Rabbit sarcoplasmic reticulum Ca(2+)-ATPase replaces yeast PMC1 and PMR1 Ca(2+)-ATPases for cell viability and calcineurin-dependent regulation of calcium tolerance

SERCA1a, the fast-twitch skeletal muscle isoform of sarco(endo)plasmic reticulum Ca(2+)-ATPase, was expressed in yeast using the promoter of the plasma membrane H(+)-ATPase. In the yeast Saccharomyces cerevisiae, the Golgi PMR1 Ca(2+)-ATPase and the vacuole PMC1 Ca(2+)-ATPase function together in Ca2+ sequestration and Ca2+ tolerance. SERCA1a expression restored growth of pmc1 mutants in media containing high Ca2+ concentrations, consistent with increased Ca2+ uptake in an internal compartment. SERCA1a expression also prevented synthetic lethality of pmr1 pmc1 double mutants on standard media. Electron microscopy and subcellular fractionation analysis showed that SERCA1a was localized in intracellular membranes derived from the endoplasmic reticulum. Finally, we found that SERCA1a ATPase activity expressed in yeast was regulated by calcineurin, a Ca2+/calmodulin-dependent phosphoprotein phosphatase. This result indicates that calcineurin contributes to calcium homeostasis by modulating the ATPase activity of Ca2+ pumps localized in intra-cellular compartments.     

Cloning, sequencing and functional expression in Escherichia coli of the gene for a P-type Na(+)-ATPase of a facultatively anaerobic alkaliphile, Exiguobacterium aurantiacum

Cloning and sequencing of the gene encoding a P-type Na(+)-ATPase of a facultatively anaerobic alkaliphile, Exiguobacterium aurantiacum, were conducted. The structural gene was composed of 2628 nucleotides. The deduced amino acid sequence (876 amino acid residues; Mr, 96,664) suggested that the enzyme possesses 10 membrane-spanning regions. When the amino acid sequences of the four putative membrane regions, M4, M5, M6 and M8, of BL77/1 ATPase were aligned with those of fungal Na(+)-ATPase, Na(+)/K(+)-ATPase, H(+)-ATPases and sarcoplasmic reticulum Ca(2+)-ATPase, it exhibited the highest homology with Ca(2+)-ATPase except M5 region. By the transformation of Escherichia coli with the expression vector (pQE30) containing the ATPase gene, the enzyme was functionally expressed in E. coli membranes.     

P(5A)-type ATPase Cta4p is essential for Ca2+ transport in the endoplasmic reticulum of Schizosaccharomyces pombe

This study establishes the role of P(5A)-type Cta4 ATPase in Ca(2+) sequestration in the endoplasmic reticulum by detecting an ATP-dependent, vanadate-sensitive and FCCP insensitive (45)Ca(2+)-transport in fission yeast membranes isolated by cellular fractionation. Specifically, the Ca(2+)-ATPase transport activity was decreased in ER membranes isolated from cells lacking a cta4(+) gene. Furthermore, a disruption of cta4(+) resulted in 6-fold increase of intracellular Ca(2+) levels, sensitivity towards accumulation of misfolded proteins in ER and ER stress, stimulation of the calcineurin phosphatase activity and vacuolar Ca(2+) pumping. These data provide compelling biochemical evidence for a P(5A)-type Cta4 ATPase as an essential component of Ca(2+) transport system and signaling network which regulate, in conjunction with calcineurin, the ER functionality in fission yeast.     

Modification of intracellular calcium concentration in cardiomyocytes by inhibition of sarcolemmal Na+/H+ exchanger

