Calcineurin-dependent Growth Control in Saccharomyces cerevisiae

Ca 2÷ ATPases deplete the cytosol of Ca 2÷ ions and are crucial to cellular Ca 2÷ homeostasis. The 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. Address all correspondence to Gerald R. Fink, Whitehead Institute, Massachusetts Institute of Technology, 9 Cambridge Center, Cambridge, MA 02142. 1. Abbreviations used in this paper: [Ca2+]c, cystolic free Ca 2+ concentration; SERCA, sarco/endoplasmic reticulum Ca 2÷ ATPase; PMCA, plasma membrane Ca 2÷ ATPase. 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 © The Rockefeller University Press, 0021-9525/94/02/351/13 $2.00 The Journal of Cell Biology, Volume 124, Number 3, February 1994 351-363 351 on July 7, 2017 jcb.rress.org D ow nladed fom 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. Materials and Methods