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Calcineurin-dependent growth control in Saccharomyces cerevisiae mutants lacking PMC1, a homolog of plasma membrane Ca2+ ATPases.

Ca2+ ATPases deplete the cytosol of Ca2+ ions and are crucial to cellular Ca2+ homeostasis. The PMC1 gene of Saccharomyces cerevisiae encodes a vacuole membrane protein that is 40% identical to the plasma membrane Ca2+ ATPases (PMCAs) of mammalian cells. Mutants lacking PMC1 grow well in standard media, but sequester Ca2+ into the vacuole at 20% of the wild-type levels. pmc1 null mutants fail to grow in media containing high levels of Ca2+, suggesting a role of PMC1 in Ca2+ tolerance. The growth inhibitory effect of added Ca2+ requires activation of calcineurin, a Ca2+ and calmodulin-dependent protein phosphatase. Mutations in calcineurin A or B subunits or the inhibitory compounds FK506 and cyclosporin A restore growth of pmc1 mutants in high Ca2+ media. Also, growth is restored by recessive mutations that inactivate the high-affinity Ca(2+)-binding sites in calmodulin. This mutant calmodulin has apparently lost the ability to activate calcineurin in vivo. These results suggest that activation of calcineurin by Ca2+ and calmodulin can negatively affect yeast growth. A second Ca2+ ATPase homolog encoded by the PMR1 gene acts together with PMC1 to prevent lethal activation of calcineurin even in standard (low Ca2+) conditions. We propose that these Ca2+ ATPase homologs are essential in yeast to deplete the cytosol of Ca2+ ions which, at elevated concentrations, inhibits yeast growth through inappropriate activation of calcineurin.

PMC1 gene of Saccharomyces cerevisiae encodes a vacuole membrane protein that is 40% identical to the plasma membrane Ca :÷ ATPases (PMCAs) of mammalian cells. Mutants lacking PMC1 grow well in standard media, but sequester Ca 2÷ into the vacuole at 20% of the wild-type levels, pmcl null mutants fail to grow in media containing high levels of Ca 2÷, suggesting a role of PMC1 in Ca ~÷ tolerance. The growth inhibitory effect of added Ca 2÷ requires activation of calcineurin, a Ca 2÷ and calmodulin-dependent protein phosphatase. Mutations in calcineurin A or B subunits or the inhibitory compounds FK506 and cyclosporin A restore growth of pmcl mutants in high Ca 2+ media.
Also, growth is restored by recessive mutations that inactivate the high-affinity Ca2+-binding sites in calmodulin. This mutant calmodulin has apparently lost the ability to activate calcineurin in vivo. These results suggest that activation of calcineurin by Ca 2+ and calmodulin can negatively affect yeast growth. A second Ca ~+ ATPase homolog encoded by the PMRI gene acts together with PMC1 to prevent lethal activation of calcineurin even in standard (low Ca 2+) conditions. We propose that these Ca ~+ ATPase homologs are essential in yeast to deplete the cytosol of Ca ~+ ions which, at elevated concentrations, inhibits yeast growth through inappropriate activation of calcineurin.

