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

Corresponding author: Jeffrey S. Flick, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232-0146, flickjs{at}ctrvax.vanderbilt.edu (E-mail).

Communicating editor: M. JOHNSTON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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.

TRANSMEMBRANE signaling in eukaryotes frequently involves activation of a phosphoinositide-specific phospholipase C (PI-PLC), which hydrolyzes phosphatidylinositol 4,5-bis phosphate (PIP₂) and generates two second messengers, inositol 1,4,5-tris phosphate (IP₃) and 1,2-diacylglycerol (DAG; LEE and RHEE 1995 ). Three classes of PI-PLC (ß, , and ) have been characterized at the molecular level; each contains a conserved catalytic domain comprised of two segments, designated X and Y, as well as nonconserved segments that confer distinct modes of regulation. Members of the PI-PLC ß family can be stimulated by binding of the G (LEE et al. 1992 ) and Gß (TOUHARA et al. 1994 ; WU et al. 1993 ) subunits released upon activation of G protein–coupled receptors. Stimulation of the PI-PLC family depends on SH2 domains, which mediate interaction with and phosphorylation by receptor-tyrosine kinases (KOCH et al. 1991 ). In contrast, regulation of the PI-PLC family is less well characterized, although it has been reported that a mammalian PI-PLC can be stimulated in vitro by a GTPase-activating protein (GAP) for the small G protein Rho (HOMMA and EMORI 1995 ) and by interaction with a novel G_h GTPase (FENG et al. 1996 ).

Cellular responses after PIP₂ hydrolysis and production of IP₃ and DAG depend on the cell type and include proliferation, differentiation, and secretion (BERRIDGE 1993 ; VALIUS and KAZLAUSKAS 1993 ; WEISS and LITTMAN 1994 ). In mammalian cells, IP₃ binds to intracellular receptors, stimulating the release of sequestered Ca²⁺, thereby activating Ca²⁺- and calmodulin-regulated protein kinases and phosphoprotein phosphatases (CLAPHAM 1995 ). DAG remains in the membrane, where it can activate members of the protein kinase C (PKC) family (NISHIZUKA 1992 ). Both second messenger pathways can elicit changes in gene expression (CLIPSTONE and CRABTREE 1992 ; FRANZ et al. 1994 ; HILL and TREISMAN 1995 ; O'KEEFE et al. 1992 ).

In animal cells, the role of any given PI-PLC isoform is difficult to assess because of the multiplicity of PI-PLC isotypes present. Moreover, the precise function of PIP₂ turnover in any given cellular response after receptor activation is complicated because of the simultaneous recruitment of additional distinct signaling systems, for example, the Ras- and PI-3 kinase–dependent pathways (VALIUS and KAZLAUSKAS 1993 ). Genetic approaches have revealed cell type–specific requirements for particular PI-PLC isozymes. A Drosophila mutant (norpA) deficient in a PI-PLC ß4 isoform lacks light-stimulated membrane potential in its photoreceptor cells and is blind (BLOOMQUIST et al. 1988 ), defining a role for PIP₂ mobilization by this PI-PLC ß isoform in insect phototransduction. Disruption of the mouse PI-PLC 1 gene results in embryonic lethality, indicating an essential requirement for this isoform in multicellular development (JI et al. 1997 ). In contrast, loss of a PI-PLC homolog in Dictyostelium discoideum did not result in any apparent phenotype (DRAYER et al. 1994 ); consequently, the role of phosphoinositide turnover by PI-PLC in this organism remains unknown.

As a means to elucidate cellular functions dependent on the activity of a PI-PLC , we have also undertaken a genetic analysis using the budding yeast Saccharomyces cerevisiae. We (FLICK and THORNER 1993 ) and others (PAYNE and FITZGERALD-HAYES 1993 ; YOKO-O et al. 1993 ) isolated a yeast gene, PLC1, that encodes a homolog of mammalian PI-PLC isoforms. Moreover, we (FLICK and THORNER 1993 ) demonstrated that Plc1p is a PI-PLC, that its activity in vitro is Ca²⁺-dependent and that, as observed for the mammalian enzymes, its substrate selectivity is influenced by the concentration of Ca²⁺. Yeast cells lacking Plc1p are viable, but grow slowly at 30° (and below), and display several other phenotypes, including temperature-sensitive lethality (at 34° and above; FLICK and THORNER 1993 ; PAYNE and FITZGERALD-HAYES 1993 ), sensitivity to hyperosmotic conditions (FLICK and THORNER 1993 ), missegregation of chromosomes during mitosis (PAYNE and FITZGERALD-HAYES 1993 ), and poor utilization of carbon sources other than glucose (FLICK and THORNER 1993 ; PAYNE and FITZGERALD-HAYES 1993 ). In at least one genetic background, a plc1 null allele is lethal (YOKO-O et al. 1993 ).

As one approach for identifying the roles that Plc1p plays in cellular physiology, we isolated and characterized two genes, which, when present in high-copy number, bypass the requirement for Plc1p activity for growth at 35°. One of the genes, PHO81, is a critical regulatory factor of the PHO regulon (SCHNEIDER et al. 1994 ). The second gene, SPL2, encodes a novel protein that is regulated in part by the PHO regulon. In S. cerevisiae, response to phosphate starvation involves the PHO81-dependent inhibition (SCHNEIDER et al. 1994 ) of a cyclin-dependent protein kinase (Cdk) comprised of the PHO80 and PHO85 gene products (KAFFMAN et al. 1994 ; SANTOS et al. 1995 ). Pho81p action reduces the inhibitory phosphorylation of the Pho4p transcription factor, thereby allowing high-level expression of genes encoding secreted acid phosphatase (PHO5), other phosphatases, phosphate transporters, and other products required for efficient phosphate assimilation (JOHNSTON and CARLSON 1992 ).