Although the Na(+)/H(+) exchanger (NHE) is considered to be involved in regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) through the Na(+)/Ca(2+) exchanger, the exact mechanisms of its participation in Ca(2+) handling by cardiomyocytes are not fully understood. Isolated rat cardiomyocytes were treated with or without agents that are known to modify Ca(2+) movements in cardiomyocytes and exposed to an NHE inhibitor, 5-(N-methyl-N-isobutyl)amiloride (MIA). [Ca(2+)](i) in cardiomyocytes was measured spectrofluorometrically with fura 2-AM in the absence or presence of KCl, a depolarizing agent. MIA increased basal [Ca(2+)](i) and augmented the KCl-induced increase in [Ca(2+)](i) in a concentration-dependent manner. The MIA-induced increase in basal [Ca(2+)](i) was unaffected by extracellular Ca(2+), antagonists of the sarcolemmal (SL) L-type Ca(2+) channel, and inhibitors of the SL Na(+)/Ca(2+) exchanger, SL Ca(2+) pump ATPase and mitochondrial Ca(2+) uptake. However, the MIA-induced increase in basal [Ca(2+)](i) was attenuated by inhibitors of SL Na(+)-K(+)-ATPase and sarcoplasmic reticulum (SR) Ca(2+) transport. On the other hand, the MIA-mediated augmentation of the KCl response was dependent on extracellular Ca(2+) concentration and attenuated by agents that inhibit SL L-type Ca(2+) channels, the SL Na(+)/Ca(2+) exchanger, SL Na(+)-K(+)-ATPase, and SR Ca(2+) release channels and the SR Ca(2+) pump. However, the effect of MIA on the KCl-induced increase in [Ca(2+)](i) remained unaffected by treatment with inhibitors of SL Ca(2+) pump ATPase and mitochondrial Ca(2+) uptake. MIA and a decrease in extracellular pH lowered intracellular pH and increased basal [Ca(2+)](i), whereas a decrease in extracellular pH, in contrast to MIA, depressed the KCl-induced increase in [Ca(2+)](i) in cardiomyocytes. These results suggest that NHE may be involved in regulation of [Ca(2+)](i) and that MIA-induced increases in basal [Ca(2+)](i), as well as augmentation of the KCl-induced increase in [Ca(2+)](i), in cardiomyocytes are regulated differentially.     

白细胞介素-2对大鼠心肌Ca2+ATPase和Na+ /K+ATPase的影响

为了探讨IL－2对心肌细胞内钙影响的可能机制,用光学法检测心肌肌浆网Ca^2 ATPase的活性,以及细胞膜Ca^2 ATPase和Na^ /K^ ATPase的活性。结果：（1）用IL－2（10、40、200、800U/ml）灌流心脏后,其肌浆网Ca^2 ATPase的活性随IL－2浓度的升高而增强;（2）在ATP浓度为0.1-4mmol/L时,Ca^2 ATPase的活性随ATP浓度的升庙则增强,由IL－2（200U／ml）灌流后的心脏获得肌浆网（SR）,其Ca^2 ATPase的活性对ATP的反应强于对照组;（3）在[Ca^2 ]为1－40μmol/L时,心脏SR Ca^2 ATPase的活性随[Ca^2 ]增加而增强,而IL－2灌流心脏后分离的SR,其Ca^2 ATPase活性在[Ca^2 ]升高时没有明显改变;（4）用nor-BNI(10nmol/L）预处理5min后,IL－2（200U／ml）灌流后不再使SR Ca^2 ATPase的活性增强;（5）用PTX（5mg/L）预处理后,IL－2对SR Ca^2 ATPase的影响减弱;（6）用磷脂酶C（PLC）抑制剂U73122（5μmol/L）处理后,IL－2不再使SR Ca^2 ATPase活性增高;（7）用IL－2直接处理从正常大鼠分离的SR后,对SR Ca^2 ATPase活性无明显影响;（8）IL－2灌流后,对心肌细胞膜Ca^2 ATPase和Na^ /K^ ATPase活性没有显著。上述结果表明,IL－2灌流心脏后使心肌肌浆网Ca^2 ATPase的活性增加,心肌细胞膜上的κ－阿片受体及其下游的G蛋白和PLC介导了IL－2的作用。尽管IL－2提高SR Ca^2 ATPase对ATP的反应性,但却抑制SR Ca^2 ATPase对钙离子的敏感性。IL－2对心肌细胞膜Ca^2 ATPase和Na^ /K^ ATPase的活性无明显影响。     

The protein kinase SOS2 activates the Arabidopsis H(+)/Ca(2+) antiporter CAX1 to integrate calcium transport and salt tolerance

The regulation of ions within cells is an indispensable component of growth and adaptation. The plant SOS2 protein kinase and its associated Ca(2+) sensor, SOS3, have been demonstrated to modulate the plasma membrane H(+)/Na(+) antiporter SOS1; however, how these regulators modulate Ca(2+) levels within cells is poorly understood. Here we demonstrate that SOS2 regulates the vacuolar H(+)/Ca(2+) antiporter CAX1. Using a yeast growth assay, co-expression of SOS2 specifically activated CAX1, whereas SOS3 did not. CAX1-like chimeric transporters were activated by SOS2 if the chimeric proteins contained the N terminus of CAX1. Vacuolar membranes from CAX1-expressing cells were made to be H(+)/Ca(2+)-competent by the addition of SOS2 protein in a dose-dependent manner. Using a yeast two-hybrid assay, SOS2 interacted with the N terminus of CAX1. In each of these yeast assays, the activation of CAX1 by SOS2 was SOS3-independent. In planta, the high level of expression of a deregulated version of CAX1 caused salt sensitivity. These findings suggest multiple functions for SOS2 and provide a mechanistic link between Ca(2+) and Na(+) homeostasis in plants.     

