A putative two pore channel AtTPC1 mediates Ca(2+) flux in Arabidopsis leaf cells.

The gene encoding voltage-gated channel with high affinity for Ca(2+) permeation has not been cloned from plants. In the present study, we isolated a full-length cDNA encoding a putative Ca(2+ )channel (AtTPC1) from Arabidopsis. AtTPC1 has two conserved homologous domains, both of which contain six transmembrane segments (S1-S6) and a pore loop (P) between S5 and S6 in each domain, and has the highest homology with the two pore channel TPC1 recently cloned from rat. The overall structure is similar to the half of the general structure of alpha-subunits of voltage-activated Ca(2+) channels from animals. AtTPC1 rescued the Ca(2+) uptake activity of a yeast mutant cch1. Sucrose-induced luminescence, which reflects a cytosolic free Ca(2+) increase in aequorin-expressing Arabidopsis leaves, was enhanced by overexpression of AtTPC1 and suppressed by antisense expression of it. Sucrose-H(+) symporters AtSUC1 and 2, which depolarize membrane potential of cells receiving sucrose, also depressed a Ca(2+) increase by their antisense expression. These results suggest that AtTPC1 mediates a voltage-activated Ca(2+ )influx in Arabidopsis leaf cells.

Calcium influx and signaling in yeast stimulated by intracellular sphingosine 1-phosphate accumulation.

In mammalian cells, intracellular sphingosine 1-phosphate (S1P) can stimulate calcium release from intracellular organelles, resulting in the activation of downstream signaling pathways. The budding yeast Saccharomyces cerevisiae expresses enzymes that can synthesize and degrade S1P and related molecules, but their possible role in calcium signaling has not yet been tested. Here we examine the effects of S1P accumulation on calcium signaling using a variety of yeast mutants. Treatment of yeast cells with exogenous sphingosine stimulated Ca(2+) accumulation through two distinct pathways. The first pathway required the Cch1p and Mid1p subunits of a Ca(2+) influx channel, depended upon the function of sphingosine kinases (Lcb4p and Lcb5p), and was inhibited by the functions of S1P lyase (Dpl1p) and the S1P phosphatase (Lcb3p). The biologically inactive stereoisomer of sphingosine did not activate this Ca(2+) influx pathway, suggesting that the active S1P isomer specifically stimulates a calcium-signaling mechanism in yeast. The second Ca(2+) influx pathway stimulated by the addition of sphingosine was not stereospecific, was not dependent on the sphingosine kinases, occurred only at higher doses of added sphingosine, and therefore was likely to be nonspecific. Mutants lacking both S1P lyase and phosphatase (dpl1 lcb3 double mutants) exhibited constitutively high Ca(2+) accumulation and signaling in the absence of added sphingosine, and these effects were dependent on the sphingosine kinases. These results show that endogenous S1P-related molecules can also trigger Ca(2+) accumulation and signaling. Several stimuli previously shown to evoke calcium signaling in wild-type cells were examined in lcb4 lcb5 double mutants. All of the stimuli produced calcium signals independent of sphingosine kinase activity, suggesting that phosphorylated sphingoid bases might serve as messengers of calcium signaling in yeast during an unknown cellular response.

Inhibition of the Ca(2+)-ATPase Pmc1p by the v-SNARE protein Nyv1p.

Pmc1p, the Ca(2+)-ATPase of budding yeast related to plasma membrane Ca(2+)-ATPases of animals, is transcriptionally up-regulated in response to signaling by the calmodulin-calcineurin-Tcn1p/Crz1p signaling pathway. Little is known about post-translational regulation of Pmc1p. In a genetic screen for potential negative regulators of Pmc1p, a vacuolar v-SNARE protein, Nyv1p, was recovered. Cells overproducing Nyv1p show decreased Ca(2+) tolerance and decreased accumulation of Ca(2+) in the vacuole, similar to pmc1 null mutants. Overexpression of Nyv1p had no such effects on pmc1 mutants, suggesting that Nyv1p may inhibit Pmc1p function. Overexpression of Nyv1p did not decrease Pmc1p levels but decreased the specific ATP-dependent Ca(2+) transport activity of Pmc1p in purified vacuoles by at least 2-fold. The effect of Nyv1p on Pmc1p function is likely to be direct because native immunoprecipitation experiments showed that Pmc1p coprecipitated with Nyv1p. Complexes between Nyv1p and its t-SNARE partner Vam3p were also isolated, but these complexes lacked Pmc1p. We conclude that Nyv1p can interact physically with Pmc1p and inhibit its Ca(2+) transport activity in the vacuole membrane. This is the first example of a Ca(2+)-ATPase regulation by a v-SNARE protein involved in membrane fusion reactions.

