Functional dependence on calcineurin by variants of the Saccharomyces cerevisiae vacuolar Ca2+/H+ exchanger Vcx1p

The Ca(2+)-dependent protein phosphatase calcineurin is an important regulator of ion transporters from many organisms, including the Saccharomyces cerevisiae vacuolar Ca(2+)/H(+) exchanger Vcx1p. In yeast and plants, cation/H(+) exchangers are important in shaping cytosolic Ca(2+) levels involved in signal transduction and providing tolerance to potentially toxic concentrations of cations such as Ca(2+), Mn(2+) and Cd(2+). Previous genetic evidence suggested Vcx1p is negatively regulated by calcineurin. By utilizing direct transport measurements into vacuolar membrane vesicles, we demonstrate that Vcx1p is a low-affinity Ca(2+) transporter and may also function in Cd(2+) transport, but cannot transport Mn(2+). Furthermore, direct Ca(2+) transport by Vcx1p is calcineurin sensitive. Using a yeast growth assay, a mutant allele of VCX1 (VCX1-S204A/L208P), termed VCX1-M1, was previously found to confer strong Mn(2+) tolerance. Here we demonstrate that this Mn(2+) tolerance is independent of the Ca(2+)/Mn(2+)-ATPase Pmr1p and results from Mn(2+)-specific vacuolar transport activity of Vcx1-M1p. This Mn(2+) transport by Vcx1-M1p is calcineurin dependent, although the localization of Vcx1-M1p to the vacuole appears to be calcineurin independent. Additionally, we demonstrate that mutation of L208P alone is enough to confer calcineurin-dependent Mn(2+) tolerance. This study demonstrates that calcineurin can positively regulate the transport of cations by VCX1-M1p.     

The product of HUM1, a novel yeast gene, is required for vacuolar Ca2+/H+ exchange and is related to mammalian Na+/Ca2+ exchangers.

Calcineurin, or PP2B, plays a critical role in mediating Ca2+-dependent signaling in many cell types. In yeast cells, this highly conserved protein phosphatase regulates aspects of ion homeostasis and cell wall synthesis. We show that calcineurin mutants are sensitive to high concentrations of Mn2+ and identify two genes, CCC1 and HUM1, that, at high dosages, increase the Mn2+ tolerance of calcineurin mutants. CCC1 was previously identified by complementation of a Ca2+-sensitive (csg1) mutant. HUM1 (for "high copy number undoes manganese") is a novel gene whose predicted protein product shows similarity to mammalian Na+/Ca2+ exchangers. hum1 mutations confer Mn2+ sensitivity in some genetic backgrounds and exacerbate the Mn2+ sensitivity of calcineurin mutants. Furthermore, disruption of HUM1 in a calcineurin mutant strain results in a Ca2+-sensitive phenotype. We investigated the effect of disrupting HUM1 in other strains with defects in Ca2+ homeostasis. The Ca2+ sensitivity of pmc1 mutants, which lack a P-type ATPase presumed to transport Ca2+ into the vacuole, is exacerbated in a hum1 mutant strain background. Also, the Ca2+ content of hum1 pmc1 cells is less than that of pmc1 cells. In contrast, the Ca2+ sensitivity of vph1 mutants, which are specifically defective in vacuolar acidification, is not significantly altered by disruption of Hum1p function. These genetic interactions suggest that Hum1p may participate in vacuolar Ca2+/H+ exchange. Therefore, we prepared vacuolar membrane vesicles from wild-type and hum1 cells and compared their Ca2+ transport properties. Vacuolar membrane vesicles from hum1 mutants lack all Ca2+/H+ antiport activity, demonstrating that Hum1p catalyzes the exchange of Ca2+ for H+ across the yeast vacuolar membrane.     