C
A2÷ plays a key role in the transduction of external signals through the cytoplasm of eukaryotic cells. Fluctuations in the cytosolic free Ca 2÷ concentration, [Ca2÷]c, ~ directly elicit a cellular response by altering the function of Ca2÷-binding proteins and their targets. Resting cells maintain [Ca2+]c at very low levels against large gradients of compartmentalized and extracellular Ca 2÷. A variety of stimuli can trigger the opening of Ca 2÷specific channels in plasma membrane or endoplasmic reticulum causing massive Ca 2÷ influx and accumulation in the cytoplasm. After stimulation, the basal [Ca2+]c levels are restored by Ca 2÷ ATPases and antiporters that transport Ca 2÷ from the cytoplasm through the plasma membrane and several internal membranes. To regulate [Ca2÷]c and effect the appropriate responses to Ca :÷ signals, cells utilize a wide array of ion transporters and sensory factors.
Ca 2÷ signaling plays an important role in the activation of T cells (Gardner, 1989). Binding of antigens to specific receptors at the surface of quiescent T cells triggers the opening of Ca 2÷ channels in the endoplasmic reticulum and plasma membrane, leading to rapid elevation in [Ca2÷]c.
The rise in [Ca~+]c is necessary for induction of many genes including IL-2 which produces an autocrine factor required for T cell proliferation. Ca 2÷ dependent transcription is blocked by the immunosuppressive drugs cyclosporin A and FK506 that act together with their respective binding proteins, cyclophilin and FKBP-12, as potent inhibitors of calcineurin (Liu et al., 1991a). A direct role for calcineurin in IL-2 expression is supported by the observation that overproduction of calcineurin partially bypasses the requirement for elevated [Ca~+]c and decreases the effectiveness of FK506 and cyclosporin A (Clipstone and Crabtree, 1992;O'Keefe et al., 1992). These findings strongly suggest that Ca2÷-dependent activation of calcineurin is a crucial step in the activation of T cells.
Recent reports have extended the range of cell types and species that respond to these drugs, suggesting that calcineurin may be a widespread component of Ca2÷-signaling mechanisms. Studies in the budding yeast Saccharomyces cerevisiae indicate that calcineurin is involved in the response to mating pheromone. Yeast cells respond to mating pheromones by arresting cell cycle progression transiently in G1 phase and inducing expression of many genes involved in conjugation. Cells that have not mated after prolonged pheromone exposure can recover and proliferate as long as sufficient Ca 2÷ is supplied in the medium (Iida et al., 1990). Recovery is inefficient in mutants lacking calcineurin activity (Cyert et al., 1991;Cyert and Thomer, 1992). The recovery defect of calcineurin mutants can be mimicked in wild-type strains by addition of cyclosporin A or FK506 (Foor et al., 1992). Although calcineurin is required for recovery from pheromone arrest, it is not essential for vegetative growth under standard conditions (Cyert et al., 1991;Liu et al., 1991b;Kuno et al., 1991;Cyert and Thorner, 1992).
Yeast calmodulin is essential for viability (Davis et al., 1986), but its ability to bind Ca 2+ with high affinity is not necessary for vegetative growth (Geiser et al., 1991). These findings can be explained by proposing that any positive functions of CaWcalmodulin and its targets such as calcineurin are redundant with those of other cellular factors during standard growth conditions. Alternatively, the vegetative functions of CaWcalmodulin and calcineurin could have gone undetected because the mutants were analyzed under conditions where [Ca2+]c is low, ~0.1-0.3 #M (Halachmi and Eilam, 1989;Ohya et al., 1991).
The functions of calmodulin and calcineurin could be better analyzed if [Ca2+]c was experimentally elevated above the basal levels. A way to accomplish this is by genetically manipulating the ion transporters involved in Ca 2+ homeostasis. The PMRI gene product, a member of the sarco/ endoplasmic reticulum (SERCA) family of Ca 2+ ATPases (Serrano, 1991), is thought to directly transport Ca 2+ into the Golgi complex to support a variety of secretory functions (Rudolph et al., 1989;Antebi and Fink, 1992). The ability ofpmrl mutants to tolerate variations in external Ca ~+ suggests that additional Ca ~+ transporters might be more important for controlling [Ca2+]c. The PMR2 gene product identified previously (Rudolph et al., 1989) is not related to known Ca ~+ ATPases any more than H + or Na+/K + ATPases (Serrano, 1991) and is required for Na + tolerance (Haro et al., 1991) but not Ca ~+ tolerance (K. W. Cunningham, unpublished data). However, a low-affinity H+/Ca 2+ antiport activity is present in isolated vacuole membranes (Ohsumi and Anraku, 1983). Most eukaryotic cells also express a plasma membrane Ca 2+ ATPase (PMCA) that is primarily responsible for maintaining [Ca2+]c submicromolar levels (Carafoli, 1992).
This study reports the identification of the PMC1 gene, which encodes a homolog of mammalian PMCAs, Genetic analysis suggests that the product of PMC1 (Pmclp) transports Ca ~+ into the vacuole and participates in the control of [Ca2+]c together with Pmrlp. High external Ca ~+ inhibits the growth of pmcl mutants because calcineurin becomes activated by Ca2+/calmodulin. Thus, CaWcalmodulin and calcineurin perform at least one function that prevents cell proliferation under these conditions. Genetic manipulation of PMC1 provides a valuable new approach to resolve the nature and functions of Ca :÷ signals.

Recombinant DNA
All procedures with recombinant DNA were performed using Escherichia coli strain DH5ot grown in Luria Broth medium with appropriate antibiotics (Maniatis et al., 1982). Protocols used for polymerase chain reaction (Boehringer-Mannheim, Mannheim, Germany) and sequencing (United States Biochemicals, Cleveland, OH) were performed according to manufacturer's directions. Plasrnid vectors were obtained from P. Hieter (Sikorski and Hieter, 1989 DNA sequencing, linearized by digestion with ClaI, and integrated into the chromosomal PMC1 locus leaving full-length PMCI::HA adjacent to a truncated pmd::URA3 allele. Plasmid pKC60 contains the complete epitopetagged PMCI::HA gene (from HindHI to BgllII at position 4,630) in the high copy vector B2205 which is a derivative of pRS306 containing a 2-pm origin of replication. Both single copy and high copy PMCI::HA alleles produced immunoreactive polypeptides and functionally complemented pmcl mutations for growth in high Ca 2+ media. The plasmid pKCll contains the PMRI locus (4.4-kb PvulI to SpeI fragment) from pL149 (Rudolph et al., 1989) inserted into 132205 (2 ~ URA3) digested with SmaI plus XbaI. The plasmids pKC52 and pKC59 used to replace the chromosomal PMC1 gene with pmcl::LEU2 and pmcl::TRP1, respectively, were constructed by three part ligations of the 0.58-kb HindlII/XbaI fragment and 0.88-kb XhoI/ HindI~ fragment of pKC45 into pRS305 and pRS304 digested with Xbal and XhoI. The plasmids were linearized before yeast transformation using HindlH (pKC52) or using StuI plus BgllI (pKC59). pKC38 was linearized with NruI to form the pmcl::HIS3 disruption.
The plasmid pKC'/3 is a derivative of pRS316-Gal (CEN ARS URA3) containing the GAL/ promoter region fused to the truncated CNAIAC allele which was amplified by PCR from W303-1A genomic DNA (from nucleotide -10 to nncleotide +1,350 relative to the initiation codon), terminating after codon 450 just before the putative calmodulin binding and autoinhibitory domains (amino acid residues 454-553) (Cyert et al., 1991;Liu et al., 1991b). The natural COOH-terminal domain was restored in pKC74 by replacing the 3' segment from SalI to Hindl]I in the polylinker with a fragment from pCNA1-201 (Cyert et al., 1991) from SalI to HindlII in the 3' noncoding region. Both pKC73 and pKC'/4 complemented cnal cna2 double mutants in media containing galactose but not glucose (see Fig. 8).

