Stimulation of calcium uptake in Saccharomyces cerevisiae by bovine protein kinase C alpha.

Ca2+ plays essential roles as a second messenger often in synergism with the calcium- and phospholipid-dependent phorbol ester receptor, protein kinase C (PKC), which stimulates Ca2+ influx in various cell types in a potential positive feedback mechanism. To address the compatibility of these mechanisms between lower eukaryotes and mammals, we have stably expressed bovine PKC alpha in the yeast Saccharomyces cerevisiae. We find that phorbol ester binding sites are created which stimulate a specific calcium- and phospholipid-dependent catalytic activity in vitro. Phorbol ester activation in vivo stimulates PKC down-regulation, uptake of extracellular Ca2+, Ca2+ dependence of cell viability, and changes in cell morphology. This may represent activation of a putative PKC-mediated signaling pathway utilized by functional yeast homologs of mammalian PKC isoforms. These are suggested by some protein data; however, their genes have not yet been characterized (Simon, A. J., Milner, Y., Saville, S. P., Dvir, A., Mochly-Rosen, D., and Orr, E. (1991) Proc. R. Soc. Lond. B 243, 165-171). Our findings indicate that bovine PKC alpha is functional in yeast and stimulates calcium uptake in a manner similar to some of its responses in mammalian cells, which suggests compatible aspects of higher and lower eukaryotic signaling pathways and the feasibility of dissecting parts of the action of common signaling mediators in a simple genetic model.

In the lower eukaryote Saccharomyces cereuisiae intracellular Ca2+ is essential for cell cycle progression (Iida et al., 1990a), which is comparable to its obligatory role in mediating G1 events in mammalian cells (reviewed by Alkon and Rasmussen (1988) and Campbell (1983)). During the late stage of the mating pheromone response Ca2+ influx is stimulated (Ohsumi and Anraku, 1985) and cell viability becomes dependent on extracellular Ca2+ (Iida et al., 1990b). In S. cereuisiae a PKC-related, diacylglycerol-stimulated but phorbol ester-unresponsive protein activity with distinct substrate specificity has been described (Ogita et al., 1990;Iwai et al., 1992), as well as putative mammalian-like PKC isoforms in a separate study (Simon et al., 1991(Simon et al., , 1992. Independently a related, essential gene PKCl has been identified with a cell cycle-specific role in osmotic stability and perhaps in bud morphogenesis, which is, however, not complemented by mammalian PKC isoforms when disrupted (Levin et al., 1990;Levin and Bartlett-Heubusch, 1992).
To begin to address the compatibility of these signaling mechanisms between higher and lower eukaryotes we have stably expressed bovine PKCa in S. cereuisiae. We find that phorbol ester binding sites are created which stimulate a specific calcium-and phospholipid-dependent catalytic activity in uitro. Phorbol ester activation in uiuo stimulates PKC down-regulation, uptake of extracellular Ca2+, Ca2+ dependence of cell viability, and changes in cell morphology, suggesting that bovine PKCa is fully functional in yeast and stimulates calcium uptake in a manner similar to some of its responses in mammalian cells.

EXPERIMENTAL PROCEDURES
cDNA Construction-The complete protein-coding region of bovine protein kinase C a (Parker et al., 1986) was isolated and joined at the NcoI site at the translation initiation codon with a synthetic HindIII-NcoI adapter 5"AGCTTAAAAAA-3' and 3"ATTTTTTGTAC-5' to optimize the sequence upstream of the ATG codon for maximum translation efficiency (Cigan and Donahue, 1987). The cDNA was with the synthetic blunt end-XbaI adaptor 5'-TAACTAACTAAT-3' truncated at the 3'-end by exonuclease Bat31 digestion and joined and 3'-ATTGATTGATTAGATC-5', which provides translation stop codons in all three reading frames. A complete protein-coding cDNA including 10 base pairs of the 3'-untranslated sequence (PKCa) and a truncated cDNA lacking coding sequences for 149 (aa 524-672) carboxyl-terminal aa (CD149) were inserted into the Hind111 and XbaI sites under control of galactose-inducible transcriptional elements in the high copy number yeast episomal expression plasmid YEp52 containing the LEU2 gene for selection (Broach et al., 1983).
After ligation plasmids were amplified in Escherichia coli DH5a and were identified and confirmed by restriction analysis and DNA sequencing.