The relationship between extramitochondrial Ca2+ concentration, respiratory rate, and membrane potential in mitochondria from brown adipose tissue of the rat

The ability of isolated mitochondria from rat brown-adipose tissue to regulate extramitochondrial Ca2+ (measured by arsenazo) was studied in relation to their ability to produce heat (measured polarographically). The energetic state of the mitochondria was expressed as a membrane potential, delta psi (estimated with safranine), and was varied semi-physiologically by the use of different GDP concentrations. In these mitochondria GDP binds to the 32-kDa polypeptide, thermogenin, which regulates coupling. Ca2+ uptake (at 5 microM extramitochondrial Ca2+) was maximal at delta psi greater than 150 mV. Basal Ca2+ release increased from 1 to 2 nmol x min-1 x mg-1 below 150 mV. Na+ -stimulated rate of Ca2+ release was stable within the investigated delta psi span (100-160 mV). Initial Ca2+ levels were maintained below 0.2 microM for 100 mV less than delta psi less than 160 mV. Ca2+ levels maintained after Ca2+ challenge (20 nmol Ca2+ x mg-1) were below 0.4 microM for delta psi greater than 135 mM. Respiration was unstimulated for delta psi greater than 150 mV and was maximal at delta psi less than or equal to 135 mV. In the presence of well-oxidised substrates, the respiration at maximally activated thermogenin was markedly below fully uncoupled respiration and was probably limited by thermogenin activity--i.e. by a limited H+ reentry (OH- exit) and therefore by a membrane potential maintained at about 135 mV. It is concluded that at membrane potentials of 135 mV and above the mitochondria exhibit full Ca2+ control and are able to regulate thermogenic output up to maximum without interfering with this Ca2+ control. Membrane potential probably does not decrease below 135 mV in vivo. Therefore, Ca2+ homeostasis and thermogenesis are non-interfering and can be hormonally independently regulated, e.g. by alpha-adrenergic and beta-adrenergic stimuli, respectively.     

The frost gene of Neurospora crassa is a homolog of yeast cdc1 and affects hyphal branching via manganese homeostasis

The Neurospora crassa mutant frost has a hyperbranching phenotype that can be corrected by adding Ca(2+), suggesting that characterization of this gene might clarify the mechanism of Ca(2+)-dependent tip growth. The wild-type allele was cloned by sib selection using protoplasts from arthroconidia. RFLP analysis revealed that the cloned DNA fragment mapped to the fr locus. The nucleotide sequence of genomic and cDNA was determined. The deduced amino acid sequence showed homology to the Saccharomyces cerevisiae CDC1 protein, implicated in manganese homeostasis. The fr mutant was sensitive to Mn(2+), and a revertant allele whose product differs by one amino acid was tolerant to Mn(2+). Mn(2+) depletion induced the wild-type strain to hyperbranch, resulting in a morphology similar to that of fr. The fr mutant was also sensitive to calcineurin inhibitors. These results suggest that fr is involved in Mn(2+) homeostasis and point to a role for Mn(2+) in Neurospora branching.     

The Ca2+ homeostasis defects in a pgm2Delta strain of Saccharomyces cerevisiae are caused by excessive vacuolar Ca2+ uptake mediated by the Ca2+-ATPase Pmc1p

Loss of the major isoform of phosphoglucomutase (PGM) causes an accumulation of glucose 1-phosphate when yeast cells are grown with galactose as the carbon and energy source. Remarkably, the pgm2Delta strain also exhibits a severe imbalance in intracellular Ca(2+) homeostasis when grown under these conditions. In the present study, we examined how the pgm2Delta mutation alters yeast Ca(2+) homeostasis in greater detail. We found that a shift from glucose to galactose as the carbon source resulted in a 2-fold increase in the rate of cellular Ca(2+) uptake in wild-type cells, whereas Ca(2+) uptake increased 8-fold in the pgm2Delta mutant. Disruption of the PMC1 gene, which encodes the vacuolar Ca(2+)-ATPase Pmc1p, suppressed the Ca(2+)-related phenotypes observed in the pgm2Delta strain. This suggests that excessive vacuolar Ca(2+) uptake is tightly coupled to these defects in Ca(2+) homeostasis. An in vitro assay designed to measure Ca(2+) sequestration into intracellular compartments confirmed that the pgm2Delta mutant contained a higher level of Pmc1p-dependent Ca(2+) transport activity than the wild-type strain. We found that this increased rate of vacuolar Ca(2+) uptake also coincided with a large induction of the unfolded protein response in the pgm2Delta mutant, suggesting that Ca(2+) uptake into the endoplasmic reticulum compartment was reduced. These results indicate that the excessive Ca(2+) uptake and accumulation previously shown to be associated with the pgm2Delta mutation are due to a severe imbalance in the distribution of cellular Ca(2+) into different intracellular compartments.     