Yeast Strains, Media, and Growth Conditions
Yeast strains were grown in standard YPD medium (2 % Difco yeast extract, 1% bacto-peptone, 2% dextrose) or YPD, pH 5.5, containing 5 mM succinic acid and supplemented with 200 mM CaC12 when indicated in the text. SC-ura and SCGal-ura media (Sherman et al., 1986) were supplemented with 400 mM CaC12 when indicated in the text.
Strain I.,4420 was derived from a cross between strains AA274 (Antebi and Fink, 1992) and YR86 (Rudolph and Fink, 1990). All yeast strains listed in Table I are derived from the isogenic strains W303-1A and W303-1B (Wallis et al., 1989) by transformation using the lithium acetate procedure or by standard genetic crosses among isogenic derivatives (Sherman et al., 1986). The pmcl::HIS3, pmcl::LEU2, and pmcl::TRPl null alleles were produced in W303-1A or W303-1B as described above from lineafized plasraids pKC38, pKC52, and pKC59. All three alleles produced identical phenotypes and are referred to as pmcl in the text.
Spontaneous revertants of pmcl were obtained by selection for growth of K473 (MATc~ pmcl::LEU2) and K482 (MATc~ pmcl::TRP1) on YPD pH 5.5, agar medium supplemented with 200 mM CaCI2. Independent revertants were picked after 5 d at 30°C, purified, and placed into complementation groups by mating the K473 and K482 derivatives in all combinations, selecting for diploids on SC-Leu-Trp, and assaying for growth on YPD, pH 5.5, plus 200 mM CaC12 or SC-Leu-1"rp plus 400 rnM CaC12. In this way, three complementation groups were identified among 42 independent recessive revertants. Based on genetic linkage and complementation testing, the first group of calcium-resistant mutants, termed crmJ, defines the CNB1 gene (Cyert and Thorner, 1992). The crm2 and crm3 groups (11 and 3 members, respectively) are not complemented by pTD59 containing the CMD1 gene (Geiser et al., 1991) and will be described in future reports.

Measurement of Exchangeable and Nonexchangeable Ca 2÷
Yeast cultures growing exponentially in YPD medium containing 0.18 mM Ca 2+ (Ohya et al., 1984) were shifted to fresh YPD medium supplemented with 45Ca 2+ (41 cpm/pmol) and grown at 30°C for 6.5 h to a final ODt00 of 1.5 (7.5 x 107 cells/ml). The total cell-associated Ca 2+ was calculated by measuring the radioactivity recovered from 0.1-ml culture aliquots which were diluted into 5 ml ice-cold buffer A (5 mM Na-Hepes, pH 6.5, 10 mM CaCI2), filtered rapidly onto 24 rnm Whatman GFF filters using a multiple vacuum filtration unit (model HV224, Hoefer Sci. Instrs., San Francisco, CA), washed three times with ice cold buffer A, dried in vacuo, and  Wallis et al., 1989Wallis et al., 1989Geiser et al., 1991Geiser et al., 1991 * All strains harbor the following additional mutations: are2-1 canl-lO0 his3-11,15 leu2-3,112 trpl-I ura3-1. ¢ Strains K444 through I(484 were constructed by transformation of the source strain. Strains I(510 through K616 were produced by crossing derivatives of W303-1A, W303-1B, and JGY41 which contained mutations in cnal, cna2, or cnbl that were introduced by transformation. processed for liquid scintillation counting. The nonexchangeable Ca 2"+ pools were determined by the same procedure except that each culture was first diluted 10-fold into prewarmed YPD medium supplemented with 20 mM CaCI2 (a 1,000-fold isotopic dilution) and incubated an additional 20 rain before filtration. The radioactivity released from the cells by this equllihration procedure represents the exchangeable Ca 2"+ pool, which was calculated as the difference between the total cell-associated Ca 2"+ and the nonexchangeable Ca 2+. All measurements were performed in duplicate and averaged.

Miscellaneous Procedures and Reagents
DAPI and immunofluorescent stainings were performed as described (Pringle et al., 1991) on early log phase cultures growing in SC-ura medium at 30"C. SDS gel electrophoresis (Laemmli, 1970) and Western blots (Johnson et al., 1984) were performed on total cell lysetes (Reid and Schatz, 1982) prepared from equivalent numbers of log phase cells grown in SC-ura medium. CaCI2 (C-7902; Sigma Immunochemicals, St. Louis, MO) and 4~CaC12 (Amersham Corp., Arlington Heights, IL) were used to supplement growth medium. FK506 was a generous gift from Vertex Pharmaceuticals, Inc. (Cambridge, MA). Cyclosporin A was provided by H. Lodish (Whitehead Institute, Cambridge, MA). All other reagents were commercially available.