Yeast Transformation and Culture Conditions-PKC expression plasmids and YEp52 (Broach et al., 1983) as a control were introduced into S. cereuisiae strain 334 (MAT a; pep4-3; prbl-1122; urd-52; leu2-3,112; regl-501; gall) (Hovland et al., 1989) by lithium acetate transformation (Ito et al., 1983). Cells were routinely grown in leucinefree synthetic medium containing 2% glucose in liquid culture or on 1.5% agar plates to select for stable propagation of the expression plasmids. Transcription of mammalian PKC cDNAs was induced for 20 h with 2% galactose, which results in up to 1000-fold induction of transcription within 6 h in strain 334 (Hovland et al., 1989). PKC activity was routinely measured in response to a single dose of 1 pM PMA or in controls (-PMA) in response to 1 p M inactive isomer 4a-PMA (both LC Services, Woburn, MA) which was added at the start of each experiment. Cells were routinely harvested from 100-ml cultures at a density of 0.5 ODm. All experiments were performed several times with comparable results, and representative data are shown.
Cell Lysis-About 5 X IOs expressing cells were washed in PBS and were resuspended in 1 ml of lysis buffer containing 1% Triton X-100, 50 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 10 pg/ml pepstatin, 40 pg/ml leupeptin, 10 pg/ml aprotinin, 200 pg/ml phenylmethylsulfonyl fluoride. Cell suspensions mixed with the same volume of acid-washed glass beads (450-500 pm) were lysed mechanically by six 30-s vortexing steps interrupted by cooling on ice for at least 30 s in 50-ml screw cap polypropylene tubes (Sambrook et al., 1989). The lysate was cleared by 4,000 X g centrifugation for 15 min and stored at -70 "C for up to several weeks.
Catalytic Activity-Phosphatidylserine with PMA or with the inactive 4a-PMA (LC Services) as a negative control was mixed in chloroform, evaporated under Nz to dryness, resuspended in 20 mM Tris, pH 7.5, mixed, and sonicated at 4 "C for 45 s before use. PKC catalytic activity was measured as described by Hu et al. (1990) by phosphorylation of 3 pg of the specific pseudosubstrate derivative [Ser-25]PKC(19-31) substrate peptide RFARKGSLRQKNV (GIBCO/BRL) in the presence of 5 pCi of [-Y-~~P]ATP at 100 p~ in 125 pl of 5-fold diluted cell extracts for 15 min at 25 "C with combinations of 1 p~ PMA, 10 mM CaClZ, and 160 pg/ml phosphatidylserine (Avanti Polar Lipids, Inc.). The effect of PKC activators and inhibitors was tested in the absence of Ca2+ with 5 p~ indolactam V(-) (LC Services), 1 p M PMA, or 1 p~ PMA and 500 pg/ml polymyxin B (Sigma). Reactions were terminated by placement under suction on phosphocellulose paper (Whatman P81), which was repeatedly washed in 75 mM phosphoric acid. Bound radioactivity was determined by liquid scintillation spectroscopy.
PKC Down-regulation-Cultures (250 ml) of PKCa-expressing cells were incubated with or without 0.5 p~ PMA for various amounts of time and were harvested at densities of 0.5-1 OD,,.
Cells were resuspended in 3 ml of 20 mM Tris, pH 6.5, 0.5 mM EGTA, 0.5 mM EDTA, 1 mM 2-mercaptoethanol and lysed by vortexing with 0.5 ml of glass beads (400-500 pm). Cell fractions were enriched on a 20/30/ 40/50% Ficoll 400 (Pharmacia) step gradient (1.5/1.5/1.5/1.5 ml) by centrifugation for 1 h at lo5 X g (Aris and Blohel, 1991). Cytosolic and membrane fractions were collected from the top fraction without Ficoll or from the interphase above the 20% layer, respectively, and stored at -70 "C for several days. Twenty pg of total protein of each fraction, which corresponds to 100 pl of the cytosolic fraction (the maximum applicable volume), was loaded and separated on a 8% SDS-polyacrylamide gel. Proteins were electrophoretically trans-ferred to nitrocellulose and analyzed by immunoblotting with PKCaspecific antibodies provided by Oliver et al. (1991). 45Ca2+ Uptake-Expressing yeast cells (lo') were washed with PBS and incubated in 100 pl of 50 mM MES, pH 6.5,5 mM MgSOl with 1 p~ PMA and/or 1 mM of NaC1, KC1, CaCl2, CdClZ, or 50 pM amiloride for 1 h at 30 "C under shaking on 96-well plates. LaCb varied from 10 to 100 p~, MnClZ from 10 p~ to 1 mM, and MgS04 in Mg-free buffer from 1 mM to 5 mM in dose-response experiments. The uptake of 2 pCi of "Ca2+ at 300 p~ or of 2 pCi of ffiRh+ at 1.4 mCi/mg in the presence of 150 p~ KC1 was measured for 1 h. Cells were transferred to Multiscreen filtration microtiter plates (Millipore) and washed four times with 200 pl of 10 mM MES, 10 mM Tris, 35 mM CaC12 pH, 6.0, filters were dried, and cell-associated radioactivity was determined by liquid scintillation spectroscopy.