The findings presented here suggest, first, that Plc1p (or, more likely, the products of the reaction it catalyzes) acts to antagonize, directly or indirectly, either the Pho80p/Pho85p Cdk or one of the downstream substrates of this protein kinase. Second, our results indicate that the target of Plc1p and Pho80p/Pho85p action is a novel factor, not the Pho4p transactivator. Finally, the genetic interactions we have uncovered among PLC1, SPL2, PHO81, and PHO80/PHO85 show that the products of these genes participate in overlapping regulatory pathways necessary for adaptation to changing nutrient and temperature conditions.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains, growth conditions, and general methods:
The yeast strains used in this study are listed in Table 1. Strain construction followed standard methods (ROSE et al. 1990 ). Yeast was grown on agar plates containing either YPGlc medium (1% yeast extract, 2% peptone, 2% glucose) or a synthetic complete medium (SCGlc) that contained (per liter): 1.7 g Yeast Nitrogen Base without amino acids or ammonium sulfate (Difco, Detroit, MI); 5 g (NH₄)₂SO₄, 20 g glucose, 20 mg each uracil and L-Arg, 30 mg each L-Tyr and L-Ile, 40 mg adenine sulfate, 50 mg L-Phe, 60 mg L-His, 100 mg each L-Glu and L-Asp, 150 mg each L-Met and L-Val, 200 mg of L-Thr, 260 mg L-Leu, and 400 mg L-Ser. Either uracil or leucine was omitted from SCGlc plates to provide selection for the maintenance of plasmids. Phosphate-depleted YPGlc and SCGlc-Ura were prepared as described elsewhere (BISSON and THORNER 1982 ). Plasmids were introduced into yeast using the lithium acetate transformation procedure in the presence of carrier DNA (GIETZ and SCHIESTL 1991 ). Conventional methods were used for the construction and manipulation of all plasmids (AUSUBEL et al. 1994 ); Escherichia coli DH5F' (GIBCO BRL, Bethesda, MD) was used for the propagation of all plasmids.

Selection of high- copy suppressors:
Strain YJF32 (plc11::HIS3) was transformed with an S. cerevisiae genomic DNA library (NASMYTH and TATCHELL 1980 ), carried in a 2-µm DNA vector (YEp13), and plated on SCGlc-Leu plates. After incubation at 23° for 12 hr, the plates were transferred to 35° for 5 days. From ~20,000 transformants, two colonies were obtained. Plasmids were rescued from these two yeast clones in bacteria (ROBZYK and KASSIR 1992 ) and were retested for their suppressor activity by transformation of YJF32 and examination of the resulting transformants for their ability to grow as isolated single colonies at 35°. One plasmid, pJF115, was able to suppress the temperature-sensitive phenotype of the plc1 cells and did not contain the PLC1 gene itself, as determined by restriction endonuclease cleavage site analysis. The insert DNA in pJF115 was designated SPL1. The selection for dosage suppressors was repeated with a different recipient strain, YJF132 (plc12::LEU2), and by using another S. cerevisiae genomic library (CARLSON and BOTSTEIN 1982 ) carried on a different 2-µm vector (YEp24). From ~18,000 transformants, three colonies grew at 35°. After recovery in bacteria and retesting, all three plasmids were found to reproducibly rescue the temperature-sensitive growth of YJF132 at 35°. As judged by restriction enzyme analysis, one plasmid carried the PLC1 gene. The other two plasmids, pJF116 and pJF117, contained overlapping fragments of genomic DNA that were not the same as either PLC1 or SPL1. The insert DNA in pJF116 and pJF117 was designated SPL2. Both SPL1- and SPL2-containing plasmids were able to suppress the temperature-sensitive phenotype of yeast containing either the plc11::HIS3 or plc12::LEU2 null allele.

Plasmid constructions:
The SPL1-containing insert in pJF115 was ~10 kb. Initial attempts to retrieve conveniently a functional subclone were unsuccessful. Therefore, restriction enzyme digestion and deletion analysis (SAMBROOK et al. 1989 ) were used to localize the complementing region within the insert. The DNA sequence adjacent to a BamHI restriction site that fell within the region responsible for suppressor activity was determined by standard methods (BIGGIN et al. 1983 ). The nucleotide sequence obtained was identical to that reported for the PHO81 gene (EMBL/GenBank accession number D13228; OGAWA et al. 1993 ). On this basis, a 4.7-kb DNA fragment containing the entire PHO81 gene and its promoter (from the HindIII site at position -1011 to the AvaII site at position +3726, relative to the first base of the ATG initiator codon of the PHO81 open reading frame) was inserted into the HindIII and SmaI sites of the vector YEp351 (HILL et al. 1986 ). The resulting plasmid was found to contain full SPL1 suppressor activity and was designated pJF243.

A 1.8-kb BamHI-Sal I fragment containing SPL2 suppressor activity was subcloned from the insert in plasmid pJF117 and ligated into the vector YEp351, which had been cleaved with BamHI and Sal I to yield plasmid pJF181. The complete nucleotide sequence on both strands of this 1.8-kb genomic segment was determined using a Sequenase kit (United States Biochemical, Cleveland, OH) and conditions recommended by the supplier from a series of nested deletions that were generated by a procedure described previously (HENIKOFF 1984 ). DNA sequences were compiled and analyzed using Lasergene software (DNASTAR Inc., Madison, WI). Databases were searched using the BLAST algorithm (ALTSCHUL et al. 1990 ).