Cloning and Expression of a Yeast Homolog of PMCA
Our strategy to control calcium signaling in yeast was to identify and manipulate the genes encoding the Ca :'+ trans- ASrFv~'f~S~C I S 1176 Figure 2. Alignment of the predicted PMC1 gene product (top sequence) with rat PMCAla (bottom sequence, [$hull and Greeb, 1988]). Gaps (-) were introduced to maximize homology (Lipman and Pearson, 1985). Predicted transmembrane domains 1-10 are indicated by underline and overline. Arrowheads (A) mark the highly conserved catalytic autophosphorylation and ATP-binding domains employed in the design of degenerate oligonucleotides for PeR analysis.
porters. Ca 2+ ATPases are members of a highly conserved family of P-type ion pumps, all of which contain highly conserved phosphorylation and ATP-binding domains (Fig. 2). At least five different yeast genes contain both these sequence motifs, as suggested by PCR analysis (see Materials and Methods). Four of these P e R products encode portions of the previously identified PMAI, PMA2, PMRI, and PMR2 genes. A novel 1.2-kbp P e R product that comigrates with the PMR2 product was cloned after amplification of genomic DNA from a pmrl pmr2 deletion strain. This clone contained an open reading frame highly homologous to a segment of mammalian PMCAs. The DNA segment was mapped by hybridization to chromosome VII very close to the PMAI gene ( Fig. 1 A), and defined a new gene termed PMC1 based on its homology to PMCAs. The entire PMC1 gene was subcloned from plasmid B1157 (see Fig. 1 A), which had been isolated previously in this laboratory during the cloning of PMA1, the major H + ATPase in the plasma membrane (Serrano et al., 1986). A long open reading frame of 1,173 codons encompassing the PCR product ( Fig. 1 B) predicts a 131-kD polypeptide showing 41% identity with rat PMCAla (Shull and Greeb, 1988), about 40 % identity with other PMCA isoforms, and less than 24 % identity with SERCA and other known P-type ion pumps. Alignment of the predicted protein sequence Pmclp with rat PMCAIa (Fig. 2) shows similarity through all 10 putative transmembane domains (M1-M10) and most hydrophilic regions except for the NH2-and COOH-terminal domains as well as several apparent insertions and deletions. The major structural differences occur in regulatory regions that are not well conserved among the known PMCA isoforms (Carafoli, 1992). The large insertion and deletion in PMCI between M2 and M3 occur in a phospholipid binding regulatory region that is subject to alternative splicing in various PMCA isoforms. Interestingly, a COOH-terminal domain that is present in most PMCA isoforms and is involved in calmodulin binding and autoinhibition is absent in Pmclp and replaced by a much smaller COOH-terminal domain lacking significant similarity to other proteins in current databases. Despite these structural differences, PMC1 appears to retain all conserved features of P-type ATPases and is the first reported fungal homolog of PMCA.
The product of PMC1 is expressed during exponential growth as judged by immunoblotting (Fig. 3) and immunofluorescence microscopy (Fig. 4). For these experi- Figure 3. Immunoblot analysis of Pmclp.
Total cell lysates prepared from exponentially grown strains W303-1A PMC1 (lane 1) or K470 PMCh:HA (lane 2) were fractionated by SDS-PAGE on 7.5% gels and analyzed by immunoblot using a 1:1,000 dilution of 12CA5 monoclonal antibody which binds specifically to HA epitopes. Immune complexes were visualized on radiographic film (Kodak) using the ECL chemiIluminescence detection kit. Protein molecular weight markers loaded in adjacent wells were marked after Ponceau S staining of the nitrocellulose blot prior to treatment with blocking agents. Figure 4. Immunofluorescence localization of Pmclp to vacuole membranes. W303-1A strains transformed with the high copy plasmid pKC60 (top), the integrating plasmid pKC55 (center), or the control plasmid B2205 (bottom) were grown to early log phase, fixed, and processed for immunofluorescence microscopy using the 12CA5 rnonoclonal antibody. The PMCI: :HA product is visible at the vacuole periphery in overexpressing strain (top) and the single copy strain (center), but only nonspecific background staining is visible in similar exposures of the strain lacking the epitope. Nomarski microscopy and DAPI staining identify the vacuoles and nucleus, respectively. menU, the PMCI::HA gene product was visualized using the 12CA5 monoclonal antibody, which recognizes a 34-amino acid insertion at codon +2 in the PMC1 open reading frame (Fig. 1 C). This epitope-tagged version of PMC1 is functional in vivo when integrated into its chromosomal locus or when expressed from high copy plasmids (see below). Immunoblotting of total cell lysates revealed a major immunoreactive polypeptide at '~130 kD, in good agreement with the predicted molecular mass of 131 kD (Fig. 3, lane 2). Only minor cross-reacting species were observed in lysates prepared from strains lacking the epitope (Fig. 3, lane 1 ). Analysis by immunofluorescence microscopy revealed that the epitope-tagged PMCI::HA gene product is localized predominantly to vacuole membranes in log-phase cells (Fig. 4). Cells producing the PMCI::HA product from its chromosomal locus displayed staining of one to three large vesicular structures corresponding to vacuoles by Nomarski optics. Expression of PMCI::HA from a multi-copy plasmid greatly increased the staining of the vacuole periphery. No staining of the nucleus, endoplasmic reticulum, mitochondria, or plasma membrane was detected in these experiments. These results suggest that the PMC1 product might function to transport Ca 2+ ions into the vacuole from the cytosol. High affinity Ca 2+ ATPase or Ca 2+ transport activities have not yet been detected in vacuole membrane vesicles (Ohsumi and Anraku, 1983).

Pmclp Sequesters Ca 2+ In Vivo
The effects of mutating the chromosomal PMC1 gene were examined to infer its function in yeast. The PMC1 gene is not essential for viability since deleting 97 % of the PMC1 open reading frame ( Fig. 1 A) had no significant effect on growth rate in rich or synthetic media at a variety of temperatures.
Additionally, disruption of PMC1 does not noticeably affect mating, sporulation, starvation, or a variety of other responses. These observations show that the function of PMC1 is not required for the execution of many processes under standard conditions.
The effect of deleting PMC1 on steady state Ca 2+ pools suggests that Pmclp functions in Ca 2+ sequestration in vivo. The yeast vacuole accumulates over 95 % of the total cellassociated calcium (Eilam et al., 1985), the majority of which is nonexchangeable in pulse/chase experiments (Eilain, 1982a,b). Accordingly, the exchangeable and nonexchangeable Ca 2+ pools were measured in exponentially growing PMC1 and pmd cells. The exchangeable Ca 2÷ pool was increased only slightly (12%) in pmcl cells relative to wild-type cells. However, the nonexchangeable Ca 2+ pool in pmcl cells was over fivefold lower than that of isogenic wildtype cells (Fig. 5). This difference reflects a large reduction in the vacuole Ca 2+ pool in pmcl mutants, and suggests that Pmclp is necessary for efficient Ca 2+ sequestration in vivo.