Subplasmalemmal mitochondria modulate the activity of plasma membrane Ca2+-ATPases

Mitochondria are dynamic organelles that modulate cellular Ca2+ signals by interacting with Ca2+ transporters on the plasma membrane or the endoplasmic reticulum (ER). To study how mitochondria dynamics affects cell Ca2+ homeostasis, we overexpressed two mitochondrial fission proteins, hFis1 and Drp1, and measured Ca2+ changes within the cytosol and the ER in HeLa cells. Both proteins fragmented mitochondria, decreased their total volume by 25-40%, and reduced the fraction of subplasmalemmal mitochondria by 4-fold. The cytosolic Ca2+ signals elicited by histamine were unaltered in cells lacking subplasmalemmal mitochondria as long as Ca2+ was present in the medium, but the signals were significantly blunted when Ca2+ was removed. Upon Ca2+ withdrawal, the free ER Ca2+ concentration decreased rapidly, and hFis1 cells were unable to respond to repetitive histamine stimulations. The loss of stored Ca2+ was due to an increased activity of plasma membrane Ca2+-ATPase (PMCA) pumps and was associated with an increased influx of Ca2+ and Mn2+ across store-operated Ca2+ channels. The increased Ca2+ influx compensated for the loss of stored Ca2+, and brief Ca2+ additions between successive agonist stimulations fully corrected subsequent histamine responses. We propose that the lack of subplasmalemmal mitochondria disrupts the transfer of Ca2+ from plasma membrane channels to the ER and that the resulting increase in subplasmalemmal [Ca2+] up-regulates the activity of PMCA. The increased Ca2+ extrusion promotes ER depletion and the subsequent activation of store-operated Ca2+ channels. Cells thus adapt to the lack of subplasmalemmal mitochondria by relying on external rather than on internal Ca2+ for signaling.     

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.     

Expression of arabidopsis CAX2 in tobacco. Altered metal accumulation and increased manganese tolerance

Metal transport from the cytosol to the vacuole is thought to be an important component of ion tolerance and of a plant's potential for use in phytoremediation. The Arabidopsis antiporter CAX2 (calcium exchanger 2) may be a key mediator of this process. CAX2 expression in yeast suppressed both Ca(2+) and Mn(2+) growth defects. A peptide-specific antibody to the antiporter reacted with a 39-kD protein from plant vacuolar membranes. Tobacco (Nicotiana tabacum) plants expressing CAX2 accumulated more Ca(2+), Cd(2+), and Mn(2+) and were more tolerant to elevated Mn(2+) levels. Expression of CAX2 in tobacco increased Cd(2+) and Mn(2+) transport in isolated root tonoplast vesicles. These results suggest that CAX2 has a broad substrate range and modulation of this transporter may be an important component of future strategies to improve plant ion tolerance.     

Manganese-stimulated phosphorylation of a rat pancreatic protein: identity with elongation factor 2

To investigate the effect of Mn2+ on pancreatic protein phosphorylation, we incubated rat pancreatic cytosol in Tris buffer (pH 7.5) with [gamma-32P]ATP. Analysis using sodium dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography revealed a single protein (p98), with an Mr of 98,000 and a pI of 6.4-6.5, which was phosphorylated in a dose-dependent manner by Mn2+. A threshold effect was observed at 35 microM, and maximal effect at 1.1 mM Mn2+. Ca2+ and calmodulin (CaM) did not cause p98 phosphorylation, but Mg2+ (10 mM) caused faint non-specific phosphorylation of p98. Ca2+ (0.03-3 mM) and CaM (1-10 micrograms/ml) significantly enhanced, whereas trifluoperazine (TFP) and Mg2+ inhibited Mn(2+)-stimulated p98 phosphorylation. Under the above incubation conditions, Mn(2+)-stimulated protein phosphorylation of p98 was also observed in isolated pancreatic acini, but not in cytosols from liver or kidney. Partial purification of p98 and amino acid sequencing of the protein band corresponding to p98 indicated complete sequence homology with rat elongation factor 2 (EF-2). Furthermore, the combination of Ca2+, Mg2+ and CaM, which is known to induce the phosphorylation of EF-2, mimicked the actions of Mn2+. Inasmuch as EF-2 is the major substrate for CaM-dependent protein kinase III (CaM-PK III), these studies suggest that in the pancreatic acinar cell Mn2+/CaM protein kinase activity is mediated via CaM-PK III and the Mn2+ participates in the regulation of this enzyme in the pancreas.     