To express the SPL2 coding sequence from the ADH1 promoter, a 0.5-kb NcoI-NdeI fragment from pJF181 (containing nucleotides -1 to +499 relative to the first base of the ATG of the SPL2 open reading frame) was converted to blunt ends by treatment with the Klenow fragment of Escherichia coli DNA polymerase I and all four deoxyribonucleotide triphosphates, and it was then inserted into the SmaI site of vector pAD4M (MARTIN et al. 1990 ). To create an in-frame fusion containing a 17-residue c-myc epitope (-IEEQKLISEEDLLRKRD-cooh; EVAN et al. 1985 ) attached to the C-terminal residue (codon 148) of Spl2, a 1.2-kb EcoRV-BspHI fragment was excised from plasmid pJF181, converted to blunt ends, and ligated into a derivative of pBluescript containing the Myc tag coding sequence (pBS-MycTag; constructed by D. MA) that had been digested with EcoRI and treated with mung bean nuclease. A 1.3-kb BamHI-Sal I fragment (nucleotides -725 to +445 of the SPL2 coding region) containing the resulting SPL2-myc fusion was subcloned into BamHI- and SalI-digested YEp352 (HILL et al. 1986 ), yielding plasmid pJF264. To create plasmid pJF184, a 3.4-kb BamHI fragment containing the entire PLC1 gene (FLICK and THORNER 1993 ) was ligated into BamHI-digested YEp351.

Physical mapping:
The SPL1 and SPL2 loci were assigned to their respective chromosomes by hybridization of appropriate ³²P-labeled internal probes corresponding to each gene (3-kb BamHI-BamHI fragment of pJF115 and 3.5-kb BamHI-BamHI fragment of pJF116, respectively) to whole yeast chromosomes that had been separated by pulsed-field gel electrophoresis (CHU et al. 1986 ) and transferred to a nylon membrane (a gift from G. ANDERSON). The positions of SPL1 and SPL2 were located more precisely by hybridization of the same probes to nitrocellulose filters (gift from L. RILES and M. OLSON) containing an ordered set of yeast genomic DNA segments inserted into a bacteriophage vector (RILES et al. 1993 ).

Construction of null mutations:
The one-step gene replacement method (ROTHSTEIN 1983 ) was used to introduce all mutant alleles into their corresponding chromosomal loci. To inactivate the PHO81 gene, plasmid pPHO81::TRP1B (gift from E. O'NEIL and E. O'SHEA) was digested with NcoI and ApaI, and the resulting fragment (in which codons 35–421 of the PHO81 coding sequence have been removed and substituted with the TRP1 gene) was used to transform YPH499 to tryptophan prototrophy. To disrupt the SPL2 gene, the 149-bp NcoI-BstEII fragment in plasmid pJF181 (codons 1–49 of the SPL2 open reading frame) was excised and replaced with a 2.7-kb fragment containing the HIS3 gene derived from plasmid pJJ217 (JONES and PRAKASH 1990 ) to create plasmid pJF230. Plasmid pJF230 was digested with EcoRV and HpaI, and the released fragment was used to transform a diploid strain, YPH501 (SIKORSKI and HIETER 1989 ), to histidine prototrophy; sporulation and tetrad dissection of a resulting diploid transformant yielded haploid segregants containing the spl2::HIS3 allele. To eliminate the PHO4 gene, plasmid pPHO4dv (gift from E. O'SHEA) was digested with SacI and XhoI, and the resulting fragment (in which codons 1–309 of the PHO4 coding sequence have been removed and substituted with the TRP1 gene) was used to transform YPH499 to tryptophan prototrophy. Proper integration of all gene transplacements 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)⁺ 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-P_i YPGlc medium. Electrophoresis, membrane blotting, and hybridization of RNA were conducted 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 +1 to +440 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-P_i 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

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Selection of dosage suppressors:
Inability of plc1 cells to grow at 35° 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 35°. This way, two separate genes, designated SPL1 and SPL2 (for suppressor of plc1), that were distinct from PLC1 were identified (Figure 1). At 35°, the growth of plc1 cells supported by high-copy SPL1 and SPL2 was less vigorous than that sustained by the authentic PLC1 gene (Figure 1). At 37°, neither SPL1 nor SPL2 could rescue the lethality of plc1 mutants. Another phenotype of plc1 cells is an inability to grow under conditions of osmotic stress (FLICK and THORNER 1993 ). High-copy SPL2, but not SPL1, was able to restore growth to the plc1 mutant on a hypertonic medium (Figure 1, bottom). Physical mapping (see MATERIALS AND METHODS) and nucleotide sequence analysis (see below) confirmed that SPL1 and SPL2 were distinct loci from PLC1.

View larger version (60K): [in this window] [in a new window]   Figure 1. —Dosage suppressors of the temperature-sensitive phenotype of plc1 cells. Strain YJF32 (plc1), transformed with vector alone (YEp351), or the same vector carrying PLC1 (pJF184), or carrying the suppressors SPL1 (pJF243) or SPL2 (pJF181), as indicated, were streaked on selective medium (SCGlc-Leu) and incubated at either 25° for 4 days (top plate) or at 35° for 5 days (middle plate) or SCGlc-Leu medium containing 1.2 M sorbitol and incubated for 5 days at 25° (bottom plate).

Identification of SPL1 as the PHO81 gene:
The nucleotide sequence of a restriction fragment derived from SPL1 plasmid insert corresponded to the published sequence of the PHO81 gene, a previously characterized locus encoding a positive regulator of PHO5 (repressible acid phosphatase) gene expression (TOH-E et al. 1973 ). Using the known PHO81 gene sequence (OGAWA et al. 1993 ), the portion of the SPL1-containing plasmid insert that contained only the PHO81 coding region and its promoter was subcloned (Figure 2). This DNA fragment carried on a high-copy vector (pJF243; Figure 2) permitted the growth of plc1 cells at 35° just as well as the original plasmid isolate. To confirm unequivocally that the SPL1 suppressor activity was provided solely by the PHO81 gene product, three mutant alleles were constructed. Two truncations of the PHO81 open reading frame (plasmids pJF202 and pJF237), even one that removed <20% of the predicted protein (codons 957–1179), eliminated SPL1 suppressor activity (Figure 2). Likewise, an allele in which we introduced a frame shift mutation at codon 557 of the PHO81 open reading frame (plasmid pJF251) also totally destroyed its SPL1 suppressor activity (Figure 2).

