ABSTRACT
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, 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 Ca2+-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.
INTRODUCTION
The budding yeast Saccharomyces cerevisiae, like other eukaryotes, actively maintains cytosolic free Ca2+ concentrations [Ca2+]i at extremely low levels in spite of very steep gradients of this ion across the plasma membrane and across intracellular membranes. It is generally believed that this asymmetric distribution avoids aggregation of Ca2+ with phosphate-containing molecules in the cytosol while still providing various organelles with sufficient Ca2+ for their proper function. Superimposed on the need for low [Ca2+]i is the requirement for Ca2+ as a second messenger in signal transduction. Transient increases in [Ca2+]i regulate a wide variety of cellular processes in other species and there is now good evidence that Ca2+ signaling is important in yeast as well. Furthermore, yeast expresses the same repertoire of signaling molecules as that used in animal cells (calmodulin and calmodulin-dependent protein kinases and phosphatases). Because of this similarity, the origin of Ca2+ signals and the individual roles of these effector molecules in yeast has become a burgeoning field (for a review, see Davis, 1994).
At the heart of this complex and highly regulated process are a battery of Ca2+ channels, antiporters and pumps, which are primarily responsible for maintaining and altering the Ca2+ levels in the various compartments. Progress in understanding the individual roles of these transporters has increased dramatically with the recent cloning and molecular characterization of several key components. The ability to manipulate genetically all of the individual Ca2+ transporters in conjunction with the downstream Ca2+ signaling factors in yeast provides a powerful new perspective on the ubiquitous problem of cellular Ca2+ homeostasis and signaling. This article summarizes many of the recent advances in our understanding of Ca2+ transporters and Ca2+ flow in yeast.
Ca2+ channels
In eukaryotic cells, Ca2+ signals are usually initiated by the triggered opening of Ca2+ channels in the plasma membrane and certain organellar membranes, which allows a rapid influx of Ca2+ into the cytosol down its concentration gradient. The massive influx of Ca2+, which typically increases [Ca2+]i 10-to 100-fold over the basal level of approximately 0.1 μmol l−1, is soon followed by channel closure and active removal of Ca2+ from the cytosol by the Ca2+ antiporters and pumps. Transient spikes and oscillations in [Ca2+]i generated by this coordinated process are known to regulate a wide variety of processes in non-excitable cells; for example, exocytosis, gene expression and cell-cycle progression. In yeast, Ca2+ signals may regulate similar processes (for reviews, see Anraku et al. 1991; Davis, 1994; Youatt, 1993). Progress in understanding Ca2+ signaling has been slow because of limitations in the direct measurement of [Ca2+]i and difficulties in quantifying Ca2+ channel activity in yeast.
Though Ca2+ channels have not been isolated or cloned from yeast, there is good evidence that these transporters exist. The patch-clamp technique has revealed a stretch-activated or mechanosensitive ion channel in the plasma membrane that passes many ions including Ca2+ (Gustin et al. 1988). Additionally, increased rates of Ca2+ influx into living yeast cells have been observed during the G1/S transition in the cell division cycle, during cell cycle arrest in late G1 caused by either mating pheromones (Ohsumi and Anraku, 1985) or certain temperature-sensitive mutations (Anand and Prasad, 1987; Prasad and Rosoff, 1992), and during the response to nutrient feeding (Eilam and Othman, 1990; Eilam et al. 1990; Nakajima et al. 1991). Another possible Ca2+ channel has been detected in purified membrane vesicles derived from the yeast vacuole (Belde et al. 1993). These vesicles accumulate Ca2+in vitro and release a small portion in response to added inositol-1,4,5-trisphosphate (InsP3), suggesting a similarity to the InsP3 receptor in the endoplasmic reticulum of animal cells. At present, it is unclear when and how the channel activities are triggered and what processes might be affected by their opening.