RESULTS
To create yeast expression constructs, we inserted the complete protein coding cDNA of bovine PKCa (Parker et al., 1986) or a truncated cDNA lacking coding sequences for 149 (CD149) carboxyl-terminal amino acids (aa) under the control of a galactose-inducible transcriptional promoter element into the high copy number episomal plasmid YEp52 (Broach et al., 1983). Yeast cells were transformed and plasmids were stably propagated by selection in leucine-free 2% glucose medium. Transcription was routinely induced with 2% galactose for 20 h in most experiments, and control-transformed cells were used to determine the experimental background.
T o test whether normal and truncated PKC protein products were properly expressed, proteins from detergent cell extracts were separated on SDS-polyacrylamide gels and analyzed with PKCa-specific antibodies in immunoblots as shown in Fig To directly test the function of the regulatory PKC domain, phorbol ester binding sites were determined after enzymatic removal of the yeast cell wall by exposure of yeast spheroplasts to [3H]phorbol-12,13-dibutyrate ( [3H]PDBu). As shown in Fig. lB, expression of normal PKCa as well as the truncation mutant CD149 resulted in [3H]PDBu binding levels that were at least 13-fold elevated over background levels of controltransformed yeast cells. A similar increase in PDBu binding has been observed upon PKC expression in transfected mammalian fibroblasts (Knopf et al., 1986). These data suggest that the truncation not unexpectedly does not interfere with the function of the PKC regulatory domain and that expression levels are comparable between both PKC forms. Scatchard analysis shown in Fig. 1C suggests lo4 PDBu-binding sites/yeast cell (10-pm diameter) with a low affinity of Kd 100 nM for normal PKCa. Affinity constants have been reported in mammalian cell types depending on the experimental conditions from subnanomolar Kd values to low affinities of Kd 60 nM in PB-3c murine mast cells (Mazurek et al., 1987), suggesting that the yeast plasma membrane may not provide an optimal environment t o detect high affinity PDBu binding.
To characterize the enzymatic activity of bovine PKCa expression products in yeast, phosphorylation of the PKCspecific [Ser-25]PKC(19-31) peptide substrate, a derivative of the pseudosubstrate sequence (House and Kemp, 1987) was measured in detergent cell extracts. Compared to control cell extracts, PKCa led to a 50-fold increase in peptide phosphorylation, which was clearly calcium- (Fig. 2 4 ) and phospholipid-dependent with a K,,, of 50 pg/ml for phosphatidylserine (Fig. 2B) comparable to the characteristics reported in A CD149 PKCa Cont. Proteins were transferred to nitrocellulose and analyzed by immunoblotting with a specific antibody to mammalian PKCn provided by Oliver et al. (1991). Size markers are indicated in kDa. B, ['HH]PDRu binding to yeast spheroplasts expressing normal and truncated PKCn.