View larger version (11K): [in this window] [in a new window]   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 (+) or inability (-) to transform strain YJF32 (plc1) to leucine prototrophy at 35°. The insert in plasmid pJF251 contains a frameshift mutation (asterisk) introduced by cleavage with BamHI, filling in with the Klenow fragment of E. coli DNA polymerase I, and religation. Restriction sites: A, AvaII; B, BamHI; E, EcoRI; H, HindIII.

PHO81 is a phosphate-regulated inhibitor of a Cdk comprised of a cyclin protein encoded by the PHO80 gene and a protein kinase catalytic subunit encoded by the PHO85 gene (SCHNEIDER et al. 1994 ). During growth in normal media (i.e., high phosphate), the PHO4 -encoded transcriptional activator is hyperphosphorylated by the Pho80p/Pho85p complex, which leads to exclusion of Pho4p from the nucleus (O'NEIL et al. 1996 ). Starvation for phosphate stimulates Pho81 protein to inhibit Pho80p/Pho85p Cdk activity, thus allowing underphosphorylated Pho4p to accumulate in the nucleus and activate transcription of genes in the PHO regulon, including PHO5 (SCHNEIDER et al. 1994 ). It has been shown previously that increased dosage of the PHO81 gene leads to partially constitutive expression of PHO5, even in media containing abundant phosphate (YOSHIDA et al. 1989 ).

SPL1 activity of PHO81 correlates with its ability to inhibit the Pho80/Pho85 Cdk:
Our results suggested that high-copy PHO81 might suppress temperature-sensitive growth of plc1 cells by inhibiting Pho80p/Pho85p, or via subsequent induction of a gene of the PHO regulon (or both). Multicopy PHO81 was indeed capable of causing derepression of the genes of the PHO regulon in plc1 mutants because, unlike plc1 cells alone, plc1 mutants carrying SPL1 plasmids secreted elevated levels of acid phosphatase, even when grown in high phosphate medium, as assessed by a colorimeteric colony overlay assay (data not shown). Domains of Pho81p required for effective inhibition of Pho80p/Pho85p in vivo, as judged by the degree of Pho5p derepression, have been mapped previously (OGAWA et al. 1993 ). The central region of Pho81p contains six tandem ankyrin repeats, flanked on their amino-terminal side by a negatively acting domain and on their C-terminal side by a positively acting domain. Tandem ankyrin repeats are also found in p16^INK4 and in other low molecular weight inhibitors of mammalian Cdk4 and Cdk6 (SERRANO et al. 1993 ). In addition to its ankyrin repeats, a C-terminal segment of Pho81p (residues 772–1179) is required for derepression of PHO5 expression (OGAWA et al. 1995 ). A plasmid, pJF237, which expressed a form of Pho81p that retained its ankyrin repeats, but truncated the C-terminal domain (removing amino acids 957–1179), failed to suppress the temperature sensitivity of plc1 cells (Figure 2) or to derepress acid phosphatase activity (data not shown). Thus, a region of Pho81p previously defined as critical for its ability to inhibit the Pho80p/Pho85p enzyme and to cause derepression of PHO5 was also required for its SPL1 activity.

Other conditions that inhibit Pho80p/Pho85p and derepress the PHO regulon suppress the temperature-sensitive growth of plc1 mutants:
To determine if PHO81 overexpression rescued the temperature-sensitive lethality of plc1 cells solely because of its inhibition of Pho80p/Pho85p and the consequent derepression of genes under its control, we examined other conditions that cause inhibition of this Cdk. First, we simply altered the P_i concentration in the growth medium. As shown previously (FLICK and THORNER 1993 ), on a medium containing high P_i (where Pho80p/Pho85p activity is high and PHO5 expression is prevented), plc1 cells failed to grow at 35° (Figure 3A, left side). In contrast, on a medium containing low P_i (where Pho80p/Pho85p activity is low and PHO5 is expressed), plc1 cells were able to propagate at high temperature (Figure 3A, right side). Thus, like PHO81 overexpression, a natural nutritional signal that leads to inhibition of Pho80p/Pho85p and PHO5 derepression also permitted the growth of plc1 cells at elevated temperature.

View larger version (107K): [in this window] [in a new window]   Figure 3. —Nutritional and mutational inactivation of the Pho80/Pho85 Cdk suppresses the temperature-sensitive phenotype of plc1 cells. (A) Normal PLC1+ 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 described in MATERIALS AND METHODS (right plate; low [Pi]), and incubated for 3 days at 36°. 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 25° for 5 days (left plate) or at 35° for 5 days (right plate).

Second, we used another genetic manipulation to inhibit the Pho80p/Pho85p enzyme. The PHO80 gene encodes the cyclin component of this Cdk. Because it is required for activity of the kinase, Pho80p acts as a negative regulator of PHO5 expression (TOH-E and SHIMAUCHI 1986 ; YOSHIDA et al. 1989 ). Indeed, pho80 mutations cause high-level constitutive derepression of PHO5 in yeast grown in high P_i media (BISSON and THORNER 1982 ), and we observed constitutive expression of acid phosphatase in plc1 pho80 double mutants (data not shown). Like plc1 cells on low P_i medium, plc1 pho80 double mutants were able to grow at 35°, even on a high P_i medium (Figure 3B). Thus, three different conditions that lead to inactivation of the Pho80p/Pho85p Cdk and concomittant derepression of the genes under its control allowed plc1 cells to grow at a temperature that would otherwise be nonpermissive for their growth.

