactions and regulatory late the novel factor, but not its ability to phosphory- circuitry involving PLC1, SPL2, and the PHO regulon. Nega- late Pho4p (whereas Pho81p may block the ability of tive (inhibitory) interactions are indicated by a bar; positive Pho80p/Pho85p to phosphorylate both this factor and (stimulatory) interactions are indicated by an arrow. See text Pho4). A somewhat analogous situation has been ob- for further explanation. served in animal cells where Cdk inhibition by Cip1/ Waf1 inhibits the function of PCNA in DNA replica- Pho85p Cdk is too high. Plc1p activity either acts to op- tion, but not in DNA repair (Li et al. 1994). pose the inhibitory action of Pho80p/Pho85p on a The interactions among PLC1, SPL2, and elements downstream target, “X,” or stimulates the function of X of the PHO regulon suggest that a factor critical for by a convergent pathway. Hence, reduction of Pho80p/ growth Ͼ34Њ is modulated by these gene products. This Pho85p activity by overproduction of either Pho81p (a same factor may also be involved in nutrient sensing known inhibitor of Pho80/Pho85) or Spl2p (a candi- and/or utilization because either a pho81⌬ or an spl2⌬ date Cdk inhibitor), by growth on low-Pi medium or by mutation prevents the growth of plc1⌬ cells on syn- introduction of a pho80⌬ mutation, all permit plc1⌬ thetic medium, but not on rich medium. In support of cells to grow at restrictive temperature. Alternatively, a connection between nutrient uptake and/or utiliza- Spl2 may act to stimulate the activity of X rather than tion and the functions of PLC1, SPL2, and the PHO reg- inhibiting Pho80p/Pho85p activity. Loss of both Plc1p ulon was our observation that a plc1⌬ pho80⌬ double and Pho81p, or both Plc1p and Spl2p, presumably mutant was sensitive to rich media, but able to grow on causes a more severe inactivation of X, explaining the synthetic medium (Figure 6B). This sensitivity could re- inability of plc1⌬ pho81⌬ and plc1⌬ spl2⌬ double mu- sult from hyperstimulation of amino acid uptake as a tants to grow on standard synthetic medium, as well as consequence of the loss of both the Pho80p/Pho85p the relief of this phenotype by a pho80⌬ mutation or by Cdk and the loss of PLC1 function. The ability pho80 augmentation with a rich growth medium. Indeed, loss mutations to promote uptake of nutrients other than of Pho81p alone has been shown to cause an elevation inorganic phosphate is highlighted by the fact that the in Pho80p/Pho85p activity in cell extracts (Schneider tup7 mutation, isolated on the basis of enhanced dTMP et al. 1994). The novel factor X, and not Pho4p, is re- uptake, is allelic to pho80 (Bisson and Thorner 1982). quired for the induction of these growth pathways be- Another connection between nitrogen metabolism and cause, for example, a pho80⌬ mutation permits growth

PIP2 turnover is suggested by a report that IP3 and DAG of plc1⌬ cells at restrictive temperature in both a levels in yeast are increased when a nitrogen source is PHO4ϩ and a pho4⌬ background. resupplied to starved cells (Schomerus and Kuntzel That Pho85 appears to interact with as many as nine 1992). other cyclins in addition to Pho80p suggests that differ- We propose the following working model (Figure 8) ent forms of Pho85p may receive differential inputs to explain the connection among PLC1, SPL2, and the from cellular metabolism, which allows the cell to as- regulators of PHO gene expression that have been un- sess globally its nutritional and physiological state so covered by the genetic interactions presented here. In the rate of growth and cell division can be adjusted ac- the absence of PLC1 function, a pathway that promotes cordingly. PIP2 has roles in membrane structure, secre- growth at elevated temperature and a pathway that