PMC1 Is Required for Ca z+ Tolerance
As pmcl mutants have decreased ability to sequester Ca 2+ in the vacuole, we tested their ability to tolerate very high or very low Ca 2+ concentrations in the growth medium. Wild-type and pmcl null mutants are indistinguishable during growth in synthetic media containing <1/~M Ca 2+ (Rudolph et al., 1989) or in complex YPD media containing varying amounts of EGTA (not shown). However, pmcl null mutants grow very poorly in YPD medium, pH 5.5, supplemented with 200 mM CaC12 (Figs. 6 and 7). The growth defect ofpmcl mutants was not observed in YPD, pH 5.5, supplemented with 200 mM MgC12 (Fig. 7). Additionally Ca :+ sensitivity was not observed in strains expressing only the epitope-tagged PMCI::HA hybrid from its chromosomal locus or from high copy plasmids (not shown). These observations demonstrate that PMC1 is necessary for tolerance to high Ca 2+ and confirm the importance of the vacuole in yeast Ca :+ homeostasis (Klionsky et al., 1990;. In liquid growth medium supplemented with 200 mM Ca 2+, wild-type strains grew exponentially after a brief lag (Fig. 6 A). In contrast, pracl mutants shifted to high Ca :+ media increased culture density at a slow linear rate that continued over at least 32 h. After 24 h of incubation, the optical density oftheprad culture increased 10-fold and the number of viable cells increased approximately 5-fold, as determined by colony forming units on agar YPD, pH 5.5, medium supplemented with 200 mM MgC12. Though the morphology of pmcl cells changed somewhat during incubation in high Ca 2+ (Fig. 6 B), they did not accumulate with a uniform morphology. Thus, the growth inhibition ofprad mutants by high Ca :+ is reversible and apparently not a consequence of a specific block in the cell division cycle.

Calcineurin Mediates Growth Inhibition of pmcl Mutants in High Ca 2÷ Medium
The mechanism by which external Ca 2÷ inhibits the growth of pmcl mutants was investigated by characterizing spontaneous revertants carrying secondary mutations that restore the ability of pmcl null mutants to proliferate in high Ca 2+ media (see Materials and Methods). Recessive mutations in three complementation groups were found to reverse the Ca 2+ sensitivity of pmcl mutants. The first group of revertants (termed crm/ for Ca 2+ resistant mutation) was found to define the CNB1 gene which encodes the Ca 2+ binding regulatory subunit of calcineurin (Kuno et al., 1991;Cyert and Thorner, 1992). The crm/alleles displayed a defect in recovery from pheromone arrest similar to cnbl null mutants (Cyert and Thorner, 1992), were genetically linked to the CNB1 locus (44:0:0, parental ditype/non-parental ditype/ tetratype), and were complemented by plasmids carrying the wild-type CNB1 gene. Additionally, disruption of CNB1 by gene replacement restores growth ofpmcl mutants (Fig. 7). Growth ofpmcl mutants is also restored by simultaneous deletion of both genes encoding the catalytic subunit of calcineurin, CNA1 and CNA2 (Fig. 7). Deletion of either CNAI alone or CNA2 alone is not sufficient to suppress the Ca 2÷ sensitivity ofpmcl. By this assay, the CNAI and CNA2 genes are functionally redundant, which explains why no spontaneous c n a / o r cna2 mutants were recovered in the initial In high Ca 2÷ liquid media (Fig. 6 A), cnbl and pmcl cnbl strains grow at faster rates (1.6 h doubling time) than an isogenie wild-type strain (2.5 h doubling time). This result suggests that calcineurin activity limits the rate of yeast growth in high Ca 2+ conditions, and is consistent with earlier observations that the CNAI, CNA2, and CNB1 genes are not required for vegetative growth in standard media (Cyert et al., 1991;Liu et al., 1991b;Cyert and Thorner, 1992). Interestingly, pmcl cnbl mutants have a larger pool of nonexchangeable Ca 2÷ than pmcl mutants (Fig. 5). If Ca 2+ sequestration into the vacuole is improved by inactivation of calcineurin, this might explain the enhanced growth rate of pmcl cnbl (and cnbl) strains in high Ca 2÷ media.