['HIPDBu binding to yeast spheroplasts transformed with control plasmids (Cont.) or expressing normal PKCn (PKCa) or truncation mutant CD149 was compared. Background binding determined in the presence of a 2000-fold excess of unlabeled PDBu has been subtracted. C, Scatchard analysis of ["HlPDRu binding to yeast spheroplasts expressing PKCn. mammalian cells (Hannun et al., 1985). As observed for PKC from mammalian cells (Hannun and Bell, 1990), a t low calcium concentrations catalytic activity was stimulated by the activators diacylglycerol (not shown) and the phorbol ester phorbol 12-myristate 13-acetate (PMA) 20-fold ( Fig. 2A). Deletion of the carboxyl-terminal 149 aa in CD149, which likely leads to inactivation of the kinase, resulted in the complete loss of catalytic activity (Fig. 2 A ) . PMA insignificantly affected the K, (from 34 to 64 p~) and caused a 30fold increase in the VmaX (from 1,800 to 57,000 cpm/min) for peptide substrate phosphorylation (Fig. 2C). Catalytic activity was also stimulated by other PKC activators including the indol alkaloid indolactam V (Fujiki et al., 1985) comparable to PMA, while the PMA response was abolished by PKC inhibitors such as the antibiotic polymyxin B (Zherelova, 1989) (Fig. 2 0 ) . These results are consistent with proper expression and calcium-and phospholipid-dependent and phorbol ester-stimulated function of bovine PKCa in yeast cells.
Upon exposure to phorbol esters, mammalian cells respond by down-regulation of PKC which may play a role in PKC action (Rodriguez-Pena and Rozengurt, 1984;Young et al., 1988;Droms and Malkinson, 1991). To test whether a compatible cellular mechanism exists in yeast, expressing cells were exposed to PMA, cytosolic and membrane cell fractions were enriched, and equal amounts of total protein of each fraction were separated on SDS-polyacrylamide gels and analyzed in immunoblots. As shown in Fig. 3A, upon PMA treatment disappearance of PKC was observed from both cell fractions. In the membrane fraction after 5 h of PMA treatment, significant loss of PKCa was observed to less than half of the original levels, and after 18 h of PMA treatment, PKCa was hardly detectable. In the cytosolic fraction much weaker PKC protein levels were detected, proportional to the lower ratio of PKC to total protein in this fraction, whereas the same amount of total protein had been loaded on the gel for each fraction. After PMA treatment for 5 or 18 h, PKC was no longer detectable. In the membrane fraction two major PKC bands of M , 80,000 and 77,000 were observed, which similarly disappeared upon phorbol ester treatment. In the down-regulation of PKCa. PKCa-expressing cells were exposed to PMA (+) or the inactive 4a-PMA (-) for the times indicated, and membrane or cytosolic cell fractions were separated on 8% SDSpolyacrylamide gels. Proteins were transferred to nitrocellulose and analyzed by immunoblotting with a specific antibody to mammalian PKCa provided by Oliver et al. (1991). Size markers are indicated in kDa. B, yeast morphology changes by PMA activation of PKCa. Cells transformed with control plasmid (Cont.) or PKCa-expressing cells (PKCa) were cultured in normal 2% galactose medium for 7.5 h in the presence of 1 IM PMA, fixed in 3.7% formaldehyde, and photographed in phase contrast. The length of the panels corresponds to 0.5 mm.
cytosolic fraction a major protein band was detected by the polyclonal antibody at M , 81,000 with constant intensity independent of PMA treatment. This band probably represents a different cytosolic protein that obscured much of the M , 80,000 PKC form, which was only marginally detectable below the background band. The traces of this M , 80,000 form disappeared in response to PMA treatment similar to the more prominent M , 77,000 PKC form in this fraction. Similar size heterogeneity has been described for PKC isolated from mammalian cells related to different levels of PKC phosphorylation (Borner et al., 1989); however, we have not addressed this question. Our findings suggest that bovine PKCa interacts with a cellular mechanism in yeast, which results in PKC down-regulation in response to phorbol esters compatible to responses observed in mammalian cells.
To test whether PKCa activation might result in other biological responses in yeast which affect cell morphology, cell proliferation was followed under the phase contrast microscope. In the absence of PMA cell morphology was always normal (not shown); however, PMA stimulation caused morphological changes similar to the mating factor-induced "shmoo" phenotype in up to 10% of PKCa-expressing cells, which were never observed in control cells (Fig. 3B).