Differential regulation of two Ca(2+) influx systems by pheromone signaling in Saccharomyces cerevisiae.

The budding yeast Saccharomyces cerevisiae generates calcium signals during the response to mating pheromones that promote survival of unmated cells. A Ca(2+) channel composed of Cch1p and Mid1p was previously shown to be necessary for the production of these calcium signals. However, we find that the Cch1p-Mid1p high-affinity Ca(2+) influx system (HACS) contributes very little to signaling or survival after treatment with alpha-factor in rich media. HACS activity was much greater after calcineurin inactivation or inhibition, suggesting the Cch1p-Mid1p Ca(2+) channel is subject to direct or indirect regulation by calcineurin. Instead a distinct low-affinity Ca(2+) influx system (LACS) was stimulated by pheromone signaling in rich medium. LACS activity was insensitive to calcineurin activity, independent of Cch1p and Mid1p, and sufficient to elevate cytosolic free Ca(2+) concentrations ([Ca(2+)]c) in spite of its 16-fold lower affinity for Ca(2+). Overexpression of Ste12p or constitutive activation of this transcription factor in dig1 dig2 double mutants had no effect on LACS activity but stimulated HACS activity when calcineurin was also inactivated. Ste12p activation had no effect on Cch1p or Mid1p abundance, suggesting the involvement of another target of Ste12p in HACS stimulation. LACS activation required treatment with mating pheromone even in dig1 dig2 double mutants and also required FAR1, SPA2, and BNI1, which are necessary for proper cell cycle arrest and polarized morphogenesis. These results show that distinct branches of the pheromone-signaling pathway independently regulate HACS and LACS activities, either of which can promote survival during long-term responses.

A homolog of voltage-gated Ca(2+) channels stimulated by depletion of secretory Ca(2+) in yeast.

In animal cells, capacitative calcium entry (CCE) mechanisms become activated specifically in response to depletion of calcium ions (Ca(2+)) from secretory organelles. CCE serves to replenish those organelles and to enhance signaling pathways that respond to elevated free Ca(2+) concentrations in the cytoplasm. The mechanism of CCE regulation is not understood because few of its essential components have been identified. We show here for the first time that the budding yeast Saccharomyces cerevisiae employs a CCE-like mechanism to refill Ca(2+) stores within the secretory pathway. Mutants lacking Pmr1p, a conserved Ca(2+) pump in the secretory pathway, exhibit higher rates of Ca(2+) influx relative to wild-type cells due to the stimulation of a high-affinity Ca(2+) uptake system. Stimulation of this Ca(2+) uptake system was blocked in pmr1 mutants by expression of mammalian SERCA pumps. The high-affinity Ca(2+) uptake system was also stimulated in wild-type cells overexpressing vacuolar Ca(2+) transporters that competed with Pmr1p for substrate. A screen for yeast mutants specifically defective in the high-affinity Ca(2+) uptake system revealed two genes, CCH1 and MID1, previously implicated in Ca(2+) influx in response to mating pheromones. Cch1p and Mid1p were localized to the plasma membrane, coimmunoprecipitated from solubilized membranes, and shown to function together within a single pathway that ensures that adequate levels of Ca(2+) are supplied to Pmr1p to sustain secretion and growth. Expression of Cch1p and Mid1p was not affected in pmr1 mutants. The evidence supports the hypothesis that yeast maintains a homeostatic mechanism related to CCE in mammalian cells. The homology between Cch1p and the catalytic subunit of voltage-gated Ca(2+) channels raises the possibility that in some circumstances CCE in animal cells may involve homologs of Cch1p and a conserved regulatory mechanism.

A conserved family of calcineurin regulators.