Cdc1p is an endoplasmic reticulum-localized putative lipid phosphatase that affects Golgi inheritance and actin polarization by activating Ca2+ signaling

In the budding yeast Saccharomyces cerevisiae, mutations in the essential gene CDC1 cause defects in Golgi inheritance and actin polarization. However, the biochemical function of Cdc1p is unknown. Previous work showed that cdc1 mutants accumulate intracellular Ca(2+) and display enhanced sensitivity to the extracellular Mn(2+) concentration, suggesting that Cdc1p might regulate divalent cation homeostasis. By contrast, our data indicate that Cdc1p is a Mn(2+)-dependent protein that can affect Ca(2+) levels. We identified a cdc1 allele that activates Ca(2+) signaling but does not show enhanced sensitivity to the Mn(2+) concentration. Furthermore, our studies show that Cdc1p is an endoplasmic reticulum-localized transmembrane protein with a putative phosphoesterase domain facing the lumen. cdc1 mutant cells accumulate an unidentified phospholipid, suggesting that Cdc1p may be a lipid phosphatase. Previous work showed that deletion of the plasma membrane Ca(2+) channel Cch1p partially suppressed the cdc1 growth phenotype, and we find that deletion of Cch1p also suppresses the Golgi inheritance and actin polarization phenotypes. The combined data fit a model in which the cdc1 mutant phenotypes result from accumulation of a phosphorylated lipid that activates Ca(2+) signaling.     

Synergistic movements of Ca(2+) and Bax in cells undergoing apoptosis.

Apoptosis is a physiological counterbalance to mitosis and plays important roles in tissue development and homeostasis. Cytosolic Ca(2+) has been implicated as a proapoptotic second messenger involved in both triggering apoptosis and regulating cell death-specific enzymes. A critical early event in apoptosis is associated with the redistribution of Bax from cytosol to mitochondria and endoplasmic reticulum (ER) membranes; however, the molecular mechanism of Bax translocation and its relationship to Ca(2+) is largely unknown. Here we provide functional evidence for a synergistic interaction between the movements of intracellular Ca(2+) and cytosolic Bax in the induction of apoptosis. Overexpression of Bax in cultured cells causes a loss of ER Ca(2+) content. Depletion of ER Ca(2+) through activation of the ryanodine receptor enhances the participation of Bax into the mitochondrial membrane. Neither Bax translocation nor Bax-induced apoptosis is affected by buffering of cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, suggesting that depletion of ER Ca(2+) rather than elevation of cytosolic Ca(2+) is the signal for cell apoptosis. This dynamic interplay of Ca(2+) and Bax movements may serve as an amplifying factor in the initial signaling steps of apoptosis.     

Loss of the major isoform of phosphoglucomutase results in altered calcium homeostasis in Saccharomyces cerevisiae