Abolishment of proton pumping and accumulation in the E1P conformational state of a plant plasma membrane H+-ATPase by substitution of a conserved aspartyl residue in transmembrane segment 6

The plasma membrane H(+)-ATPase AHA2 of Arabidopsis thaliana, which belongs to the P-type ATPase superfamily of cation-transporting ATPases, pumps protons out of the cell. To investigate the mechanism of ion transport by P-type ATPases we have mutagenized Asp(684), a residue in transmembrane segment M6 of AHA2 that is conserved in Ca(2+)-, Na(+)/K(+)-, H(+)/K(+)-, and H(+)-ATPases and which coordinates Ca(2+) ions in the SERCA1 Ca(2+)-ATPase. We describe the expression, purification, and biochemical analysis of the Asp(684) --> Asn mutant, and provide evidence that Asp(684) in the plasma membrane H(+)-ATPase is required for any coupling between ATP hydrolysis, enzyme conformational changes, and H(+)-transport. Proton pumping by the reconstituted mutant enzyme was completely abolished, whereas ATP was still hydrolyzed. The mutant was insensitive to the inhibitor vanadate, which preferentially binds to P-type ATPases in the E(2) conformation. During catalysis the Asp(684) --> Asn enzyme accumulated a phosphorylated intermediate whose stability was sensitive to addition of ADP. We conclude that the mutant enzyme is locked in the E(1) conformation and is unable to proceed through the E(1)P-E(2)P transition.     

Stimulatory pathways of the Calcium-sensing receptor on acid secretion in freshly isolated human gastric glands.

Gastric acid secretion is not only stimulated via the classical known neuronal and hormonal pathways but also by the Ca(2+)-Sensing Receptor (CaSR) located at the basolateral membrane of the acid-secretory gastric parietal cell. Stimulation of CaSR with divalent cations or the potent agonist Gd(3+) leads to activation of the H(+)/K(+)-ATPase and subsequently to gastric acid secretion. Here we investigated the intracellular mechanism(s) mediating the effects of the CaSR on H(+)/K(+)-ATPase activity in freshly isolated human gastric glands. Inhibition of heterotrimeric G-proteins (G(i) and G(o)) with pertussis toxin during stimulation of the CaSR with Gd(3+) only partly reduced the observed stimulatory effect. A similar effect was observed with the PLC inhibitor U73122. The reduction of the H(+)/K(+)-ATPase activity measured after incubation of gastric glands with BAPTA-AM, a chelator of intracellular Ca(2+), showed that intracellular Ca(2+) plays an important role in the signalling cascade. TMB-8, a ER Ca(2+)store release inhibitor, prevented the stimulation of H(+)/K(+)-ATPase activity. Also verapamil, an inhibitor of L-type Ca(2+)-channels reduced stimulation suggesting that both the release of intracellular Ca(2+) from the ER as well as Ca(2+) influx into the cell are involved in CaSR-mediated H(+)/K(+)-ATPase activation. Chelerythrine, a general inhibitor of protein kinase C, and Go 6976 which selectively inhibits Ca(2+)-dependent PKC(alpha) and PKC(betaI)-isozymes completely abolished the stimulatory effect of Gd(3+). In contrast, Ro 31-8220, a selective inhibitor of the Ca(2+)-independent PKCepsilon and PKC-delta isoforms reduced the stimulatory effect of Gd(3+) only about 60 %. On the other hand, activation of PKC with DOG led to an activation of H(+)/K(+)-ATPase activity which was only about 60 % of the effect observed with Gd(3+). Incubation of the parietal cells with PD 098059 to inhibit ERK1/2 MAP-kinases showed a significant reduction of the Gd(3+) effect. Thus, in the human gastric parietal cell the CaSR is coupled to pertussis toxin sensitive heterotrimeric G-Proteins and requires calcium to enhance the activity of the proton-pump. PLC, ERK 1/2 MAP-kinases as well as Ca(2+) dependent and Ca(2+)-independent PKC isoforms are part of the down-stream signalling cascade.     

Adenosine triphosphatases during maturation of cultured human skeletal muscle cells and in adult human muscle.