Ca2+ influx has been reported in response to PKC activation in a variety of eukaryotic cells such as Nitella syncarpa plasmalemma (Zherelova, 1989), Aplysia bag cell neurons (Strong et al., 1987), rat ventricular myocytes (Lacerda et al., 1988), and rat pituitary cells (Stojilkovic et al., 1988). To investigate whether mammalian PKCa expression leads to a similar response in yeast, 4sCa2+ uptake was measured after PMA stimulation (Fig. 4). While control cells did not respond, PMA-activation of PKCa led to an up to 4-fold increase, which was abolished by a defective catalytic domain (CD149) lacking 149 carboxyl-terminal aa as expected, suggesting that this response depends on activation of the kinase (Fig. 4A). PMA did not affect uptake of tracer "Rb+ mixed with K' , indicating that the ion uptake response is specific for divalent Ca2+ ions as opposed to monovalent ions (Fig. 4B). At the same time glucose stimulates uptake of divalent as well as monovalent ions (Fig. 4B) by a mechanism including hyperpolarization of the cell membrane (Borst- Pauwels, 1981;Eilam and Othman, 1990).
The PMA stimulation of PKCa-mediated Ca2+ uptake was abolished by La'+ ( Fig. 4C), an inhibitor of carrier mediated Ca2+ influx (Eilam and Grossowicz, 1982), but it was largely unaffected by other divalent ions such as Mn2+ (not shown). Increasing Mg2' concentrations resulted in decreased basal Ca2+ uptake (not shown), consistent with the idea that both ions may compete with a shared transport system in yeast (Borbolla and Pena, 1980). Our findings indicate that we are observing specific ion uptake rather than uptake due to permeabilization or other damage of cell membranes (Borbolla and Pena, 1980;Eilam and Othman, 1990). As shown in Fig.  40, PMA stimulation of PKCa-mediated Ca2+ uptake was little affected by the monovalent ions Na+ and K+ or by amiloride which blocks Na+/H+ and Na+/Ca2+ exchange mechanisms, but the response was significantly increased even in control cells by Cd", which raises the cation permeability of the yeast cell membrane and results in intracellular acidification (Borbolla and Pena, 1980;Kessels et al., 1987). Only PMA-stimulated responses have been shown in Fig. 4 0 for control and PKCa-expressing cells. Responses of control or PKCa-expressing cells in the absence of PMA have been omitted for clarity, since they were indistinguishable from PMA-stimulated control cells as indicated in Fig. 4A. Part of the Ca2+ uptake response was measured in DEAE-dextranpermeabilized cells (not shown) suggesting that it involves Ca2+ uptake into vacuoles, the major cellular stores for Ca2+

TABLE I Ca2+ dependence of yeast cell growth and viability after PMA activation of PKCa
Control or PKCn expressing cells were cultured with 2% galactose in the absence (-PMA) or presence (+PMA) of 1 PM PMA in Ca2+supplemented (1 mM) (+Ca2+) or Caz+-free (-Ca2+) synthetic minimal medium (Iida et al., 1990b). Loss of cell viability (% of stained cells) was evaluated after 10 h by vital staining in 0.01% methylene blue, 2% sodium citrate (Rose, 1975). Cell growth was measured hy following the cell density photometrically for 2 weeks and a ODm reading of 10-fold diluted cultures is shown. in yeast (Theuvenet et al., 1986). Our findings suggest that activation of PKCa catalytic activity by phorbol ester stimulates a yeast Ca2+ net uptake mechanism, which may involve a proposed cell surface cation channel (Eilam et al., 1990).
During the late stage of the yeast mating pheromone response cell viability becomes dependent on extracellular Ca2+ (Iida et al., 1990b) and its influx into yeast cells is stimulated (Ohsumi and Anraku, 1985). To investigate whether bovine PKCa activation may cause Ca2+ dependence of yeast viability, cell proliferation was studied in Ca*+-free synthetic medium and cells were evaluated by vital staining. Although the growth or viability of control cells was unaffected by phorbol ester or lack of extracellular Ca", PMA-activation of PKCa caused complete lack of growth and led to accelerated loss of cell viability in up to 35% of cells as early as 7.5 h after PMA induction in Ca2+-free liquid medium as shown in Table I. In these experiments cell growth was measured by ODGoo reading for up to 2 weeks. Both responses, cell growth and viability, were restored in the presence of 1 mM extracellular Caz+ as shown in Table I.

DISCUSSION
Our findings indicate that bovine PKCa is fully functionally expressed in yeast and creates cellular phorbol ester binding sites. Expression products display substrate-specific calcium-, phospholipid-, and phorbol ester-responsive enzymatic activity in cell extracts in vitro and result in proportional biological consequences. Four responses which were stimulated by phorbol esters in vivo (PKC down-regulation, 4sCa2+ net uptake, Ca2+ dependence of cell viability, and altered cell morphology) all correlate well with the enzymatic activities measured in vitro.