Vacuolar H+/Ca2+ antiport
Biochemical experiments indicate that the yeast vacuolar membrane actively transports Ca2+via H+/Ca2+ antiport (Dunn et al. 1994; Ohsumi and Anraku, 1983; Okorokov et al. 1985). Ca2+ uptake activity into purified vacuoles and vacuole membrane vesicles is completely dependent on the transmembrane pH gradient ΔpH (interior acid) that is normally produced by the vacuolar H+ V-ATPase, though some uptake still occurs in the absence of ATP if the ΔpH is generated by chemical ion diffusion gradients (Dunn et al. 1994). A potential difference Δ Ψ (interior positive) did not promote Ca2+ uptake. Uptake into isolated vacuoles is saturable by cytosolic Ca2+ and displays an apparent Km for Ca2+ at 25–50 μmol l−1, which is much higher than the [Ca2+]i observed in living cells (approximately 0.15 μmol l−1). The antiporter has not been isolated, its structural gene has not been cloned and no mutants are available to address its specific functions and roles in yeast. Recently though, we have isolated a yeast gene whose predicted product is homologous to the retinal Na+/Ca2+,K+ and cardiac Na+/Ca2+ exchangers from mammals and appears to be required for optimal Ca2+ sequestration into the vacuole in vivo (K. W. Cunningham and G. R. Fink, in preparation). Therefore, it is possible that the cloned gene encodes the previously characterized low-affinity H+/Ca2+ antiporter or possibly some other type of Ca2+ transporter.
Pmc1p: a vacuolar Ca2+ pump
The yeast vacuole membrane also contains a putative high-affinity Ca2+ pump, Pmc1p, which is the product of the PMC1 gene (Cunningham and Fink, 1994). Pmc1p is approximately 40% identical to plasma membrane Ca2+-ATPases (PMCAs) and is much less similar to other P-type ion pumps (Fig. 1). Pmc1p apparently lacks the calmodulin-binding domain at the C terminus, but otherwise appears to be a functional ion pump localized predominantly to the vacuole membrane. By analogy to the animal enzyme, Pmc1p is expected to catalyze the high-affinity (Km approximately 1 μmol l−1) and ATP-dependent transport of Ca2+ into the vacuole. Such an activity was not observed in previous experiments using isolated vacuole membrane vesicles (Dunn et al. 1994; Ohsumi and Anraku, 1983). It is possible that the Ca2+ transport activity of Pmc1p was obscured by the much greater H+/Ca2+ antiport activity in these experiments. Alternatively, Pmc1p may have unexpected properties, such as instability, low Ca2+ affinity, sensitivity to protonophores, or other characteristics that prevented its earlier detection. In support of its function as a Ca2+ transporter in living yeast cells, mutants lacking Pmc1p accumulate Ca2+ in the vacuole at less than 20% of the wild-type rate during growth in standard medium (Cunningham and Fink, 1994). The pmc1 null mutants also display a severe sensitivity to Ca2+ supplements in the growth medium. Although it is likely that Pmc1p transports Ca2+ into the vacuole, further biochemical experiments are necessary to demonstrate its activities. The biochemical properties of Pmc1p should be resolved through studies of isolated Pmc1p or of vacuole membrane vesicles prepared from mutants specifically lacking the H+/Ca2+ antiport activity.