Na+/K(+)-ATPase, Mg(2+)-ATPase and sarcoplasmic reticulum (SR) Ca(2+)-ATPase are examined in cultured human skeletal muscle cells of different maturation grade and in human skeletal muscle. Na+/K(+)-ATPase is investigated by measuring ouabain binding and the activities of Na+/K(+)-ATPase and K(+)-dependent 3-O-methylfluorescein phosphatase (3-O-MFPase). SR Ca(2+)-ATPase is examined by ELISA, Ca(2+)-dependent phosphorylation and its activities on ATP and 3-O-methylfluorescein phosphate. Na+/K(+)-ATPase and SR Ca(2+)-ATPase are localized by immunocytochemistry. The activities of Na+/K(+)-ATPase and SR Ca(2+)-ATPase show a good correlation with the other assayed parameters of these ion pumps. All ATPase parameters investigated increase with the maturation grade of the cultured muscle cells. The number of ouabain-binding sites and the activities of Na+/K(+)-ATPase and K(+)-dependent 3-O-MFPase are significantly higher in cultured muscle cells than in muscle. The Mg(2+)-ATPase activity, the content of SR Ca(2+)-ATPase and the activities of SR Ca(2+)-ATPase and Ca(2+)-dependent 3-O-MFPase remain significantly lower in cultured cells than in muscle. The ouabain-binding constant and the molecular activities of Na+/K(+)-ATPase and SR Ca(2+)-ATPase are equal in muscle and cultured cells. During ageing of human muscle the activity as well as the concentration of SR Ca(2+)-ATPase decrease. Thus the changes of the activities of the ATPases are caused by variations of the number of their molecules. Na+/K(+)-ATPase is localized in the periphery of fast- and slow-twitch muscle fibers and at the sarcomeric I-band. SR Ca(2+)-ATPase is predominantly confined to the I-band, whereas fast-twitch fibers are much more immunoreactive than slow-twitch fibers. The presence of cross-striation for Na+/K(+)-ATPase and SR Ca(2+)-ATPase in highly matured cultured muscle cells indicate the development and subcellular organization of a transverse tubular system and SR, respectively, which resembles the in vivo situation.     

Role of transmembrane segment M8 in the biogenesis and function of yeast plasma-membrane H(+)-ATPase

Of the four transmembrane helices (M4, M5, M6, and M8) that pack together to form the ion-binding sites of P(2)-type ATPases, M8 has until now received the least attention. The present study has used alanine-scanning mutagenesis to map structure-function relationships throughout M8 of the yeast plasma-membrane H(+)-ATPase. Mutant forms of the ATPase were expressed in secretory vesicles and at the plasma membrane for measurements of ATP hydrolysis and ATP-dependent H(+) pumping. In secretory vesicles, Ala substitutions at a cluster of four positions near the extracytoplasmic end of M8 led to partial uncoupling of H(+) transport from ATP hydrolysis, while substitution of Ser-800 (close to the middle of M8) by Ala increased the apparent stoichiometry of H(+) transport. A similar increase has previously been reported following the substitution of Glu-803 by Gln (Petrov, V. et al., J. Biol. Chem. 275:15709-15718, 2000) at a position known to contribute directly to Ca(2+) binding in the Ca(2+)-ATPase of sarcoplasmic reticulum (Toyoshima, C., et al., Nature 405: 647-655, 2000). Four other mutations in M8 interfered with H(+)-ATPase folding and trafficking to the plasma membrane; based on homology modeling, they occupy positions that appear important for the proper bundling of M8 with M5, M6, M7, and M10. Taken together, these results point to a key role for M8 in the biogenesis, stability, and physiological functioning of the H(+)-ATPase.     

NHE6 protein possesses a signal peptide destined for endoplasmic reticulum membrane and localizes in secretory organelles of the cell