The protein phosphatase calcineurin mediates many cellular responses to calcium signals. Using a genetic screen in yeast, we identified a new family of proteins conserved in fungi and animals that inhibit calcineurin function when overexpressed. Overexpression of the yeast protein Rcn1p or the human homologs DSCR1 or ZAKI-4 inhibited two independent functions of calcineurin in yeast: The activation of the transcription factor Tcn1p and the inhibition of the H(+)/Ca(2+) exchanger Vcx1p. Purified recombinant Rcn1p and DSCR1 bound calcineurin in vitro and inhibited its protein phosphatase activity. Signaling via calmodulin, calcineurin, and Tcn1p induced Rcn1p expression, suggesting that Rcn1p operates as an endogenous feedback inhibitor of calcineurin. Surprisingly, rcn1 null mutants exhibited phenotypes similar to those of Rcn1p-overexpressing cells. This effect may be due to lower expression of calcineurin in rcn1 mutants during signaling conditions. Thus, Rcn1p levels may fine-tune calcineurin signaling in yeast. The structural and functional conservation between Rcn1p and DSCR1 suggests that the mammalian Rcn1p-related proteins, termed calcipressins, will modulate calcineurin signaling in humans and potentially contribute to disorders such as Down Syndrome.

DSCR1, overexpressed in Down syndrome, is an inhibitor of calcineurin-mediated signaling pathways.

Down syndrome is one of the major causes of mental retardation and congenital heart malformations. Other common clinical features of Down syndrome include gastrointestinal anomalies, immune system defects and Alzheimer's disease pathological and neurochemical changes. The most likely consequence of the presence of three copies of chromosome 21 is the overexpression of its resident genes, a fact which must underlie the pathogenesis of the abnormalities that occur in Down syndrome. Here we show that DSCR1, the product of a chromosome 21 gene highly expressed in brain, heart and skeletal muscle, is overexpressed in the brain of Down syndrome fetuses, and interacts physically and functionally with calcineurin A, the catalytic subunit of the Ca(2+)/calmodulin-dependent protein phosphatase PP2B. The DSCR1 binding region in calcineurin A is located in the linker region between the calcineurin A catalytic domain and the calcineurin B binding domain, outside of other functional domains previously defined in calcineurin A. DSCR1 belongs to a family of evolutionarily conserved proteins with three members in humans: DSCR1, ZAKI-4 and DSCR1L2. We further demonstrate that overexpression of DSCR1 and ZAKI-4 inhibits calcineurin-dependent gene transcription through the inhibition of NF-AT translocation to the nucleus. Together, these results suggest that members of this newly described family of human proteins are endogenous regulators of calcineurin-mediated signaling pathways and as such, they may be involved in many physiological processes.

Functional expression of mung bean Ca2+/H+ antiporter in yeast and its intracellular localization in the hypocotyl and tobacco cells.

The Ca2+-transport activity and intracellular localization of the translation product of cDNA for mung bean Ca2+/H+ antiporter (VCAX1) were examined. When the cDNA was expressed in Saccharomyces cerevisiae that lacked its own genes for vacuolar Ca2+-ATPase and the antiporter, VCAX1 complemented the active Ca2+ transporters, and the microsomal membranes from the transformant showed high activity of the Ca2+/H+ antiporter. Treatment of the vacuolar membranes with a cross-linking reagent resulted in a clear band of the dimer detected with antibody specific for VCAX1p. The antibody was also used for immunolocalization of the antiporter in fractions obtained by sucrose-density-gradient centrifugation of the microsomal fraction from mung bean. The immunostained band was detected in the vacuolar membrane fraction and the slightly heavy fractions that exhibited activity of the Golgi marker enzyme. A fusion protein of VCAX1p and green fluorescent protein was expressed in tobacco cells. The green fluorescence was clearly observed on the vacuolar membrane and, in some cases, in the small vesicles. The subcellular fractionation of transformed tobacco cells confirmed the vacuolar membrane localization of the fusion protein. These results confirm that VCAX1p functions in the vacuolar membrane as a Ca2+/H+ antiporter and also suggest that VCAX1p may exist in the Golgi apparatus.

The Saccharomyces cerevisiae RanGTP-binding protein msn5p is involved in different signal transduction pathways.

In eukaryotes, control of transcription by extracellular signals involves the translocation to the nucleus of at least one component of the signal transduction pathway. Transport through the nuclear envelope requires the activity of an import or export receptor that interacts with the small GTPase Ran. We have cloned the MSN5 gene of the yeast Saccharomyces cerevisiae that is postulated to encode one of these receptors. Msn5p belongs to a family of proteins with a conserved N-terminal sequence that acts as a RanGTP-binding domain. The results presented here provide genetic data supporting Msn5p involvement in several different signal transduction pathways. All of these pathways include changes in gene expression, and regulated nucleocytoplasmic redistribution of a component in response to external conditions has already been described in some of them. We have cloned MSN5 following two different strategies. Msn5p was constitutively localized in the nucleus. Phenotypic analysis of the msn5 mutant demonstrated that this protein participates in processes such as catabolite repression, calcium signaling, mating, and cell proliferation, as well as being involved in previously characterized phosphate utilization. Therefore, Msn5p could be a receptor for several proteins involved in different signaling pathways.