Disruption of the genes for several other Pho85-associated cyclins does not suppress plc1:
Pho85p has recently been shown to associate with nine other putative cyclin proteins, in addition to Pho80p, including: Pcl1p, Pcl2p, Pcl5p, Pcl6p, Pcl7p, Pcl8p, Pcl9p, Pcl10p, and Clg1p (MEASDAY et al. 1997 ). To determine if loss of other Pho85 Cdk complexes can bypass the temperature sensitivity caused by the plc1 mutation, we generated double mutants of plc1 with pcl1, pcl2, pcl5, and clg1. Unlike plc1 pho80 cells, none of these double mutants was able to grow at a restrictive temperature, indicating that suppression was not a general result of perturbed Pho85p activity or altered cyclin ratios. A plc1 pho85 double mutant also failed to grow at restrictive temperature (data not shown). This finding might mean that a Pho85p-independent activity of overproduced Pho81p (or of loss of Pho80p) is responsible for suppression of the plc1 mutation. It is more likely, however, that alteration or reduction of Pho85p activity is able to suppress the plc1 temperature-sensitive defect, whereas total elimination of all Pho85p-dependent Cdk activity has such a deleterious effect on cells as to preclude growth of a plc1 pho85 double mutant under any condition. In support of this view, pho85 mutants, which lack 10 different Cdk complexes, grow quite poorly even at 30° (GILLIQUET and BERBEN 1993 ), show defects in glycogen metabolism (TIMBLIN et al. 1996 ), and display abnormal cellular morphology (MEASDAY et al. 1997 ).

Molecular characterization of the SPL2 gene:
The SPL2 suppressor locus was delimited to a 1.8-kb BamHI-Sal I fragment by subcloning restriction fragments from one of the original plasmid isolates and retesting for suppressor activity (Figure 4A). Within this DNA fragment, nucleotide sequence analysis (EMBL/GenBank accession number P38839) delineated a 148-codon open reading frame (YHR136c) that originated from the right arm of chromosome 8 (JOHNSTON et al. 1994). Two different approaches were taken to confirm that the 148-codon open reading frame encoded the SPL2 suppressor activity. First, a frameshift mutation introduced at codon 49 abolished the suppressor activity of the subcloned BamHI-Sal I fragment (plasmid pJF217; Figure 4A). Second, expression of just the 148-codon sequence from a different promoter (ADH 1) on a high copy plasmid vector was able to suppress the temperature-sensitive growth defect of plc1 cells (plasmid pJF235; Figure 4A). Furthermore, our subsequent characterization (see below) confirmed that SPL2 encodes the predicted protein.

View larger version (97K): [in this window] [in a new window]   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 5' 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 (+) or inability (-) to transform YJF32 (plc1) to leucine prototrophy at 35°. 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 (#). Restriction sites: B, BamHI; Bs, BspHI; Bt, BstEII; N, NcoI; Nd, NdeI; R, EcoRV; and, S, Sal I. (B) Nucleotide sequence (where +1 indicates the first base of the ATG initiator codon; EMBL/GenBank 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 substitutions (TANAKA et al. 1990 ) (A=G=P=S; L=I=V=M; R=K=H; D=E; N=Q ; F=Y=W; S=T; N=D; Q=E) between Spl2p and any of the other proteins listed are given as white-on-black letters.

The deduced SPL2 gene product (Figure 4B) has a calculated molecular mass of 17 kD and, allowing for conservative amino acid replacements, is similar to the sequences of mammalian low molecular weight Cdk inhibitors and also to the ankyrin repeat region in Pho81p (Figure 4C). Similarity between Spl2p and the other proteins is as follows: p16 (29%), p18 (33%), p19 (30%), and Pho81 (27%). By comparison, Far1p, a demonstrated inhibitor of the Cdc28/Cln2 Cdk (PETER and HERSKOWITZ 1994 ), does not bear detectable homology to any mammalian Cdk inhibitor yet identified. We noted that the N-terminal 20 residues of Spl2p are comprised almost exclusively of hydrophobic and uncharged amino acids, and that Gly2 is a potential target for N-myristoylation, although Spl2 lacks other consensus residues found in efficiently myristoylated proteins (TOWLER et al. 1988 ). The remainder of the protein can be divided into two regions: residues 32–97 constitute a highly basic segment (net charge +9), and residues 98–148 comprise a highly acidic segment (net charge -11; Figure 4B). Potential phosphorylation sites for several classes of protein kinase are also present in Spl2, including cAMP-dependent protein kinase (Ser59 and Ser86), proline-directed protein kinases (Thr5, Ser30 and Thr81), and casein kinase II (Ser142 and Ser143). The functional significance of these sites has not been explored.

SPL2 gene expression and Spl2 protein level are regulated by phosphate:
The SPL2 promoter region contains three matches to the consensus (5'-CACGTG-3') for binding of the Pho4p transactivator located at positions -147, -90, and -31 (where +1 is the ATG; Figure 4B). When P_i is limiting, Pho4p is in the nucleus, binds to such sites in the upstream regions of PHO5 and the other genes of the PHO regulon, and activates transcription (FISHER et al. 1991 ). To determine if its expression is regulated by phosphate, a ³²P-labeled SPL2 probe was hybridized to size-fractionated RNA prepared from yeast grown in either a high or a low P_i medium. The internal control for loading was a probe for CMD1 mRNA, that encodes calmodulin. The SPL2 transcript was polyadenylated and long enough (~0.45 kb) to encode the SPL2 open reading frame (Figure 5A). In cells grown in high P_i medium, SPL2 mRNA was expressed at ~30% of the level of the CMD1 mRNA. After shift to low P_i medium for 2.5 hr, the steady-state level of SPL2 mRNA increased 2.7-fold (when normalized to CMD1 RNA), indicating that SPL2 expression is induced and suggesting that its potential Pho4-binding sites have a functional role. In further support that induction occurs at the transcriptional level, expression of an SPL2-lacZ fusion (containing SPL2 sequences from -724 to +3) in wild-type cells increased 4.4-fold after transfer to a low P_i medium, whereas in pho80 cells, expression was constitutively high and did not increase significantly upon transfer to low P_i medium, as expected if SPL2 is regulated by Pho4p (data not shown).