Activation of Calcineurin by CaZ+/Calmodulin Is Necessary for Inhibition of pmcl Mutants
The protein phosphatase activity of manunalian calcineurin in vitro is greatly stimulated by binding of Ca 2* and calmodulin (Stewart et al., 1982). Similarly, calmodulin binds to the CNA1 and CNA2 gene products only in the presence of Ca 2+ (Cyert et al., 1991;Liu et al., 1991b). Mutant forms of calmodulin that are unable to bind Ca 2+ fail to activate target enzymes in vitro (Hurwitz et al., 1988), but still provide functions necessary for yeast viability (Geiser et al., 1991). Using cmdl-3 strains, which express a calmodulin mutant containing two amino acid substitutions in each of Figure 8. Expression of COOH-terminal truncated CNA1 (CNA1AC) bypasses cmdl-3 inpmcl mutants. Strains K473 (lines I and 2), KSI0 (lines 3 and 4), K559 (lines 5 and 6), and K607 (lines 7 and 8) were transformed with low copy plasmids containing derivatives of CNAI (pKC74, lines 1, 3, 5, and 7) or CNAIAC (pKC73, lines 2, 4, 6, and 8) under control of the galactose-inducible promoter of GALL Strain KS10 (lines 9 and 10) was also transformed with a control plasmid pUN50, or pTD59 containing the CMD1 gene (Geiser et al., 1991). Strain K473 was also transformed with the high copy control plasmids B2205 or pKC11 containing the PMRI gene. Serial fivefold dilutions of saturated cultures were spotted onto complete medium lacking uracil (to maintain selection for the plasmids) supplemented with the 2% glucose or galactose and 400 mM CaClz as indicated. The plates were photographed after 3 d incubation at 30°C. High Ca 2+ inhibits growth of pmcl cmdl-3 and pmcl cnal cna2 but not pmcl crrbl mutants expressing CNAI AC. High Ca 2+ does not inhibit growth of pmcl cmdl-3 or pmcl cnbl mutants expressing CNA1.
the three high affinity Ca2+-binding domains, it is possible to test whether Ca 2÷ binding to calmodulin is necessary for growth inhibition of pmcl mutants by high Ca 2÷. In contrast topmcl mutants, apmcl cmdl-3 double mutant grows well on high Ca 2÷ media (Fig. 7). Introduction of a low copy plasmid carrying the wild-type CMD1 gene into the pmcl cmdl-3 strain restored Ca 2÷ sensitivity (Fig. 8, lines 9 and 10), indicating that the mutant calmodulin has not gained a function (dominant over wild type), but rather has specifically lost a function required for Ca2÷-induced growth inhibition. Identical results were obtained with apmcl::HIS3 cmdl-6 strain (K445), which contains only three point mutations in the CMD1 gene (data not shown). Thus, a mutant with amino acid substitutions in the high affinity Ca 2÷ binding sites of calmodulin has the same phenotype as a mutant that has lost calcineurin function. Ca2÷/calmodulin activates calcineurin in vitro by binding to a COOH-terminal domain of the catalytic subunit and relieving autoinhibition by this domain (Stewart et al., 1982;Hashimoto et al., 1990). Proteolytic removal of the autoinhibitory domain causes calmodulin-independent stimulation of protein phosphatase activity (Manaian and Klee, 1983).
These biochemical data predict that a CNAI truncation mutant lacking the autoinhibitory domain would become activated independent of Ca2+/calmodulin and would prevent growth of pmcl cmdl-3 strains in high Ca 2÷ media. As predicted, a pmcl cmdl-3 strain expressing the truncated CNAIAC allele from an inducible promoter (GAL/) fails to grow in high Ca 2÷ medium (Fig. 8, line 4). In contrast, expression of wild-type CNA1 from this promoter does not inhibit growth of the pmcl cmdl-3 strain in high Ca 2÷ medium (Fig. 8, line 3). The GAL/-induced expression of CNA1 and CNA1AC does not inhibit growth of yeast strains in the absence of added Ca 2÷ (not shown). Furthermore, expression of either CNA1 or CNA1AC inhibits growth of a pmcl cnal cna2 strain (Fig. 8, lines 5 and 6) but not a pmcl cnbl strain (lines 7 and 8) in high Ca 2÷ media. Thus, removing the COOH-terminal autoinhibitory domain of calcineurin A specifically bypasses the requirement for Ca2+/calmodulin to promote growth arrest, though calcineurin B and Ca 2+ are still required for this arrest. These data suggest that the major role of Ca2+/calmodulin in growth inhibition by Ca 2+ is to activate calcineurin via its COOH-terminal region.
The cmdl-3 mutant calmodulin is unable to perform this function.

FKS06 and Cyclosporin A Inhibit Yeast Calcineurin In Vivo
The immunosuppressant drug FKS06 binds tightly to a complex of proteins present in wild-type yeast strains and absent in extracts made from strains lacking calcineurin A, calcineurin B, or the yeast homolog of FKBP-12 encoded by FPR1 (Foor et al., 1992). A similar complex formed in mammalian cells results in the complete inhibition of calcineurin activity towards phosphopeptide substrates (Liu et al., 1991a). These considerations prompted us to test whether FK506 would restore growth ofpmcl mutants in high Ca 2+ media by inhibiting yeast calcineurin.
Addition of FK506 dramatically restores growth of pmcl mutants in high Ca 2÷ medium, but has no effect on pmcl fprl strains lacking FKBP-12 (Fig. 9 A). In this assay, the  (Table I)  50% effective dosage (EDs0) of FK506 was estimated at 25 ng/ml which is over 1,000 times lower than the 50% lethal dosage (LDs0) of ,,050 #g/ml (Heitman et al., 1991). There is also a direct correlation between the effectiveness of the drug and the dosage of calcineurin A genes, pmcl cnal and pmcl cna2 double mutants each required approximately half as much FK506 (12 ng/ml) when compared to the pmcl strain. These results fully support the hypothesis that a direct target of FK506/FKBP-12 in yeast is activated calcineurin. Cyclosporin A, a structurally distinct inhibitor of calcineurin acting through its own binding protein (Liu et al., 1991a), also restores growth of pmcl mutants in high Ca 2÷ media ( Fig. 9 B). The EDs0 of cyclosporin A in this assay (1-2 #g/ml) is much lower than its LDs0 (,,o200/zg/ml), is directly proportional to CNA gene dosage, and is independent of FKBP-12. The effectiveness of both drugs can be explained by a model where a growth inhibitory complex of Ca2÷/calmodulin/calcineurin forms in pmcl mutants during Figure 10. The synthetic lethality ofpmcl pmrl double mutants is reversed by a cnbl mutation or by addition of FK506. Strains K610 (pmrl), K612 (pmrl cnbl), K614 (pmrlpmcl), and K616 (pmrlpmcl cnbl) were streaked onto YPD agar medium supplemented with FK506 (0.4/zg/ml) and/or CaCI2 (20 mM) as indicated. The strains were initially recovered by germinating ascopores produced from a triply heterozygous diploid on YPD+FK506+CaCI2 medium where all genotypes are viable. The colonies were photographed after 4 d of incubation at 30°C. The pmrl pmcl double mutants did not form visible colonies on YPD or YPD plus 20 mM CaC12. growth in high Ca 2÷ media, and that this complex is inhibited by the stoichiometric binding of FK506/FKBP-12 or cyclosporin A/cyclophilin (Foor et al., 1992). This model implies that [Ca2+]c is elevated in pmcl mutants during incubation in high Ca 2+ media, in agreement with the genetic data suggesting that Ca2+/calmodulin but not apo-calmodulin (as produced by cmdl-3) is required for the inhibitory effect of calcineurin.