We have detected PKCa in yeast whole cell detergent extracts (Fig. 1A) as well as in cytosolic and membrane fractions (Fig. 3A) consistent with its localization in mammalian cells (Kraft and Anderson, 1983). We have demonstrated disappearance of the protein from yeast membrane and cytosolic fractions upon PMA stimulation, which is consistent with PKC down-regulation. This has been described in mammalian cells and involves PKC proteolysis (Rodriguez-Pena and Rosengurt, 1984;Young et al., 1988). These findings indicate a compatible mechanism in yeast, which interacts with PKCa to cause its down-regulation, This may play a role in PKC action in mammalian cells, although this has not been demonstrated (Droms and Malkinson, 1991;Young et al., 1988). Ca2+ influx has been described in response to PKC stimulation in various eukaryotic experimental systems (Strong et al., 1987;Lacerda et al., 1988;Stojilkovic et al., 1988;Zherelova, 1989). In higher eukaryotic cells, including mammalian cells, the activation of specific ion channels has been implicated in this response, with durations ranging from readily reversible (<5 min) to persistent (>30 min) PKC effects by Bovine Protein Kinase C a in Yeast 3461 (Shearman et al., 1989). In yeast an energy-independent cell surface Ca2+ uptake mechanism has been reported that exploits an electrochemical gradient (Eilam et al., 1990). Ca2+ is subsequently transported into the yeast vacuole in an energydependent process via the Ca2+/H+ antiporter (Ohsumi and Anraku, 1983), which is fueled by a proton gradient generated b y the vacuolar H+-ATPase (Eilam et al., 1990). The reported yeast Ca2+ uptake mechanism is not inconsistent with our own measurements in yeast cells. In our assay we measure the accumulation of extracellularly added 45Ca2+ in the yeast cell from the technical short time limit of our experiments (10 min, not shown) up to 2 h. This accumulation is proportional to the activity of expressed PKCa. It is increased by phorbol ester stimulation and decreased to background levels if the kinase is inactivated as shown in Fig. 4A.
Part of the Ca2+ uptake response was measured in DEAEdextran-permeabilized cells (not shown), suggesting that it involves Ca2+ uptake into vacuoles, the major cellular stores for Caz+ in yeast (Theuvenet et al., 1986). Our standard assay does not allow us to discriminate between mechanisms that may control ion fluxes across the plasma membrane or between the cytosol and yeast vacuole. It also does not formally address the direction of the ion fluxes modulated by PKC. However, the net accumulation of extracellularly added 45Ca2+ we are observing in the cell suggests an underlying Ca2+ flux across the cell membrane into the cytosol and subsequently into the vacuole. PKCa may directly modulate 45Ca2+ influx into the cell and/or vacuole. Alternatively, our data are consistent with a constitutive influx mechanism, where 45Ca2+ efflux from the vacuole or across the membrane would be modulated by PKCa. Future unidirectional studies will be required to dissect the modulation of these ion fluxes in detail.
As shown in Fig. 4B, the PMA stimulation of PKC-mediated Ca2+ net uptake is specific for divalent Ca2+ ions as opposed t o monovalent Rb+/K+ ions. It remains largely unaffected by competition with monovalent ions Na+ and K+ and is not abolished by competition with divalent ions Mn2+ and Mg2+ (not shown). Increasing M$+ concentrations lead to reduced basal Ca2+ uptake, which results in a maximum PMA stimulation of up to 4-fold at 5 mM M e . This may be due to a partially shared transport system for the divalent ions Mg2+ and Ca2+ in yeast (Borbolla and Pena, 1980). The lack of M e suppression of Ca2+ uptake by PMA-stimulated PKC may be due to modulation of a different mechanism, which remains to be identified and may involve vacuolar Ca2+ transport. Amiloride, a blocker of Na+/H+ and Na+/Ca2+ exchange mechanisms, does not affect PKC-mediated Ca2+ uptake, whereas La3+, an inhibitor of carrier-mediated Ca2+ influx, abolishes the response and Cd" substantially increases the uptake even in control cells by raising the cation permeability of the yeast cell membrane (Borbolla and Pena, 1980;Eilam and Grossowicz, 1982;Kessels et al., 1987) as shown in Fig. 4. Our findings suggest that activation of PKCa catalytic activity by phorbol esters stimulates net Ca2+ uptake into the yeast cell. The mechanisms involved may share some characteristics of a proposed cell surface channel and may include Ca2+ fluxes between the cytosol and the yeast vacuole, the major cellular stores for Caz+ in yeast (Eilam et al., 1990;Theuvenet et al., 1986).