Pmr1p: a secretory Ca2+ pump
Another putative Ca2+ pump localizes to the Golgi complex or related secretory compartments and is encoded by the PMR1 gene (Antebi and Fink, 1992; Rudolph et al. 1989). Pmr1p is approximately 50% identical to a P-type ion pump of unknown function that is expressed in many animal tissues (Gunteski-Hamblin et al. 1992), approximately 30% identical to members of the SERCA sub-family, which are Ca2+-ATPases found in the sarcoplasmic/endoplasmic reticulum of animal cells, and less than 25% identical to other ion pumps (Fig. 1). The biochemical activities of Pmr1p have not been investigated, but several lines of genetic evidence suggest that it functions as a primary Ca2+ transporter supplying Ca2+ to compartments in the secretory pathway (Antebi and Fink, 1992; Rudolph et al. 1989). Mutants lacking Pmr1p function secrete abnormal proteins that have not been proteolytically cleaved by a Ca2+-dependent protease located in a late Golgi compartment, though this defect and others can be remedied by supplementing the growth medium with Ca2+ concentrations greater than 10 mmol l−1. Decreasing extracellular Ca2+ to below 1 μmol l−1 causes a severe growth defect in pmr1 null mutants (Antebi and Fink, 1992; Rudolph et al. 1989). These and other results (see below) strongly suggest that Pmr1p functions as a primary Ca2+ transporter that supplies the Golgi with the Ca2+ required for specific secretory functions.
Ca2+ transport in the endoplasmic reticulum
Ca2+ is generally thought to play important roles in protein traffic in the endoplasmic reticulum (ER) and related secretory compartments (Sambrook, 1990). To date, there is no evidence in yeast for the existence of authentic SERCA-type Ca2+ pumps. With the possible exception of Pmr1p, the known P-type ATPases in yeast are not homologous to the SERCA family members from plants and animals and have functions unrelated to Ca2+ transport. The PMA1 gene encodes the major P-type H+-ATPase of the plasma membrane (Serrano et al. 1986) and PMA2 encodes a transporter of unknown function that is 90% identical to the PMA1 gene product (Schlesser et al. 1988). Pmr2p, which is encoded by at least four tandemly repeated genes, is likely to be a plasma membrane ion pump involved in Na+ and Li+ efflux but not in Ca2+ transport (Garciadeblas et al. 1993; Haro et al. 1991; Rudolph et al. 1989). The predicted products of PMR2 genes are more than 97% identical to each other and have high degree of similarity to the CTA3 gene product of the fission yeast Schizosaccharomyces pombe (Fig. 1) that has been implicated in Ca2+ metabolism (Ghislain et al. 1990; Halachmi et al. 1992). Finally, the DRS2 gene is expected to encode a highly divergent P-type ion pump of unknown catalytic function (Ripmaster et al. 1993). Whether any of these proteins (Pma1p, Pma2p, Pmr2p or Drs2p) is involved in Ca2+ transport into the ER or other membrane compartments is not known, but unidentified Ca2+ transporters have been measured in some membrane preparations (Hiraga et al. 1991; Okorokov et al. 1993).
Ca2+-sensitive mutants
A genetic approach towards identifying important factors in Ca2+ metabolism has been to isolate yeast mutants with altered responses to Ca2+ in the growth medium (Ohya et al. 1984, 1986). Wild-type yeast strains can grow in media containing more than 100 mmol l−1 Ca2+, but recessive mutations in at least 18 genes abolish growth under these conditions (Ohya et al. 1986). Many of these genes appear to be necessary for maintaining the proper structure or function of the vacuole. Mutations that inactivate subunits of the vacuolar H+ V-ATPase or other factors necessary for acidification of the vacuole lumen cause extreme sensitivity to added CaCl2 and cause about a sixfold elevation in [Ca2+]i in standard media as measured in single cells using the fluorescent indicator Fura-2 (Ohya et al. 1991). Deactivation of the vacuolar H+/Ca2+ antiporter has been proposed to explain these effects, but other indirect mechanisms are also possible.