The Inviability of a pmrl pmcl Double Mutant Is Suppressed by Inactivation of Calcineurin
The role of PMC1 in Ca ~+ tolerance may be shared by PMRI (Rudolph et al., 1989;Antebi and Fink, 1992) Figure 11. A working model for Ca 2+ transport in yeast and Ca2+/calcineurin dependent inhibition of growth. The cytoplasmic pool of Ca 2+ is increased by influx of external Ca 2+ and decreased by sequestration activities of Pmclp and Pmrlp which directly pump Ca 2÷ into the vacuole and Golgi complex, respectively. Other Ca ~+ transporters may also contribute to control of cytoplasmic Ca 2+. Elevation of the cytosolic free Ca 2÷ concentration, [Ca2+]c, activates calcineurin by increasing the binding of Ca 2+ and calmodulin. Inappropriate activation of calcineurin, attained by increased Ca 2÷ influx and/or decreased sequestration, dephosphorylates protein substrates whose phosphorylation is necessary for growth during these conditions. The downstream targets of calcineurin and their roles in growth control are unknown. It is possible that calcineurin negatively regulates a Ca2+-sequestering mechanism distinct from Pmclp (Fig. 5).
pmcl mutants restores growth in high Ca 2÷ media (Fig. 8,  lines 11 and 12). Moreover, simultaneous deletion of both PMC1 and PMR1 causes a phenotype more severe than either mutation alone. Strains containing both pmrl pmcl null mutations are inviable on all media regardless of the external Ca 2÷ levels (from 0.2 to 200 mM). However, the lethality of pmrl pmcl strains is prevented by inactivation of calcineurin with either FK506 (400 ng/ml) or the crib1 null mutation (Fig. 10). Thus, PMC1 andPMR1 are redundant for an essential function only when calcineurin can be activated, The simplest model consistent with these findings (see Fig. 11) is that both PMC1 and PMR1 gene products transport Ca 2÷ from the cytosol into internal compartments. Adequate Ca 2÷ ATPase activity is essential for yeast growth in order to prevent the inhibitory activation of calcineurin by calmodulin in response to elevated [Ca2+]c. Calcineurin activation may inhibit growth ofpmcl mutants by a variety of mechanisms, such as decreasing the activity of unknown factors necessary for growth or Ca 2÷ tolerance.

PMC1 Encodes a Vacuolar Ca z÷ ATPase Involved in Ca ~÷ Sequestration and Regulation of [Ca2÷]c
Several lines of evidence indicate that Pmclp transports Ca 2÷ from the cytoplasm into the vacuole. First, the pre-dicted product of PMC1 contains all the conserved features of P-type ion pumps and is highly homologous to mammalian PMCAs (41% identical through 81% of its sequence). Second, the phenotypes of pmcl mutants are consistent with a direct role for Pmclp in Ca 2+ transport. Although pmcl null mutants show no obvious growth or morphological phenotypes on standard media, they accumulate significantly less Ca 2÷ in the vacuole and are intolerant of high Ca 2÷ in the growth medium. Third, the behavior ofpmcl mutants is dramatically affected by modifications of a Ca~÷-sensitive regulatory pathway consisting of calmodulin and calcineurin. Finally, PMC1 shares an essential function with PMRI, a member of SERCA-type Ca 2÷ pumps (Serrano, 1991). All these properties are consistent with a general model of Ca 2+ transport and signaling thought to exist in most eukaryotic cells (Fig. 11). However, the study of Ca 2÷ metabolism in yeast has revealed some noteworthy distinctions.
Ca 2÷ uptake into the yeast vacuole in vitro is predominantly due to low affinity H+/Ca ~÷ antiport activity (Ohsumi and Anraku, 1983). However, during growth in 0.18 mM Ca ~+ YPD medium (Ohya et al., 1984), the majority of Ca ~+ sequestered in the vacuole requires Pmclp (Fig. 5). Thermodynamic considerations also argue that Ca 2+ ATPases are necessary to account for the high levels of Ca ~+ sequestered in fungal vacuoles (Miller et al., 1990). In the absence of Pmclp, the residual Ca 2+ sequestration can be attributed to the vacuolar antiporter and/or Pmrlp in the Golgi complex. PMR1 is required for viability ofpmcl mutants and overexpression of PMRI restores Ca 2+ tolerance to prad mutants. The simplest interpretation of these findings is that Pmclp and Pmrlp both transport Ca 2÷ from the cytoplasm into internal compartments and therefore function redundantly in controlling cytoplasmic Ca 2+ levels. This effect is not unlike the roles of PMCA and SERCA in returning [Ca2+]c to submicromolar levels after Ca 2+ influx is induced by external stimuli (Carafoli, 1987). The major distinction is that yeast utilize Pmclp for Ca 2÷ sequestration into the vacuole whereas mammalian cells utilize PMCA for Ca 2+ efflux through the plasma membrane.