Calcium influx factor is synthesized by yeast and mammalian cells depleted of organellar calcium stores.

Depletion of endoplasmic reticulum Ca2+ stores leads to the entry of extracellular Ca2+ into the cytoplasm, a process termed capacitative or store-operated Ca2+ entry. Partially purified extracts were prepared from the human Jurkat T lymphocyte cell line and yeast in which Ca2+ stores were depleted by chemical and genetic means, respectively. After microinjection into Xenopus laevis oocytes, the extracts elicited a wave of increased cytoplasmic free Ca2+ ([Ca2+]i) that spread from the point of injection across the oocyte. Extracts from cells with replete organellar Ca2+ stores were inactive. The increases depended on extracellular Ca2+, were unaffected by the inositol 1,4,5-trisphosphate (IP3) inhibitor heparin or an anti-IP3 receptor antibody and were unchanged when the endoplasmic reticulum was segregated to the hemisphere opposite the injection site by centrifugation. Confocal microscopy revealed that [Ca2+]i increases were most pronounced at the periphery of the oocyte. The patterns of [Ca2+]i increases were replicated by computer simulations based on a diffusible messenger of about 700 Da that directly activates Ca2+ influx. In addition, ICRAC, a Ca2+ release-activated Ca2+ current monitored in Jurkat cells by whole-cell patch clamp recordings, was more rapidly activated when active extracts were included in the patch pipette than by the inclusion of a Ca2+ chelator or IP3. These data support the existence in yeast and mammalian cells depleted of Ca2+ stores of a functionally conserved diffusible calcium influx factor that directly activates Ca2+ influx.

Intracellular sequestration of sodium by a novel Na+/H+ exchanger in yeast is enhanced by mutations in the plasma membrane H+-ATPase. Insights into mechanisms of sodium tolerance.

Sodium tolerance in yeast is disrupted by mutations in calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, which is required for modulation of Na+ uptake and efflux mechanisms. Five Na+-tolerant mutants were isolated by selecting for suppressors of calcineurin mutations, and mapped to the PMA1 gene, encoding the plasma membrane H+-ATPase. One mutant, pma1-alpha4, which has the single amino acid change Glu367 --> Lys at a highly conserved site within the catalytic domain of the ATPase, was analyzed in detail to determine the mechanism of Na+ tolerance. After exposure to Na+ in the culture medium, 22Na influx in the pma1 mutant was reduced 2-fold relative to control, consistent with a similar decrease in ATPase activity. Efflux of 22Na from intact cells was relatively unchanged in the pma1 mutant. However, selective permeabilization of the plasma membrane revealed that mutant cells retained up to 80% of intracellular Na+ within a slowly exchanging pool. We show that NHX1, a novel gene homologous to the mammalian NHE family of Na+/H+ exchangers, is required for Na+ sequestration in yeast and contributes to the Na+-tolerant phenotype of pma1-alpha4.

ECA1 complements yeast mutants defective in Ca2+ pumps and encodes an endoplasmic reticulum-type Ca2+-ATPase in Arabidopsis thaliana.

To understand the structure, role, and regulation of individual Ca2+ pumps in plants, we have used yeast as a heterologous expression system to test the function of a gene from Arabidopsis thaliana (ECA1). ECA1 encoded a 116-kDa polypeptide that has all the conserved domains common to P-type Ca2+ pumps (EC 3.6.1.38). The amino acid sequence shared more identity with sarcoplasmic/endoplasmic reticulum (53%) than with plasma membrane (32%) Ca2+ pumps. Yeast mutants defective in a Golgi Ca2+ pump (pmr1) or both Golgi and vacuolar Ca2+ pumps (pmr1 pmc1 cnb1) were sensitive to growth on medium containing 10 mM EGTA or 3 mM Mn2+. Expression of ECA1 restored growth of either mutant on EGTA. Membranes were isolated from the pmr1 pmc1 cnb1 mutant transformed with ECA1 to determine if the ECA1 polypeptide (ECA1p) could be phosphorylated as intermediates of the reaction cycle of Ca2+-pumping ATPases. In the presence of [gamma-32P]ATP, ECA1p formed a Ca2+-dependent [32P]phosphoprotein of 106 kDa that was sensitive to hydroxylamine. Cyclopiazonic acid, a blocker of animal sarcoplasmic/endoplasmic reticulum Ca2+ pumps, inhibited the formation of the phosphoprotein, whereas thapsigargin did not. Immunoblotting with an antibody against the carboxyl tail showed that ECA1p was associated mainly with the endoplasmic reticulum membranes isolated from Arabidopsis plants. The results support the model that ECA1 encodes an endoplasmic reticulum-type Ca2+ pump in Arabidopsis. The ability of ECA1p to restore growth of mutant pmr1 on medium containing Mn2+, and the formation of a Mn2+-dependent phosphoprotein suggested that ECA1p may also regulate Mn2+ homeostasis by pumping Mn2+ into endomembrane compartments of plants.