A second search for mutants specifically sensitive to Ca2+ revealed the CSG2 gene (Beeler et al. 1994). In response to elevated external [Ca2+], csg2 mutants accumulate Ca2+ into an exchangeable pool rather than into the non-exchangeable (vacuolar) pool. Therefore, Csg2p may normally function to promote Ca2+ efflux from this unidentified compartment, to inhibit Pmr1p or another non-vacuolar Ca2+ transporter, or to influence cellular Ca2+ flow by a more indirect mechanism (Beeler et al. 1994). CSG2 is identical to CLS2 identified in the screen for Ca2+-sensitive mutants described above (Y. Takita, Y. Ohya and Y. Anraku, in preparation). The predicted product of CSG2/CLS2 has no significant similarity to other protein sequences, but contains multiple membrane-spanning domains and is localized to the ER (Y. Takita, Y. Ohya and Y. Anraku, in preparation). Further biochemical and genetic analyses may clarify the function of this interesting protein and define the roles of the other CLS genes in Ca2+ tolerance.
Ca2+ flow and dynamics
A working model of Ca2+ metabolism that takes into account the new findings is depicted in Fig. 2. Yeast cells growing in standard media (approximately 0.2 mmol l−1 Ca2+) would take up Ca2+ from the medium via the opening of unidentified Ca2+ channels in the plasma membrane or through other unspecified mechanisms. The steady-state [Ca2+]i of about 0.1 μmol l−1 is maintained through the combined action of Pmr1p in the Golgi complex, Pmc1p in the vacuole, the H+/Ca2+ antiporter in the vacuole and possibly unidentified transporters in other organelles such as the plasma membrane. It also seems likely that vesicle-mediated transport processes ultimately contribute to the sequestration of Ca2+ in the vacuole and export of Ca2+ from the cell concomitant with protein targeting. Since Pmr1p and other Ca2+ transporters are presumably synthesized in the ER and then sorted to their final destinations, their operation within secretory compartments might significantly affect the overall flow of Ca2+ in the cell (Fig. 2). Yeast mitochondria accumulate little Ca2+ and their role in Ca2+ metabolism is poorly understood (Carafoli et al. 1970; Uribe et al. 1992).
Pulse-chase experiments using 45Ca2+ have revealed two major intracellular ‘pools’ that accumulate Ca2+. The smaller pool is highly exchangeable with external Ca2+ with a half-time of approximately 2 min (Cunningham and Fink, 1994; Eilam, 1982a,b) and probably reflects the portion of Ca2+ in the cytosol or in secretory organelles that can be exported from the cell. More than 90% of the total cell-associated Ca2+ accumulates in a non-exchangeable pool in growing cells and this pool is largely confined to the vacuole (Eilam et al. 1985; Ohsumi et al. 1988). Estimates of vacuolar Ca2+ content range from 1 to 4 mmol l−1 in wild-type cells grown in standard media, but it is likely that intravacuolar free Ca2+ concentrations [Ca2+]v are effectively much lower due to buffering by soluble inorganic polyphosphates (Dunn et al. 1994). Ca2+ can be completely released from isolated vacuoles or from whole cells using the ionophores A23187 or ionomycin, suggesting that the non-exchangeable pool of Ca2+ is soluble. As expected for vacuolar Ca2+ transporters, accumulation of Ca2+ into the non-exchangeable pool is decreased fivefold in pmc1 null mutants relative to PMC1 strains (Cunningham and Fink, 1994).
Conversely, the non-exchangeable pool is significantly increased in pmr1 null mutants, presumably as a consequence of decreased Ca2+ accumulation in the secretory pathway and export (K. W. Cunningham and G. R. Fink, in preparation). Together, these findings suggest that the vacuole is a major Ca2+ sink in yeast.