Calcineurin Activation by Caz+ /Calmodulin Is Growth Inhibitory in pmcl Mutants
Our results show that the inviability ofpmcl strains in high Ca 2+ media requires calcineurin activation by Ca2+/calmodulin. The growth defects ofpmcl strains in high Ca 2+ media are completely reversed in strains lacking either the catalytic or regulatory subunits of calcineurin or in strains containing mutant forms of calmodulin with defective Ca 2+ binding sites. Expression of a truncated calcineurin A lacking the autoinhibitory domain bypasses the requirement for Ca2+/calmodulin, preventing growth ofpmcl cmdl-3 double mutants in high Ca 2÷ medium. These in vivo data are consistent with the mechanism of calcineurin activation in vitro where maximal stimulation of phosphopeptide phosphatase activity requires binding of Ca2÷/calmodulin to relieve autoinhibition by the COOH-terminal domain of the catalytic A subunit.
The clear effect of inactivating calcineurin inpmcl mutants was exploited here in a bioassay for potential antagonists of yeast calcineurin. The immunosuppressant drugs FK506 and cyclosporin A, together with their respective immunophilin binding proteins, form inactive complexes with yeast and mammalian calcineurin (Liu et al., 1991a;Foor et al., 1992). As demonstrated here, both of these compounds mimic the effect of calcineurin mutations, restoring growth to pmcl mutants in high Ca 2+ media. Moreover, the effective dosages of these drugs decreased about twofold when the gene dosage of calcineurin A was reduced by half. These findings provide strong evidence that calcineurin is the direct target of FK506 and cyclosporin A in yeast cells. Based on these results, we propose that calcineurin activation occurs during incubation of pmcl mutants in high Ca 2+ media.
Calcineurin activation also occurs in standard media in pmclpmrl double mutants, and therefore is not restricted to conditions of high external Ca 2+. Calcineurin-dependent growth inhibition can be observed in pmcl pmrl strains in media containing 0.18-200 mM Ca ~÷. Because Pmclp and Pmrlp function in Ca ~+ sequestration, their shared essential function (in >0.18 mM Ca :÷) is most likely to maintain sufficiently low [Ca2+]c to prevent activation of calcineurin. Though growth inhibition by calcineurin activation was most evident in pmclpmrl and pmcl strains, it is possible that calcineurin inhibits growth of wild-type strains in response to high Ca 2÷ environments (Fig. 6) or in response to natural Ca ~+ signals. Transient Ca 2÷ signals might be generated during some situations by the physiological modulation of Pmclp and Pmrlp activities or other Ca 2+ transporters.

Calcineurin-dependent Signaling Pathways
The results reported here suggest that calcineurin activation can have a net negative effect on growth, which itself might obscure other positive effects. A positive (growth enhancing) role for calcineurin in yeast has been observed in response to prolonged cell cycle arrest by mating pheromones (Cyert et al., 1991;Cyert and Thorner, 1992). The process of recovery from pheromone arrest is inefficient in calcineurin mutants and in strains treated with FK506 or cyclosporin A (Foor et al., 1992). Thus, calcineurin activation can affect yeast proliferation in a positive or negative way depending on the circumstances. The net effect of activated calcineurin may be a consequence of differences in the activity or availability of phosphoprotein substrates, in the nature of the cytosolic Ca 2+ signal, or possibly the input of additional regulatory signals.
Calcineurin-dependent signaling in human cells can also have a net positive or negative effect depending upon the cell status. Cyclosporin A and FK506 can prevent activation and proliferation of mature T cells, whereas these compounds block the induced suicide of immature T cells (Bierer et al., 1991). The calcineurin-dependent inhibition of yeast growth reported here is reversible, occurs at many stages of the cell cycle, and is therefore distinct from the type of cell death observed in immature T cells. A more complete understanding of the different responses will require the identification of distinguishing calcineurin targets.
Our results show that in pmcl mutants, calcineurin function decreases tolerance to external Ca 2÷ (Fig. 6) and decreases sequestration of Ca 2+ into the nonexchangeable (vacuolar) pool (Fig. 5). It is unlikely that the latter effect is due to increased influx or decreased efflux of Ca 2÷ since both these models would predict decreased tolerance to external Ca 2+. An alternative explanation is that another enzyme involved in Ca 2÷ transport into the vacuole, such as the H+/Ca 2+ antiporter (Ohsumi and Anraku, 1983), is negatively regulated by calcineurin. This explanation would also account for the calcineurin-dependent growth inhibition of pmcl mutants in high Ca 2+ conditions. Given the remarkable interspecies conservation of the mechanism by which calcineurin is activated and inhibited, it is plausible that the physiological roles of calcineurin are similar in human and yeast cells. The bioassays reported here will be useful to identify the downstream targets and functions of yeast calcineurin as a framework for understanding Ca2+-signaling mechanisms and responses in human cells.