tion (through modulation of the activity of Arf pro- controls nutrient uptake and/or utilization cannot teins; Brown et al. 1993; Waksman et al. 1996), and function properly because the activity of the Pho80p/ actin cytoskeleton dynamics (through binding to the 46 J. S. Flick and J. Thorner yeast homologs of profilin, cofilin, and gelsolin; Pol- cineurin as a key signaling enzyme in T-lymphocyte activation. lard et al. 1994). Therefore, cells may monitor the sup- Nature 357: 695–697. Creasy, C. L., S. L. Madden and L. W. Bergman, 1993 Molecular ply of PIP2 via its Plc1p-mediated turnover and inte- analysis of the PHO81 gene of Saccharomyces cerevisiae. Nucleic Ac- grate this information with the activity of the Pho80p/ ids Res. 21: 1975–1982. Davis, T. N., M. S. Urdea, F. R. Masiarz and J. Thorner, 1986 Pho85p Cdk. In this way, proper coordinate regulation Isolation of the yeast calmodulin gene: calmodulin is an essential of the rate of cell growth (which requires amino acids protein. Cell 47: 423–431. for protein synthesis and lipids for membrane expan- Drayer, A. L., J. Van der Kaay, G. W. Mayr and P. Van Haastert, 1994 Role of phospholipase C in Dictyostelium: formation of sion) and commitment to the cell cycle may be achieved. inositol 1,4,5-trisphosphate and normal development in cells lacking phospholipase C activity. EMBO J. 13: 1601–1609. We thank Erin O’Shea, Lawrence Bergman, Vivian Measday, Elder, R. T., E. Y. Loh and R. W. Davis, 1983 RNA from the yeast Brenda Andrews and Kim Arndt for the generous gifts of plasmids transposable element Ty1 has both ends in the direct repeats, a and strains, J. Michael Bishop for the gift of hybridoma cell line structure similar to retrovirus RNA. Proc. Natl. Acad. Sci. USA c-myc 9E10, Linda Riles, Maynard Olson, and Mark Johnston for 80: 2432–2436. Evan, G. I., G. K. Lewis, G. Ramsay J. M. Bishop, information and advice during the physical mapping of SPL1/PHO81 and 1985 Isolation and SPL2, Mark Lindsay for excellent technical assistance, Kathi of monoclonal antibodies specific for human c-myc proto-onco- Glauner gene product. Mol. Cell. Biol. 5: 3610–3616. for assistance in the analysis of SPL2 gene expression, Fankhauser, H., M. Schweingruber, E. Edenharter M. Randy Schekman and for the use of his Phosphorimager, and members Schweingruber, 1995 Growth of a mutant defective in a puta- Chau V. Huynh of the Thorner lab for helpful discussions, especially tive phosphoinositide-specific phospholipase C of Schizosaccharo- and Elisabeth A. Schnieders. myces pombe is restored by low concentrations of phosphate and This work was supported by a postdoctoral fellowship (DRG-1163) inositol. Curr. Genet. 28: 199–203. from the Cancer Research Fund of the Damon Runyon-Walter Feng, J.-F., S. G. Rhee and M.-J. Im, 1996 Evidence that phospholi- Winchell Foundation, funds from the Department of Biochemistry, pase ␦1 is the effector in the Gh(transglutaminase II)-mediated Vanderbilt University, and a National Scientist Development Grant signalling. J. Biol. Chem. 271: 16451–16454. Fisher, F., P.-S. Jayaraman C. Goding, (AHA-9630327N) from the American Heart Association (to J.S.F.), by and 1991 C-Myc and the yeast transcription factor PHO4 share a common CACGTG-bind- funds provided by the Berkeley campus Lucille P. Markey Program in ing motif. Oncogene 6: 1099–1104. Biomolecular Structure and Design, by facilities supplied by the Can- Flick, J. S., and M. Johnston, 1990 Two systems of glucose repres- cer Research Laboratory of the University of California, Berkeley, and sion of the GAL1 promoter in Saccharomyces cerevisiae. Mol. Cell. by National Institutes of Health research grant GM21841 (to J.T.). Biol. 10: 4757–4769. Flick, J., and J. Thorner, 1993 Genetic and biochemical characterization of a phosphatidyl-inositol-specific phospholipase C in Sac- charomyces cerevisiae. Mol. Cell. Biol. 13: 5861–5876. LITERATURE CITED Franz, B., E. Norby, G. Bren, N. Steffan, C. Paya et al., 1994 Calcineurin acts in synergy with PMA to inactivate IkB/MAD3, Altschul, S., W. Gish, W. Miller, E. Myers and D. Lipman, an inhibitor of NF-kB. EMBO J. 13: 861–870. 