Phosphoglucomutase (PGM) is a key enzyme in glucose metabolism, where it catalyzes the interconversion of glucose 1-phosphate (Glc-1-P) and glucose 6-phosphate (Glc-6-P). In this study, we make the novel observation that PGM is also involved in the regulation of cellular Ca(2+) homeostasis in Saccharomyces cerevisiae. When a strain lacking the major isoform of PGM (pgm2Delta) was grown on media containing galactose as sole carbon source, its rate of Ca(2+) uptake was 5-fold higher than an isogenic wild-type strain. This increased rate of Ca(2+) uptake resulted in a 9-fold increase in the steady-state total cellular Ca(2+) level. The fraction of cellular Ca(2+) located in the exchangeable pool in the pgm2Delta strain was found to be as large as the exchangeable fraction observed in wild-type cells, suggesting that the depletion of Golgi Ca(2+) stores is not responsible for the increased rate of Ca(2+) uptake. We also found that growth of the pgm2Delta strain on galactose media is inhibited by 10 microM cyclosporin A, suggesting that activation of the calmodulin/calcineurin signaling pathway is required to activate the Ca(2+) transporters that sequester the increased cytosolic Ca(2+) load caused by this high rate of Ca(2+) uptake. We propose that these Ca(2+)-related alterations are attributable to a reduced metabolic flux between Glc-1-P and Glc-6-P due to a limitation of PGM enzymatic activity in the pgm2Delta strain. Consistent with this hypothesis, we found that this "metabolic bottleneck" resulted in an 8-fold increase in the Glc-1-P level compared with the wild-type strain, while the Glc-6-P and ATP levels were normal. These results suggest that Glc-1-P (or a related metabolite) may participate in the control of Ca(2+) uptake from the environment.     

Alteration in calcium handling at the subcellular level in mdx myotubes

In this study, we have tested the hypothesis that augmented [Ca(2+)] in subcellular regions or organelles, which are known to play a key role in cell survival, is the missing link between Ca(2+) homeostasis alterations and muscular degeneration associated with muscular dystrophy. To this end, different targeted chimeras of the Ca(2+)-sensitive photoprotein aequorin have been transiently expressed in subcellular compartments of skeletal myotubes of mdx mice, the animal model of Duchenne muscular dystrophy. Direct measurements of the [Ca(2+)] in the sarcoplasmic reticulum, [Ca(2+)](sr), show a higher steady state level at rest and a larger drop after KCl-induced depolarization in mdx compared with control myotubes. The peaks in [Ca(2+)] occurring in the mitochondrial matrix of mdx myotubes are significantly larger than in controls upon KCl-induced depolarization or caffeine application. The augmented response of mitochondria precedes the alterations in the Ca(2+) responses of the cytosol and of the cytoplasmic region beneath the membrane, which become significant only at a later stage of myotube differentiation. Taking into account the key role played by mitochondria Ca(2+) handling in the control of cell death, our data suggest that mitochondria are potential targets of impaired Ca(2+) homeostasis in muscular dystrophy.     

An endoplasmic reticulum-bound Ca(2+)/Mn(2+) pump,ECA1, supports plant growth and confers tolerance to Mn(2+) stress

Plants can grow in soils containing highly variable amounts of mineral nutrients, like Ca(2+) and Mn(2+), though the mechanisms of adaptation are poorly understood. Here, we report the first genetic study to determine in vivo functions of a Ca(2+) pump in plants. Homozygous mutants of Arabidopsis harboring a T-DNA disruption in ECA1 showed a 4-fold reduction in endoplasmic reticulum-type calcium pump activity. Surprisingly, the phenotype of mutant plants was indistinguishable from wild type when grown on standard nutrient medium containing 1.5 mM Ca(2+) and 50 microM Mn(2+). However, mutants grew poorly on medium with low Ca(2+) (0.2 mM) or high Mn(2+) (0.5 mM). On high Mn(2+), the mutants failed to elongate their root hairs, suggesting impairment in tip growth processes. Expression of the wild-type gene (CAMV35S::ECA1) reversed these conditional phenotypes. The activity of ECA1 was examined by expression in a yeast (Saccharomyces cerevisiae) mutant, K616, which harbors a deletion of its endogenous calcium pumps. In vitro assays demonstrated that Ca(2+), Mn(2+), and Zn(2+) stimulated formation of a phosphoenzyme intermediate, consistent with the translocation of these ions by the pump. ECA1 provided increased tolerance of yeast mutant to toxic levels of Mn(2+) (1 mM) and Zn(2+)(3 mM), consistent with removal of these ions from the cytoplasm. These results show that despite the potential redundancy of multiple Ca(2+) pumps and Ca(2+)/H(+) antiporters in Arabidopsis, pumping of Ca(2+) and Mn(2+) by ECA1 into the endoplasmic reticulum is required to support plant growth under conditions of Ca(2+) deficiency or Mn(2+) toxicity.     