Several of the biological responses we observed after phor-bo1 ester stimulation of PKCa, stimulation of net Ca2+ uptake, dependence on extracellular Caz+ for cell viability, and changes in cell morphology have been reported upon activation of the mating pheromone response pathway (Marsh and Herskowitz, 1988;Iida et al., 1990b). Partial activation ofthis pathway at the initial receptor/G protein level has been shown after co-expression of mammalian &adrenergic receptors with mammalian G, a protein subunits and removal of inter-fering yeast G a subunits (King et al., 1990). This resulted in the "shmoo" phenotype and in induction of the FUSl gene promoter; however, actual cell mating was not stimulated, a result consistent with multiple roles of yeast pheromone receptors (King et al., 1990).
Since this is the only major signal transduction pathway characterized in yeast to date, we explored whether mammalian PKC might specifically interact with this pathway. We were unable to detect other established responses to this pathway upon PKCa stimulation, in particular, cell agglutination and induction of pathway-specific genes such as FUSl.
Weak PKCa responses were occasionally observed in PKCtransformed, diploid yeast cells (not shown), whereas the mating factor response is specific to haploid cells. These findings demonstrate that bovine PKCa stimulation does not activate the mating pheromone response pathway. Cellular responses to PKCa activation may be stimulated by other mechanisms, and signaling pathways may exist in yeast which have not yet been established. PKCa activation did not affect yeast cell viability in normal culture medium; however, it rendered cells calcium-dependent, since no cell growth was observed in calcium-free medium after PKC activation ( Table  I). The lack of growth correlated with decreased cell viability as determined by vital staining. PMA treatment lowered cell viability 3-fold in PKC-expressing cells. The calcium dependence of cell viability and ea2+ net uptake, which are both induced by PKCa activation, may be functionally related and involve common regulatory mechanisms that remain to be identified. Stimulation of net Caz+ uptake appears physiologically meaningful in a state of Ca2+ dependence.
The endogenous PKC background in yeast is still controversial. We have not observed any evidence for phorbol esterresponsive yeast PKC activity in any of our assays. This is consistent with a described PKC-related diacylglycerol-stimulated yeast protein activity, which does not significantly respond to phorbol esters and displays distinct substrate specificity (Ogita et al., 1990;Iwai et al., 1992). In combination with the specific responses observed in our assays, these findings suggest that mammalian PKC isoforms can be selectively activated in yeast by phorbol esters and studied in the presently undefined yeast cellular background. The related, essential yeast gene PKCl plays a role in cell cycle-specific osmotic stability; however, it is not complemented by mammalian PKC isoforms, including a, when disrupted (Levin et al., 1990;Levin and Bartlett-Heubusch, 1992) and consequently does not represent a functional yeast homolog of mammalian PKCa. The function of PKCl characterized in yeast suggests a role in a common pathway with genes of the SKcd and BCKl locus, possibly in bud morphogenesis (Fields and Thorner, 1991;Levin and Bartlett-Heubusch, 1992;Lee and Levin, 1992).
Since the functional counterparts of mammalian PKC genes have not been identified in yeast we have expressed an available mammalian PKC isoform to exploit this advantageous, rapid, lower eukaryotic experimental model to study mammalian PKC signaling. The normal catalytic function of bovine PKCa and its biological responses in yeast suggest that it may, in response to phorbol esters, participate in a pathway shared with putative functional yeast homologs to mammalian PKC isoforms that have not yet been characterized at the gene level. The report of mammalian-like PKC isoforms in yeast suggests that they may exist (Simon et al., 1991;, and bovine PKCa may activate the putative signaling pathway they participate in. The future dissection of these interactions should be facilitated by the experimental advantages of the yeast model. The knowledge gained should help to elucidate the yeast signaling pathway involved as well as the molecular mechanisms of mammalian PKC action. Our Calcium Uptake by Bovine Protein Kinase C cy in Yeast results suggest compatible aspects of higher and lower eukaryotic signaling pathways and support the perspective of dissecting parts of the action of common signaling mediators in a simple genetic model.