1990 Basic local alignment search tool. J. Mol. Biol. 215: 403– Gietz, R. D., and R. H. Schiestl, 1991 Applications of high effi- 410. ciency lithium acetate transformations of intact yeast cells using Ausubel, F., R. Brent, R. Kingston, J. Moore, J. Seidman et al., single-stranded nucleic acids as carrier. Yeast 7: 253–263. 1994 Current Protocols in Molecular Biology, Greene Publishing Gilliquet, V., and G. Berben, 1993 Positive and negative regula- Associates/Wiley-Interscience, New York. tors of the Saccharomyces cerevisiae “PHO system” participate in Benton, B. M., J.-H. Zang and J. Thorner, 1994 A novel FK506- several cell functions. FEBS Lett. 108: 333–340. and rapamycin-binding protein (FPR3 gene product) in the Guan, K., C. Jenkins, Y. Li, M. Nichols, X. Wu et al., 1994 Growth yeast Saccharomyces cerevisiae is a proline rotamase localized to the suppression by p18, a p16INK4/MTS1- and p14INK4B/MTS2- nucleolus. J. Cell Biol. 127: 623–639. related CDK6 inhibitor, correlates with wild-type pRb function. Berridge, M. J., 1993 Inositol trisphosphate and calcium signal- Genes Dev. 8: 2939–2952. ling. Nature 361: 315–325. Henikoff, S., 1984 Unidirectional digestion with exonuclease III Biggin, M. D., T. J. Gibson and G. F. Hong, 1983 Buffer gradient creates targeted breakpoints for DNA sequencing. Gene 28: gels and 35S label as an aid to rapid DNA sequence determina- 351–359. tion. Proc. Natl. Acad. Sci. USA 80: 3963–3965. Hill, C., and R. Treisman, 1995 Transcriptional regulation by ex- Bisson, L., and J. Thorner, 1982 Mutations in the PHO80 gene tracellular signals: mechanisms and specificity. Cell 80: 199–211. confer permeability to 5Ј-mononucleotides in Saccharomyces cere- Hill, J. E., A. M. Myers, T. J. Koerner and A. Tzagoloff, visiae. Genetics 102: 341–359. 1986 Yeast/E. coli shuttle vectors with multiple unique restric- Bloomquist, B. T., R. D. Shortridge, S. Schneuwly, M. Perdew, C. tion sites. Yeast 2: 163–167. Montrell et al., 1988 Isolation of a putative phospholipase C Homma, Y., and Y. Emori, 1995 A dual functional signal mediator gene of Drosophilia, norpA, and its role in phototransduction. showing RhoGAP and phospholipase C-␦ stimulating activities. Cell 54: 723–733. EMBO J. 14: 286–291. Brown, H., S. Gutowski, C. Moomaw, C. Slaughter and P. Stern- Ji, Q.-S., G. Winnier, K. Niswender, D. Horstman, R. Wisdom et al., weis, 1993 ADP-ribosylation factor, a small GTP-dependent 1997 Essential role of the tyrosine kinase substrate phospholi- regulatory protein, stimulates phospholipase D activity. Cell 75: pase C-␥1 in mammalian development. Proc. Natl. Acad. Sci. 1137–1144. USA 94: 2999–3003. Carlson, M., and D. Botstein, 1982 Two differentially regulated Johnston, M., and M. Carlson, 1992 Regulation of carbon and mRNAs with different 5Ј ends encode secreted with intracellular phosphate utilization, pp. 193–282 in The Molecular and Cellular forms of yeast invertase. Cell 28: 145–154. Biology of the Yeast Saccharomyces, edited by E. Jones, J. Pringle Chan, F., J. Zhang, L. Cheng, D. Shapiro and A. Winoto, 1995 and J. Broach. Cold Spring Harbor Laboratory Press, Cold Identification of human and mouse p19, a novel CDK 4 and Spring Harbor, NY. CDK6 inhibitor with homology to p16INK4. Mol. Cell. Biol. 15: Jones, E. W., 1991 Tackling the protease problem in Saccharomyces 2682–2698. cerevisiae. Methods Enzymol. 194: 428–453. Chu, G., D. Vollrath and R. W. Davis, 1986 Separation of large Jones, J. S., and L. Prakash, 1990 Yeast Saccharomyces cerevisiae se- DNA molecules by contour-clamped homogeneous electric lectable markers in pUC18 polylinkers. Yeast 6: 363–366. fields. Science 234: 1582–1585. Kaffman, A., I. Herskowitz, R. Tjian and E. O’Shea, 1994 Phos- Clapham, D. E., 1995 Calcium signaling. Cell 80: 259–268. phorylation of the transcription factor PHO4 by a cyclin-CDK Clipstone, N. A., and G. R. Crabtree, 1992 Identification of cal- complex, PHO80-PHO85. Science 263: 1153–1156. Plc1p Function and the Pho80p/Pho85p Cdk in Yeast 47