View larger version (34K): [in this window] [in a new window]   Figure 5. —SPL2 mRNA expression, Spl2 protein production and subcellular fractionation. (A) Total RNA (20 µg; lanes 1 and 2) or of poly(A)+ RNA (1 µg; lane 3) were prepared from strain YPH499 grown at 30° 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.

For detection, sequences encoding a c-Myc epitope were fused in-frame to the C terminus of the SPL2 coding region. The epitope used is recognized by the monoclonal antibody 9E10. The epitope-tagged Spl2 (Spl2myc) expressed from the SPL2 promoter suppressed the temperature sensitivity of plc1 cells as efficiently as untagged Spl2 (data not shown), demonstrating that Spl2myc was functional. Extracts of yeast expressing Spl2myc from the SPL2 promoter on a multicopy plasmid were analyzed by SDS-PAGE and immunoblotting (Figure 5B). Spl2myc migrated with a mobility corresponding to an apparent molecular mass of ~23 kD, which was somewhat larger than its calculated molecular mass (19.2 kD). However, anomalous migration has been observed for other polypeptides that are also very highly charged (BENTON et al. 1994 ; SWARTZMAN et al. 1996 ). As judged by densitometry, Spl2myc level increased about threefold in cells grown in a low P_i medium compared to its level in an equivalent amount of extract from cells grown on the same medium containing high P_i (Figure 5B), which is consistent with the increase observed in the level of the SPL2 mRNA and SPL2-lacZ expression under the same conditions.

Despite its apparent homology to other small Cdk inhibitors, the hydrophobic nature of the N terminus of Spl2 and its potential myristoylation site suggested that this segment of the protein might serve as a signal sequence or act as a membrane anchor. To determine whether Spl2 was a secretory protein or localized to a membrane-bound compartment, total extracts of cells expressing Spl2myc from its own promoter on a multicopy plasmid were fractionated by differential centrifugation into soluble and particulate material. As expected for a soluble protein, the majority of Spl2myc remained in the supernatant fraction even after prolonged centrifugation at 100,000 g (Figure 5B). A small amount of the Spl2myc was found in the pellet from the 100,000 g centrifugation. When this material was resuspended in buffer containing 1% Triton X-100 and subjected to recentrifugation, none of the Spl2myc was extracted, suggesting that the particulate Spl2myc was not membrane-associated (data not shown). While the amount of soluble Spl2myc increased in cells grown in low-P_i medium, the amount in the particulate fraction did not increase (Figure 5B).

Genetic interactions of plc1, pho81, and spl2 mutations:
One mechanism to explain how increased expression of PHO81 and SPL2 can compensate for the loss of PLC1 to permit growth at 35° is that these gene products might be components in a pathway required for growth at elevated temperature that lies downstream of PLC1 and that requires PLC1 function for its full activity. If so, then one might expect that null mutations in PHO81 and/or SPL2 might result in phenotypes that resemble those of a plc1 mutant. Unlike a plc1 mutant, however, pho81 and spl2 single mutants and pho81 spl2 double mutants grew as well as wild-type cells on SCGlc at 35° (data not shown), on hyperosmotic medium (data not shown), on synthetic medium at 25° (Figure 6A), and on glycerol-containing medium (data not shown). The pho81 mutant failed to derepress acid phosphatase activity and was unable to grow on low-P_i medium (data not shown), as observed previously by others (TOH-E et al. 1973 ). By contrast, the plc1 mutant (Figure 3A) and the spl2 mutant (data not shown) grew well on low-P_i medium and, when limited for phosphate, displayed levels of secreted acid phosphatase comparable to those seen in derepressed wild-type cells (data not shown). The lack of similarity in phenotypes between a plc1 mutant and the pho81 and spl2 single and double mutants suggests that neither PHO81 nor SPL2 function in a strictly linear pathway downstream of PLC1.

View larger version (114K): [in this window] [in a new window]   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 25° 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 25° for 4 and 5 days, respectively.

An alternative hypothesis to explain suppression of the temperature sensitivity of plc1 cells by high-level expression of PHO81 or SPL2 is that PHO81 and SPL2 act in an independent pathway(s) that is necessary for growth at elevated temperature and that overlaps (or is partially redundant in function) with the PLC1-dependent pathway. If so, double mutants in which both pathways are disrupted should display more severe defects than single mutants in which only one pathway is nonfunctional. To test this possibility, plc1 pho81 and plc1 spl2 double mutants were constructed and examined, along with the plc1 pho80 double mutant (Figure 3B). On rich medium (YPGlc) at 25°, the plc1 pho81 and plc1 spl2 double mutants grew (Figure 6B) at a rate similar to that displayed by the plc1 single mutant (Figure 6A). Unlike the plc1 single mutant (Figure 6A), however, neither the plc1 pho81 double mutant nor the plc1 spl2 double mutant was able to form visible colonies on the synthetic medium (SCGlc), even after incubation for 5 days at the same temperature (Figure 6B). In contrast, as might have been anticipated from the ability of a pho80 mutation to suppress the temperature sensitivity of a plc1 mutation (Figure 3B), the plc1 pho80 double mutant grew on the synthetic medium at a rate similar to that of the plc1 single mutant. Surprisingly, the plc1 pho80 double mutant was unable to form single colonies on rich medium after 5 days at 25°. Thus, with respect to growth on synthetic medium, the plc1 pho81 and plc1 spl2 double mutants did indeed display a much more severe phenotype than a plc1 single mutant.