Reduced loading of intracellular Ca(2+) stores and downregulation of capacitative Ca(2+) influx in Bcl-2-overexpressing cells

The mechanism of action of the oncogene bcl-2, a key regulator of the apoptotic process, is still debated. We have employed organelle-targeted chimeras of the Ca(2+)-sensitive photoprotein, aequorin, to investigate in detail the effect of Bcl-2 overexpression on intracellular Ca(2+) homeostasis. In the ER and the Golgi apparatus, Bcl-2 overexpression increases the Ca(2+) leak (while leaving Ca(2+) accumulation unaffected), hence reducing the steady-state [Ca(2+)] levels. As a direct consequence, the [Ca(2+)] increases caused by inositol 1,4,5 trisphosphate (IP3)-generating agonists were reduced in amplitude in both the cytosol and the mitochondria. Bcl-2 overexpression also reduced the rate of Ca(2+) influx activated by Ca(2+) store depletion, possibly by an adaptive downregulation of this pathway. By interfering with Ca(2+)-dependent events at multiple intracellular sites, these effects of Bcl-2 on intracellular Ca(2+) homeostasis may contribute to the protective role of this oncogene against programmed cell death.     

Manganese specificity determinants in the Arabidopsis metal/H+ antiporter CAX2

In plants and fungi, vacuolar transporters help remove potentially toxic cations from the cytosol. Metal/H(+) antiporters are involved in metal sequestration into the vacuole. However, the specific transport properties and the ability to manipulate these transporters to alter substrate specificity are poorly understood. The Arabidopsis thaliana cation exchangers, CAX1 and CAX2, can both transport Ca(2+) into the vacuole. There are 11 CAX-like transporters in Arabidopsis; however, CAX2 was the only characterized CAX transporter capable of vacuolar Mn(2+) transport when expressed in yeast. To determine the domains within CAX2 that mediate Mn(2+) specificity, six CAX2 mutants were constructed that contained different regions of the CAX1 transporter. One class displayed no alterations in Mn(2+) or Ca(2+) transport, the second class showed a reduction in Ca(2+) transport and no measurable Mn(2+) transport, and the third mutant, which contained a 10-amino acid domain from CAX1 (CAX2-C), showed no reduction in Ca(2+) transport and a complete loss of Mn(2+) transport. The subdomain analysis of CAX2-C identified a 3-amino acid region that is responsible for Mn(2+) specificity of CAX2. This study provides evidence for the feasibility of altering substrate specificity in a metal/H(+) antiporter, an important family of transporters found in a variety of organisms.     

Role of mitochondrial Ca2+ in the regulation of cellular energetics

Calcium is an important signaling molecule involved in the regulation of many cellular functions. The large free energy in the Ca(2+) ion membrane gradients makes Ca(2+) signaling inherently sensitive to the available cellular free energy, primarily in the form of ATP. In addition, Ca(2+) regulates many cellular ATP-consuming reactions such as muscle contraction, exocytosis, biosynthesis, and neuronal signaling. Thus, Ca(2+) becomes a logical candidate as a signaling molecule for modulating ATP hydrolysis and synthesis during changes in numerous forms of cellular work. Mitochondria are the primary source of aerobic energy production in mammalian cells and also maintain a large Ca(2+) gradient across their inner membrane, providing a signaling potential for this molecule. The demonstrated link between cytosolic and mitochondrial Ca(2+) concentrations, identification of transport mechanisms, and the proximity of mitochondria to Ca(2+) release sites further supports the notion that Ca(2+) can be an important signaling molecule in the energy metabolism interplay of the cytosol with the mitochondria. Here we review sites within the mitochondria where Ca(2+) plays a role in the regulation of ATP generation and potentially contributes to the orchestration of cellular metabolic homeostasis. Early work on isolated enzymes pointed to several matrix dehydrogenases that are stimulated by Ca(2+), which were confirmed in the intact mitochondrion as well as cellular and in vivo systems. However, studies in these intact systems suggested a more expansive influence of Ca(2+) on mitochondrial energy conversion. Numerous noninvasive approaches monitoring NADH, mitochondrial membrane potential, oxygen consumption, and workloads suggest significant effects of Ca(2+) on other elements of NADH generation as well as downstream elements of oxidative phosphorylation, including the F(1)F(O)-ATPase and the cytochrome chain. These other potential elements of Ca(2+) modification of mitochondrial energy conversion will be the focus of this review. Though most specific molecular mechanisms have yet to be elucidated, it is clear that Ca(2+) provides a balanced activation of mitochondrial energy metabolism that exceeds the alteration of dehydrogenases alone.     