Koch, C. A., D. Anderson, M. F. Moran, C. Ellis and T. Pawson, ing: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1991 SH2 and SH3 domains: elements that control interac- Cold Spring Harbor, NY. tions of cytoplasmic signalling proteins. Science 252: 668–674. Santos, R. C., N. C. Waters, C. L. Creasy and L. W. Bergman, Lee, C. H., D. Park, D. Wu, S. G. Rhee and M. I. Simon, 1992 Mem- 1995 Structure-function relationships of the yeast cyclin-depen- bers of the Gq ␣ subunit gene family activate phospholipase C ␤ dent protein kinase, PHO85. Mol. Cell. Biol. 15: 5482–5491. isozymes. J. Biol. Chem. 267: 16044–16047. Schneider, K. R., R. L. Smith and E. K. O’Shea, 1994 Phosphate- Lee, S. B., S. G. Rhee, and 1995 Significance of PIP2 hydrolysis and regulated inactivation of the kinase PHO80-PHO85 by the CDK regulation of phospholipase C isozymes. Curr. Opin. Cell Biol. 7: inhibitor PHO81. Science 266: 122–125. 183–189. Schomerus, C., and H. Kuntzel, 1992 CDC25-dependent induc- Li, R., S. Waga, G. Hannon, D. Beach and B. Stillman, 1994 Dif- tion of inositol 1,4,5-trisphosphate and diacylglycerol in Saccharo- ferential effects by the p21 CDK inhibitor on PCNA-dependent myces cerevisiae by nitrogen. FEBS Letts. 307: 249–252. DNA replication and repair. Nature 371: 534–537. Schurr, A., and E. Yagil, 1971 Regulation and characterization of Martin, G., D. Viskochil, G. Bollag, P. McCabe, W. Crosier et al., acid and alkaline phosphatase in yeast. J. Gen. Microbiol. 65: 1990 The GAP-related domain of the neurofibromatosis type 1 291–303. gene product interacts with ras p21. Cell 63: 843–849. Serrano, M., G. J. Hannon and D. Beach, 1993 A new regulatory Measday, V., L. Moore, R. Retnakaran, J. Lee, M. Donoviel et al., motif in cell-cycle control causing specific inhibition of cyclin 1997 A family of cyclin-like proteins that interact with the D/CDK4. Nature 366: 704–707. Pho85 cyclin-dependent kinase. Mol. Cell. Biol. 17: 1212–1223. Sherr, C. J., and J. M. Roberts, 1995 Inhibitors of mammalian G1 Nasmyth, K., and K. Tatchell, 1980 The structure of transposable cyclin- dependent kinases. Genes Dev. 9: 1149–1163. yeast mating type loci. Cell 19: 753–764. Sikorski, R. S., and P. Hieter, 1989 A system of shuttle vectors and Nishizuka, Y., 1992 Intracellular signaling by hydrolysis of phos- yeast host strains designed for efficient manipulation of DNA in pholipids and activation of protein kinase C. Science 258: 607– Saccharomyces cerevisiae. Genetics 122: 19–27. 614. Swartzman, E. E., M. N. Viswanathan and J. Thorner, 1996 The O’Keefe, S., J. Tamura, R. Kincaid, M. Tocci and E. O’Neil, PAL1 gene product is a peroxisomal ABC transporter in the yeast 1992 FK506- and CsA-sensitive activation of the interleukin-2 Saccharomyces cerevisiae. J. Cell Biol. 132: 549–563. promoter by calcineurin. Nature 357: 692–694. Tanaka, K., M. Nakafuku, T. Satoh, J. Marshall, J. Gibbs et al., O’Neil, E., A. Kaffman, E. Jolly and E. O’Shea, 1996 Regulation 1990 S. cerevisiae genes IRA1 and IRA2 encode proteins that of PHO4 nuclear localization by the PHO80-PHO85 cyclin-CDK may be functionally equivalent to mammalian ras GTPase activat- complex. Science 271: 209–212. ing proteins. Cell 60: 803–807. Ogawa, N., K. Noguchi, Y. Yamashita, T. Yasuhara, N. Hayashi et Timblin, B. K., K. Tatchell and L. W. Bergman, 1996 Deletion of al., 1993 Promoter analysis of the PHO81 gene encoding a 134 the gene encoding the cyclin-dependent kinase Pho85 alters gly- kDa protein bearing ankyrin repeats in the phosphatase regulon cogen metabolism in Saccharomyces cerevisae. Genetics 143: 57–66. of Saccharomyces cerevisiae. Mol. Gen. Genet. 