The NHE6 protein is a unique Na(+)/H(+) exchanger isoform believed to localize in mitochondria. It possesses a hydrophilic N-terminal portion that is rich in positively charged residues and many hydrophobic segments. In the present study, signal sequences in the NHE6 molecule were examined for organelle localization and membrane topogenesis. When the full-length protein was expressed in COS7 cells, it localized in the endoplasmic reticulum and on the cell surface. Furthermore, the protein was fully N-glycosylated. When green fluorescent protein was fused after the second (H2) or third (H3) hydrophobic segment, the fusion proteins were targeted to the endoplasmic reticulum (ER) membrane. The localization pattern was the same as that of fusion proteins in which green fluorescent protein was fused after H2 of NHE1. In an in vitro system, H1 behaved as a signal peptide that directs the translocation of the following polypeptide chain and is then processed off. The next hydrophobic segment (H2) halted translocation and eventually became a transmembrane segment. The N-terminal hydrophobic segment (H1) of NHE1 also behaved as a signal peptide. Cell fractionation studies using antibodies against the 15 C-terminal residues indicated that NHE6 protein localized in the microsomal membranes of rat liver cells. All of the NHE6 molecules in liver tissue possess an endoglycosidase H-resistant sugar chain. These findings indicate that NHE6 protein is targeted to the ER membrane via the N-terminal signal peptide and is sorted to organelle membranes derived from the ER membrane.     

The C-terminal 165 amino acids of the plasma membrane Ca(2+)-ATPase confer Ca2+/calmodulin sensitivity on the Na+,K(+)-ATPase alpha-subunit.

The C-terminal 165 amino acids of the rat brain plasma membrane (PM) Ca(2+)-ATPase II containing the calmodulin binding auto-inhibitory domain was connected to the C-terminus of the ouabain sensitive chicken Na+,K(+)-ATPase alpha 1 subunit. Expression of this chimeric molecule in ouabain resistant mouse L cells was assured by the high-affinity binding of [3H]ouabain. In the presence of Ca2+/calmodulin, this chimeric molecule exhibited ouabain inhibitable Na+,K(+)-ATPase activity; the putative chimeric ATPase activity was absent in the absence of Ca2+/calmodulin and activated by Ca2+/calmodulin in a dose-dependent manner. Furthermore, this chimeric molecule could bind monoclonal IgG 5 specific to the chicken Na+,K(+)-ATPase alpha 1 subunit only in the presence of Ca2+/calmodulin, suggesting that the epitope for IgG 5 in this chimera is masked in the absence of Ca2+/calmodulin and uncovered in their presence. These results propose a direct interaction between the calmodulin binding auto-inhibitory domain of the PM Ca(2+)-ATPase and the specific regions of the Na+,K(+)-ATPase alpha 1 subunit that are structurally homologous to the PM Ca(2+)-ATPase. A comparison of the deduced amino acid sequences revealed several possible regions within the Na+,K(+)-ATPase that might interact with the auto-inhibitory domain of the PM Ca(2+)-ATPase.     

Influence of the 53 kDa glycoprotein on the cooperativity of the Ca(2+)-ATPase of the sarcoplasmic reticulum.

Previous results from this laboratory suggest that the 53 kDa glycoprotein (GP-53) of rabbit skeletal muscle sarcoplasmic reticulum membrane (SR) may influence coupling between Ca2+ transport and ATP hydrolysis by the Ca(2+)-ATPase. Here we report evidence that GP-53 may influence the cooperative behavior of the Ca(2+)-ATPase. The ATPase activity of the Ca(2+)-ATPase displays negative cooperative dependence (Hill coefficient n less than 1) on [MgATP] and has positive cooperative dependence (n greater than 1) on [Ca2+]free. We have determined the degree of cooperativity for native SR vesicles, SR preincubated with antiserum against GP-53 or preimmune serum, and SR partially extracted with KCl-cholate. Our results show that SR preincubated with preimmune serum or SR treated with cholate in 50 mM KCl (yielding membranes rich in GP-53) demonstrate a cooperative dependence of Ca(2+)-ATPase activity on both [ATP] and [Ca2+] similar to that of untreated SR. SR preincubated with anti-GP-53 antiserum (which causes an uncoupling of Ca2+ transport from ATP hydrolysis) or SR extracted with cholate in 1 M KCl (yielding membranes depleted of GP-53) displays decreased positive cooperative dependence on [Ca2+] and decreased negative cooperative dependence on [ATP]. The results are consistent with the interpretation that GP-53 may influence the cooperative behavior of the Ca(2+)-ATPase.     

Asymmetrical, agonist-induced fluctuations in local extracellular [Ca(2+)] in intact polarized epithelia.