The Golgi Ca2+-ATPase KlPmr1p function is required for oxidative stress response by controlling the expression of the heat-shock element HSP60 in Kluyveromyces lactis

The Golgi P-type Ca2+-ATPase, Pmr1p, is the major player for calcium homeostasis in yeast. The inactivation of KlPMR1 in Kluyveromyces lactis leads to high pleiotropic phenotypes that include reduced glycosylation, cell wall defects, and alterations of mitochondrial metabolism. In this article we found that cells lacking KlPmr1p have a morphologically altered mitochondrial network and that mitochondria (m) from Klpmr1delta cells accumulate Ca2+ more slowly and reach a lower [Ca2+]m level, when exposed to [Ca2+] < 5 microM, than wild-type cells. The Klpmr1delta cells also exhibit traits of ongoing oxidative stress and present hyperphosphorylation of KlHog1p, the hallmark for the activation of stress response pathways. The mitochondrial chaperone KlHsp60 acts as a multicopy suppressor of phenotypes that occur in cells lacking the Ca2+-ATPase, including relief from oxidative stress and recovery of cell wall thickness and functionality. Inhibition of KlPMR1 function decreases KlHSP60 expression at both mRNA and protein levels. Moreover, KlPRM1 loss of function correlates with both decreases in HSF DNA binding activity and KlHSP60 expression. We suggest a role for KlPMR1 in HSF DNA binding activity, which is required for proper KlHSP60 expression, a key step in oxidative stress response.     

Protein kinase C contributes to the maintenance of contractile force in human ventricular cardiomyocytes

Prolonged Ca(2+) stimulations often result in a decrease in contractile force of isolated, demembranated human ventricular cardiomyocytes, whereas intact cells are likely to be protected from this deterioration. We hypothesized that cytosolic protein kinase C (PKC) contributes to this protection. Prolonged contracture (10 min) of demembranated human cardiomyocytes at half-maximal Ca(2+) resulted in a 37 +/- 5% reduction of active force (p < 0.01), whereas no decrease (2 +/- 3% increase) was observed in the presence of the cytosol (reconstituted myocytes). The PKC inhibitors GF 109203X and G? 6976 (10 micromol/liter) partially antagonized the cytosol-mediated protection (15 +/- 5 and 9 +/- 2% decrease in active force, p < 0.05). Quantitation of PKC isoform expression revealed the dominance of the Ca(2+)-dependent PKCalpha over PKCdelta and PKCepsilon (189 +/- 31, 7 +/- 3, and 7 +/- 2 ng/mg protein, respectively). Ca(2+) stimulations of reconstituted human cardiomyocytes resulted in the translocation of endogenous PKCalpha, but not PKCbeta1, delta, and epsilon from the cytosol to the contractile system (PKCalpha association: control, 5 +/- 3 arbitrary units; +Ca(2+), 39 +/- 8 arbitrary units; p < 0.01, EC(50,Ca) = 645 nmol/liter). One of the PKCalpha-binding proteins were identified as the thin filament regulatory protein cardiac troponin I (TnI). Finally, the Ca(2+)-dependent interaction between PKCalpha and TnI was confirmed using purified recombinant proteins (binding without Ca(2+) was only 28 +/- 18% of that with Ca(2+)). Our data suggest that PKCalpha translocates to the contractile system and anchors to TnI in a Ca(2+)-dependent manner in the human heart, contributing to the maintenance of contractile force.