238: 444–454. Toh-e, A., and T. Shimauchi, 1986 Cloning and sequencing of the Ogawa, N., K.-I. Noguchi, H. Sawai, Y. Yamashita, C. Yompakdee PHO80 gene and CEN15 of Saccharomyces cerevisaie. Yeast 2: 129– et al., 1995 Functional domains of Pho81p, an inhibitor of 139. Toh-e, A., Y. Ueda, S. Kakimoto Y. Oshima, Pho85p protein kinase, in the transduction pathway of Pi signals and 1973 Isolation and in Saccharomyces cerevisiae. Mol. Cell. Biol. 15: 977–1004. characterization of acid phosphatase mutants in Saccharomyces Payne, W., and M. Fitzgerald-Hayes, 1993 A mutation in PLC1, a cerevisiae. J. Bacteriol. 122: 727–738. candidate phosphoinositide-specific phospholipase C gene from Touhara, K., J. Inglese, J. Pitcher, G. Shaw and R. Lefkowitz, Saccharomyces cerevisiae, causes aberrant mitotic chromosome seg- 1994 Binding of G protein ␤␥-subunits to pleckstrin homology regation. Mol. Cell. Biol. 13: 4351–4364. domains. J. Biol. Chem. 269: 10217–10220. Peter, M., and I. Herskowitz, 1994 Direct inhibition of the yeast Towler, D., J. Gordon, S. Adams and L. Glaser, 1988 The biology cyclin-dependent kinase Cdc28-Cln by Far1. Science 265: 1228– and enzymology of eukaryotic protein acylation. Annu. Rev. Bio- 1231. chem. 57: 69–99. Pollard, T., S. Almo, S. Quirk, V. Vinson and E. Lattman, Valius, M., and A. Kazlauskas, 1993 Phospholipase C-␥1 and phos- 1994 Structure of actin-binding proteins: insights about func- phatidylinositol 3 kinase are the downstream mediators of the tion at atomic resolution. Annu. Rev. Cell. Biol. 10: 207–249. PDGF receptor’s mitogenic signal. Cell 73: 321–334. Riles, L., J. E. Dutchik, A. Baktha, B. K. McCauley, E. C. Thayer et Waksman, M., Y. Eli, M. Liscovitch and J. Gerst, 1996 Identifica- al., 1993 Physical maps of the six smallest chromosomes of Sac- tion and characterization of a gene encoding phospholipase D charomyces cerevisiae at a resolution of 2.6 kilobase pairs. Genetics activity in yeast. J. Biol. Chem. 271: 2361–2364. 134: 81–150. Weiss, A., and D. R. Littman, 1994 Signal transduction by lympho- Robzyk, K., and Y. Kassir, 1992 A simple and highly efficient pro- cyte antigen receptors. Cell 76: 263–274. cedure for rescuing autonomous plasmids from yeast. Nucleic Wu, D., A. Katz and M. Simon, 1993 Activation of phospholipase C Acids Res. 20: 3790. ␤2 by the ␣ and ␤␥ subunits of trimeric GTP-binding protein. Rose, M. D., F. Winston and P. Hieter, 1990 Methods in Yeast Genet- Proc. Natl. Acad. Sci. USA 90: 5297–5301. ics: A Laboratory Course Manual, Cold Spring Harbor Laboratory Yoko-o, T., Y. Matsui, H. Yagisawa, H. Nojima, I. Uno et al., Press, Cold Spring Harbor, NY. 1993 The putative phosphoinositide-specific phospholipase C Rothstein, R. J., 1983 One-step gene disruption in yeast. Methods gene, PLC1, of the yeast Saccharomyces cerevisiae is important for Enzymol. 101: 202–211. cell growth. Proc. Natl. Acad. Sci. USA 90: 1804–1808. Salama, S. R., K. B. Hendricks and J. Thorner, 1994 G1 cyclin Yoshida, K., N. Ogawa and Y. Oshima, 1989 Function of the PHO degradation: the PEST motif of yeast Cln2 is necessary, but not regulatory genes for repressible acid phosphatase synthesis in sufficient, for rapid protein turnover. Mol. Cell. Biol. 14: 7953– Saccharomyces cerevisiae. Mol. Gen. Genet. 217: 40–46. 7966. Sambrook, J., E. F. Fritsch and T. Maniatis, 1989 Molecular Clon- Communicating editor: M. Johnston