Tcn1p/Crz1p, a calcineurin-dependent transcription factor that differentially regulates gene expression in Saccharomyces cerevisiae.

Ca2+ signals regulate gene expression in animal and yeast cells through mechanisms involving calcineurin, a protein phosphatase activated by binding Ca2+ and calmodulin. Tcn1p, also named Crz1p, was identified as a transcription factor in yeast required for the calcineurin-dependent induction of PMC1, PMR1, PMR2A, and FKS2 which confer tolerance to high Ca2+, Mn2+, Na+, and cell wall damage, respectively. Tcn1p was not required for other calcineurin-dependent processes, such as inhibition of a vacuolar H+/Ca2+ exchanger and inhibition of a pheromone-stimulated Ca2+ uptake system, suggesting that Tcn1p functions downstream of calcineurin on a branch of the calcium signaling pathway leading to gene expression. Tcn1p contains three zinc finger motifs at its carboxyl terminus resembling the DNA-binding domains of Zif268, Swi5p, and other transcription factors. When fused to the transcription activation domain of Gal4p, the carboxy terminal domain of Tcn1p directed strong calcineurin-independent expression of PMC1-lacZ and other target genes. The amino-terminal domain of Tcn1p was found to function as a calcineurin-dependent transcription activation domain when fused to the DNA-binding domain of Gal4p. This amino-terminal domain also formed Ca2+-dependent and FK506-sensitive interactions with calcineurin in the yeast two-hybrid assay. These findings suggest that Tcn1p functions as a calcineurin-dependent transcription factor. Interestingly, induction of Tcn1p-dependent genes was found to be differentially controlled in response to physiological Ca2+ signals generated by treatment with mating pheromone and high salt. We propose that different promoters are sensitive to variations in the strength of Ca2+ signals generated by these stimuli and to effects of other signaling pathways.

Glucose repression affects ion homeostasis in yeast through the regulation of the stress-activated ENA1 gene.

In this report we show that the ENA1/PMR2A gene is under glucose repression. The SNF1 protein kinase, acting independently from the HOG and calcineurin pathways, is essential to release ENA1 from glucose repression. The transcriptional repressor Ssn6p negatively regulates ENA1 expression and, like other glucose repressible genes, this repression is mediated in part by Mig1p. Deletion of a fragment from the ENA1 promoter that includes two Mig1p consensus binding sites gives a high level of expression in glucose without added salt. We suggest that regulation of ENA1 by the SNF1 pathway could be part of a general mechanism through which yeast cells respond to carbon source starvation by activating protective systems against different types of stress.

CAX1, an H+/Ca2+ antiporter from Arabidopsis.

Reestablishment of the resting state after stimulus-coupled elevations of cytosolic-free Ca2+ requires the rapid removal of Ca2+ from the cytosol of plant cells. Here we describe the isolation of two genes, CAX1 and CAX2, from Arabidopsis thaliana that suppress a mutant of Saccharomyces cerevisiae that has a defect in vacuolar Ca2+ accumulation. Both genes encode polypeptides showing sequence similarities to microbial H+/Ca2+ antiporters. Experiments on vacuolar membrane-enriched vesicles isolated from yeast expressing CAX1 or CAX2 demonstrate that these genes encode high efficiency and low efficiency H+/Ca2+ exchangers, respectively. The properties of the CAX1 gene product indicate that it is the high capacity transporter responsible for maintaining low cytosolic-free Ca2+ concentrations in plant cells by catalyzing pH gradient-energized vacuolar Ca2+ accumulation.

Calcineurin inhibits VCX1-dependent H+/Ca2+ exchange and induces Ca2+ ATPases in Saccharomyces cerevisiae.