Yeast cells grow very well at a wide range of environmental Ca2+ concentrations from less than 1 μmol l−1 to more than 100 mmol l−1 and can adapt to large and rapid fluctuations in extracellular [Ca2+] (Anraku et al. 1991). Exponentially growing cells arrest transiently in the G1 phase of the cell division cycle after addition of A23187 plus EGTA (a chelator of Ca2+ and other ions) to the medium (Iida et al. 1990a). In response to increasing extracellular [Ca2+], wild-type cells dramatically increase the non-exchangeable pool of Ca2+ (Beeler et al. 1994; Dunn et al. 1994), which probably reflects an increased rate of Ca2+ sequestration due to elevated levels of [Ca2+]i (Halachmi and Eilam, 1993). Mutants lacking PMC1 grow poorly in media containing a high [Ca2+], although growth can be restored by overexpression of PMR1 or the cloned antiporter gene, which implies that the rate of Ca2+ sequestration is growth-limiting under these conditions (Cunningham and Fink, 1994; K. W. Cunningham and G. R. Fink, in preparation). At Ca2+ concentrations below 10 μmol l−1, mutants lacking PMR1 fail to grow (Rudolph et al. 1989), but growth can be restored by overexpression of PMC1. Strains simultaneously deleted for PMC1 and PMR1 are inviable at all Ca2+ concentrations. These results are consistent with a model in which Pmc1p and Pmr1p function redundantly in Ca2+ sequestration, although they have distinct essential roles in response to either high or low extracellular [Ca2+], respectively. The simple model of Ca2+ metabolism (Fig. 2) is sufficient to accomplish the cellular goals of maintaining [Ca2+]i at tolerable levels and supplying Ca2+ to various internal compartments at a wide range of extracellular Ca2+ concentrations. Almost certainly, though, this model will be amended as new transporters are identified and as the modes of transporter regulation become understood.
Ca2+ signaling
Until recently, Ca2+ signals and signaling factors were thought to have only minor effects on cellular processes in yeast. Mutants expressing a defective calmodulin that is unable to bind Ca2+ with high affinity do not display any obvious defects in growth, mating, sporulation or various stress responses, suggesting that any signaling mediated by this factor is not required for any of these processes (Geiser et al. 1991). Initial reports did not identify any phenotype of mutants lacking calmodulin-dependent protein kinases (Ohya et al. 1991; Pausch et al. 1991) and identified only subtle effects of inactivating calcineurin (Cyert et al. 1991; Cyert and Thorner, 1992; Foor et al. 1992). However, clear effects of calcineurin mutations have now been observed in several new conditions. Calcineurin function appears to be necessary for growth in media containing high levels of Na+ and Li+ (Nakamura et al. 1993) and for the maximum induction of the PMR2 gene in response to these conditions (Mendoza et al. 1994). Additionally, the Ca2+-and calmodulin-dependent activation of calcineurin appears to inhibit growth of pmc1 null mutants in high-Ca2+ medium (Cunningham and Fink, 1994) and to induce the expression of several other genes (K. W. Cunningham and G. R. Fink, in preparation). Identification of the targets of activated calcineurin should provide not only a useful reporter for Ca2+ signaling events but also crucial information about the processes wherein Ca2+ signaling plays important roles.
Powerful methods of monitoring [Ca2+]i in living yeast cells are now available. The fluorescent indicator Indo-1 (Halachmi and Eilam, 1989; Halachmi and Eilam, 1993) and the luminescent protein aequorin (Nakajima et al. 1991) have been used successfully to estimate [Ca2+]i in cell suspensions, whereas Fura-2 has been employed to image Ca2+ in single cells (Iida et al. 1990b; Ohya et al. 1991). Despite the technical difficulties associated with these techniques, the ability to combine molecular genetics and cell physiology offers great promise for the future.
Future prospects
Although the key participants in Ca2+ transport and signaling in yeast are rapidly becoming reasonable well understood, many important questions remain to be answered. Are the yeast Ca2+ channels similar to those of other species? When and how are natural Ca2+ signals produced? What are the physiological responses to these signals? Are these processes related to Ca2+ metabolism and signaling in plant and animal cells? The complete understanding of the catalytic and regulatory factors that act in a coordinated manner to produce Ca2+ signals and control Ca2+ metabolism will ultimately require the concerted application of many different approaches. The genetic and molecular tools available in yeast promise to add an exciting new perspective to the basic mechanisms of Ca2+ transport and signaling.