If the loss of SPL2, like the loss of PHO81, leads to increased activity of the PHO80/PHO85-encoded Cdk, then the inability of the plc1 pho81 and plc1 spl2 double mutants to grow on SCGlc might be caused by hyperelevation of the Pho80/Pho85 Cdk. To test this possibility, a plc1 spl2 pho80 triple mutant was constructed. Indeed, this strain was able to grow on the synthetic medium (Figure 6B) just as well as a plc1 single mutant (Figure 6A). Conversely, this triple mutant was also able to grow well on rich medium (Figure 6B), suggesting that the inability of the plc1 pho80 cells to grow on YPGlc might be caused, directly or indirectly, by the derepression of SPL2. Taken all together, these results suggest that, in addition to their role in supporting growth at elevated temperature, the functions of PLC1, PHO81, SPL2, and the PHO80/PHO85-encoded Cdk all converge on a pathway required for proper response to elevated temperature and changing nutrient levels.

Epistasis relationships in plc1 suppression:
Raising the dosage of either PHO81 or SPL2 suppressed the temperature-sensitive growth of a plc1 mutant (Figure 1). Because SPL2 is a phosphate-regulated gene (Figure 4 and Figure 5), and because overexpression of PHO81 derepresses expression of other genes in the PHO regulon (YOSHIDA et al. 1989 ), it was possibile that high-copy PHO81 suppressed the plc1 mutation solely because of stimulation of SPL2 expression. By this model, suppression of a plc1 mutation by a pho80 mutation (Figure 3) should also require SPL2 activity. To test this hypothesis, the phenotype of the plc1 pho80 spl2 triple mutant was compared to that of an otherwise isogenic plc1 pho80 double mutant. As observed before, the plc1 pho80 double mutant was able to grow at 34°, a temperature that is nonpermissive for the plc1 single mutant (Figure 7). Likewise, the plc1 pho80 spl2 triple mutant was also able to grow at 34°, demonstrating that SPL2 is not essential for the ability of a pho80 mutation to bypass the plc1 mutation. This result further suggests that when the Pho80p/Pho85p Cdk is inactivated, the activity of a factor(s) other than SPL2 is stimulated that allows the Plc1-deficient cells to grow at an elevated temperature. On the other hand, growth of the plc1 pho80 spl2 triple mutant at 34° and 35° was discernibly weaker than that of the plc1 pho80 double mutant, indicating that elevated SPL2 expression, while not obligatory, contributes to suppression.

View larger version (111K): [in this window] [in a new window]   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.

When the Pho80p/Pho85p Cdk is inactivated, there is a dramatic increase in the transcription of PHO4 - dependent genes. Because SPL2 was not essential for suppression of the temperature-sensitive growth of a plc1 mutant by a pho80 mutation, it seemed reasonable to assume that elevated expression of another PHO4 - dependent gene(s) was responsible for the suppression since, to date, Pho4p is the only known in vivo substrate of the Pho80p/Pho85p Cdk. To determine if any genes under Pho4p control were required for suppression, a plc1 pho80 pho4 triple mutant was constructed. As expected, this strain failed to grow on a low-P_i medium and did not express detectable acid phosphatase activity (data not shown). In marked contrast, the plc1 pho80 pho4 triple mutant grew at 34° and at a rate comparable to that of the plc1 pho80 spl2 triple mutant (Figure 7). Thus, PHO4-dependent transcription was not required for suppression of the plc1 mutation when the Pho80p/Pho85p Cdk was inactivated by a pho80 mutation. Furthermore, the fact that plc1 pho80 pho4 and plc1 pho80 spl2 strains grew similarly and somewhat more weakly than the plc1 pho80 double mutant (Figure 7) provides an additional demonstration that SPL2 is under PHO4 control, and that SPL2 expression contributes to the ability of cells to grow at elevated temperatures. Nonetheless, the fact that PHO4 is not required for suppression suggests that loss of the Pho80p/Pho85p Cdk suppresses a plc1 mutation because the Pho80p/Pho85p normally antagonizes the function of an as yet unidentified factor that is required for the ability of cells to grow at elevated temperatures.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

On the basis of the pleiotropic phenotypes displayed by yeast cells lacking a PLC1 gene, we proposed that Plc1p deficiency causes defects in nutritional and stress-related responses (FLICK and THORNER 1993 ). As shown here, two genes, PHO81 and SPL2, when overexpressed, can bypass the need for PLC1 to permit growth >34°. Because elevated dosage of either gene suppressed a plc1 null allele, Pho81p and Spl2p might represent components in a pathway directly downstream of Plc1p. However, the genetic interactions of the plc1, pho81, and spl2 mutants (Figure 6) suggest that Plc1p, Pho81p, and Spl2p act independently, but in a partially redundant or overlapping manner. Thus, the functions of Plc1p, Pho81p, and Spl2p appear to converge on a common target that is required for growth at elevated temperature and proper response to nutrient levels.

When P_i is limiting, Pho81p inhibits the Pho80p/Pho85p Cdk (SCHNEIDER et al. 1994 ). Although the inhibitory action of Pho81p apparently is enhanced by low-P_i conditions, overexpression of PHO81 causes partially constitutive derepression of PHO5, even when cells are grown in high-P_i medium (YOSHIDA et al. 1989 ). Thus, Pho81p must be capable, even in its basal state, of inhibiting Pho80p/Pho85p. In fact, it has been shown by others that overexpression of Pho81p does reduce Pho80p/Pho85p activity in vitro, that growth in low-P_i medium causes a PHO81-dependent reduction in Pho80p/Pho85p activity in vitro, and that a pho80 mutation totally inactivates a PHO85-dependent kinase activity (KAFFMAN et al. 1994 ; SCHNEIDER et al. 1994 ). Hence, the observed suppression of the temperature-sensitive phenotype of plc1 cells by high-copy PHO81 (Figure 1), by growth on low-P_i medium (Figure 3A), and by a pho80 mutation (Figure 3B) is most likely caused by inhibition of Pho80p/Pho85p activity. Therefore, in a formal genetic sense, Plc1p activity may either negatively regulate the Pho80p/Pho85p Cdk or produce a signal that opposes the effect of Pho80p/Pho85p on a downstream target. The interaction of PI-PLC activity with a phosphate-regulated pathway appears to be conserved. In Schizosaccharomyces pombe, a mutation in the plc1⁺ gene, encoding a PI-PLC homolog, is partially suppressed by growth on low-P_i media (FANKHAUSER et al. 1995 ).