The PMC1 gene in Saccharomyces cerevisiae encodes a vacuolar Ca2+ ATPase required for growth in high-Ca2+ conditions. Previous work showed that Ca2+ tolerance can be restored to pmc1 mutants by inactivation of calcineurin, a Ca2+/calmodulin-dependent protein phosphatase sensitive to the immunosuppressive drug FK506. We now report that calcineurin decreases Ca2+ tolerance of pmc1 mutants by inhibiting the function of VCX1, which encodes a vacuolar H+/Ca2+ exchanger related to vertebrate Na+/Ca2+ exchangers. The contribution of VCX1 in Ca2+ tolerance is low in strains with a functional calcineurin and is high in strains which lack calcineurin activity. In contrast, the contribution of PMC1 to Ca2+ tolerance is augmented by calcineurin activation. Consistent with these positive and negative roles of calcineurin, expression of a vcx1::lacZ reporter was slightly diminished and a pmc1::lacZ reporter was induced up to 500-fold by processes dependent on calcineurin, calmodulin, and Ca2+. It is likely that calcineurin inhibits VCX1 function mainly by posttranslational mechanisms. Activities of VCX1 and PMC1 help to control cytosolic free Ca2+ concentrations because their function can decrease pmc1::lacZ induction by calcineurin. Additional studies with reporter genes and mutants indicate that PMR1 and PMR2A, encoding P-type ion pumps required for Mn2+ and Na+ tolerance, may also be induced physiologically in response to high-Mn2+ and -Na+ conditions through calcineurin-dependent mechanisms. In these situations, inhibition of VCX1 function may be important for the production of Ca2+ signals. We propose that elevated cytosolic free Ca2+ concentrations, calmodulin, and calcineurin regulate at least four ion transporters in S. cerevisiae in response to several environmental conditions.

Mutations in PMR1 suppress oxidative damage in yeast cells lacking superoxide dismutase.

Mutants of Saccharomyces cerevisiae lacking a functional SOD1 gene encoding Cu/Zn superoxide dismutase (SOD) are sensitive to atmospheric levels of oxygen and are auxotrophic for lysine and methionine when grown in air. We have previously shown that these defects of SOD-deficient yeast cells can be overcome through mutations in either the BSD1 or BSD2 (bypass SOD defects) gene. In this study, the wild-type allele of BSD1 was cloned by functional complementation and was physically mapped to the left arm of chromosome VII. BSD1 is identical to PMR1, encoding a member of the P-type ATPase family that localizes to the Golgi apparatus. PMR1 is thought to function in calcium metabolism, and we provide evidence that PMR1 also participates in the homeostasis of manganese ions. Cells lacking a functional PMR1 gene accumulate elevated levels of intracellular manganese and are also extremely sensitive to manganese ion toxicity. We demonstrate that mutations in PMR1 bypass SOD deficiency through a mechanism that depends on extracellular manganese. Collectively, these findings indicate that oxidative damage in a eukaryotic cell can be prevented through alterations in manganese homeostasis.

Ca2+ transport in Saccharomyces cerevisiae.

Cytosolic free Ca2+ is maintained at submicromolar levels in budding yeast by the activity of Ca2+ pumps and antiporters. We have recently identified the structural genes for two Ca2+ pumps, PMC1 [correction of PCM1] and PMR1, which are required for Ca2+ sequestration into the vacuole and secretory organelles, respectively. The function of either Ca2+ pump is sufficient for yeast viability, but deletion of both genes is lethal because of elevation of cytosolic [Ca2+] and activation of calcineurin, a Ca(2+)- and calmodulin-dependent protein phosphatase. Calcineurin activation decreases Ca2+ sequestration in the vacuole by a putative Ca2+ antiporter and may also increase Ca2+ pump activity. These regulatory processes can affect the ability of yeast strains to tolerate high extracellular [Ca2+]. We propose a model in which the cellular response to changes in the environmental levels of Ca2+ is mediated by calmodulin and calcineurin which, in turn, modulate the various types of Ca2+ transporters.

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.

Yeast KEX2 protease and mannosyltransferase I are localized to distinct compartments of the secretory pathway.

The KEX2 protease (product of the KEX2 gene) functions late in the secretory pathway of Saccharomyces cerevisiae by cleaving the polypeptide chains of prepro-killer toxin and prepro-alpha-factor at paired basic amino acid residues. The intracellular vesicles containing KEX2 protease sedimented in density gradients to a position distinct from those containing mannosyltransferase I (product of the MNN1 gene), a marker enzyme for the Golgi complex. The recovery of intact compartments containing these enzymes approached 80% after sedimentation. We propose that the KEX2 protease and mannosyltransferase I reside within distinct compartments.