Pho4p is the only known in vivo target of the Pho80p/Pho85p Cdk, and phosphorylation of Pho4p by Pho80p/Pho85p prevents it from stimulating transcription by excluding Pho4p from the nucleus (O'NEIL et al. 1996 ). Hence, inactivation of Pho80p/Pho85p could perhaps allow growth of plc1 cells at elevated temperature because it permits Pho4p to stimulate the expression of a gene(s) that are required for growth at 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 (34°) that is nonpermissive for a plc1 single mutant (Figure 7). These results suggest that Pho80p/Pho85p-dependent inhibition (presumably by direct phosphorylation) of another factor, different from Pho4p, is responsible for blocking growth of plc1 cells at elevated temperature. In further support of this conclusion, high-copy PHO4 causes robust constitutive expression of PHO5 in plc1 cells, as in wild-type cells (YOSHIDA et al. 1989 ), yet it is only a very weak suppressor of the temperature sensitivity and does not suppress at all in a plc1 pho81 double mutant (J. FLICK, unpublished results). These observations indicate that the weak suppression by multicopy PHO4 is caused by elevated expression of PHO81 and possibly SPL2, which are both PHO4 -dependent genes (YOSHIDA et al. 1989 ; CREASY et al. 1993 ; Figure 5), and the resulting inhibition of the Pho80p/Pho85p Cdk (rather than caused by induction of other PHO genes).

The second plc1 suppressor was the SPL2 gene (Figure 4). SPL2 encodes a 17-kD soluble protein. Although both SPL2 mRNA (Figure 5A) and Spl2 protein (Figure 5B) appear to be expressed at a significant basal level, growth of cells in low-P_i medium caused a readily detectable induction (approximately threefold). However, suppression of the temperative-sensitive defect of plc1 cells by high-copy SPL2 did not require stimulation by low-P_i conditions because suppression was observed on high-P_i medium (Figure 1). An spl2 mutant had no readily discernible growth phenotype (Figure 6), and computer searches determined that no homolog or related gene exists in the S. cerevisiae genome. 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 ROBERTS 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 phosphorylate the novel factor, but not its ability to phosphorylate Pho4p (whereas Pho81p may block the ability of Pho80p/Pho85p to phosphorylate both this factor and Pho4). A somewhat analogous situation has been observed in animal cells where Cdk inhibition by Cip1/Waf1 inhibits the function of PCNA in DNA replication, but not in DNA repair (LI et al. 1994 ).

The interactions among PLC1, SPL2, and elements of the PHO regulon suggest that a factor critical for growth >34° is modulated by these gene products. This same factor may also be involved in nutrient sensing and/or utilization because either a pho81 or an spl2 mutation prevents the growth of plc1 cells on synthetic medium, but not on rich medium. In support of a connection between nutrient uptake and/or utilization and the functions of PLC1, SPL2, and the PHO regulon was our observation that a plc1 pho80 double mutant was sensitive to rich media, but able to grow on synthetic medium (Figure 6B). This sensitivity could result from hyperstimulation of amino acid uptake as a consequence of the loss of both the Pho80p/Pho85p Cdk and the loss of PLC1 function. The ability pho80 mutations to promote uptake of nutrients other than inorganic phosphate is highlighted by the fact that the tup7 mutation, isolated on the basis of enhanced dTMP uptake, is allelic to pho80 (BISSON and THORNER 1982 ). Another connection between nitrogen metabolism and PIP₂ turnover is suggested by a report that IP₃ and DAG levels in yeast are increased when a nitrogen source is resupplied to starved cells (SCHOMERUS and KUNTZEL 1992 ).

We propose the following working model (Figure 8) to explain the connection among PLC1, SPL2, and the regulators of PHO gene expression that have been uncovered by the genetic interactions presented here. In the absence of PLC1 function, a pathway that promotes growth at elevated temperature and a pathway that controls nutrient uptake and/or utilization cannot function properly because the activity of the Pho80p/Pho85p Cdk is too high. Plc1p activity either acts to oppose the inhibitory action of Pho80p/Pho85p on a downstream target, "X," or stimulates the function of X by a convergent pathway. Hence, reduction of Pho80p/Pho85p activity by overproduction of either Pho81p (a known inhibitor of Pho80/Pho85) or Spl2p (a candidate Cdk inhibitor), by growth on low-P_i medium or by introduction of a pho80 mutation, all permit plc1 cells to grow at restrictive temperature. Alternatively, Spl2 may act to stimulate the activity of X rather than inhibiting Pho80p/Pho85p activity. Loss of both Plc1p and Pho81p, or both Plc1p and Spl2p, presumably causes a more severe inactivation of X, explaining the inability of plc1 pho81 and plc1 spl2 double mutants to grow on standard synthetic medium, as well as the relief of this phenotype by a pho80 mutation or by augmentation with a rich growth medium. Indeed, loss of Pho81p alone has been shown to cause an elevation in Pho80p/Pho85p activity in cell extracts (SCHNEIDER et al. 1994 ). The novel factor X, and not Pho4p, is required for the induction of these growth pathways because, for example, a pho80 mutation permits growth of plc1 cells at restrictive temperature in both a PHO4⁺ and a pho4 background.