We recently proposed that extracellular Ca(2+) ions participate in a novel form of intercellular communication involving the extracellular Ca(2+)-sensing receptor (CaR). Here, using Ca(2+)-selective microelectrodes, we directly measured the profile of agonist-induced [Ca(2+)]ext changes in restricted domains near the basolateral or luminal membranes of polarized gastric acid-secreting cells. The Ca(2+)-mobilizing agonist carbachol elicited a transient, La(3+)-sensitive decrease in basolateral [Ca(2+)] (average approximately 250 microM, but as large as 530 microM). Conversely, carbachol evoked an HgCl2-sensitive increase in [Ca(2+)] (average approximately 400 microM, but as large as 520 microM) in the lumen of single gastric glands. Both responses were significantly reduced by pre-treatment with sarco-endoplasmic reticulum Ca(2+) ATPase (SERCA) pump inhibitors or with the intracellular Ca(2+) chelator BAPTA-AM. Immunofluorescence experiments demonstrated an asymmetric localization of plasma membrane Ca(2+) ATPase (PMCA), which appeared to be partially co-localized with CaR and the gastric H(+)/K(+)-ATPase in the apical membrane of the acid-secreting cells. Our data indicate that agonist stimulation results in local fluctuations in [Ca(2+)]ext that would be sufficient to modulate the activity of the CaR on neighboring cells.     

Ca(2+) sequestering in the early-branching amitochondriate protozoan Tritrichomonas foetus: an important role of the Golgi complex and its Ca(2+)-ATPase

Total membrane vesicles isolated from Tritrichomonas foetus showed an ATP-dependent Ca(2+) uptake, which was not sensitive to 10 microM protonophore FCCP but was blocked by orthovanadate, the inhibitor of P-type ATPases (I(50)=130 microM), and by the Ca(2+)/H(+) exchanger, A-23187. The Ca(2+) uptake was prevented also by thapsigargin, an inhibitor of the SERCA Ca(2+)-ATPases. The sensitivity of the Ca(2+) uptake by the protozoan membrane vesicles to thapsigargin was similar to that of Ca(2+)-ATPase from rabbit muscle sarcoplasmic reticulum. Fractionation of the total membrane vesicles in sucrose density gradient revealed a considerable peak of Ca(2+) transport activity that co-migrated with the Golgi marker guanosine diphosphatase (GDPase). Electron microscopy confirmed that membrane fractions of the peak were enriched with the Golgi membranes. The Golgi Ca(2+)-ATPase contributed to the Ca(2+) uptake by all membrane vesicles 80-85%. We conclude that: (i) the Golgi and/or Golgi-like vesicles form the main Ca(2+) store compartment in T. foetus; (ii) Ca(2+) ATPase is responsible for the Ca(2+) sequestering in this protozoan, while Ca(2+)/H(+) antiporter is not involved in the process; (iii) the Golgi pump of this ancient eukaryotic microorganism appears to be similar to the enzymes of the SERCA family by its sensitivity to thapsigargin.     

Functional expression of plant plasma membrane H(+)-ATPase in yeast endoplasmic reticulum.

Recombinant plant plasma membrane H(+)-ATPase has been produced in a yeast expression system comprising a multicopy plasmid and the strong promoter of the yeast PMA1 gene. Western blotting with a specific monoclonal antibody showed that the plant ATPase is one of the major membrane proteins made by the transformed cells, accounting for about 1% of total yeast protein. The plant ATPase synthesized in yeast is fully active. It hydrolyzes ATP, pumps protons, and the reaction cycle involves a phosphorylated intermediate. Phosphorylation is possible from both ATP and Pi. Unlike the situation in plants, however, most of the plant ATPase is not expressed in the yeast plasma membrane. Rather, the enzyme appears to remain trapped at a very early stage of secretory pathway: insertion into the endoplasmic reticulum. This organelle was observed to proliferate in the form of stacked membranes surrounding the yeast nucleus in order to accommodate the large amount of plant ATPase produced. In this location, the plant ATPase can be purified with high yield (70 mg from 1 kg of yeast) from membranes devoid of endogenous yeast plasma membrane H(+)-ATPase. This convenient expression system could be useful for other eukaryotic membrane proteins and ATPases.     

Long-term stabilization and crystallization of (Ca2+ + Mg2+)-ATPase of detergent-solubilized erythrocyte plasma membrane.

Conditions which were optimal for the stabilization of Ca2(+)-transporting ATPase in solubilized sarcoplasmic reticulum membranes (Piku?la, S., Mullner, N., Dux, L. and Martonosi, A. (1988) J. Biol. Chem. 263, 5277-5286) were also found conducive for preservation of (Ca2+ + Mg2+)-ATPase activity in detergent-solubilized erythrocyte plasma membrane for up to 60 days. Of particular importance for the stabilization of calmodulin-stimulated Ca2(+)-dependent activity of (Ca2+ + Mg2+)-ATPase of solubilized erythrocyte plasma membrane was the presence of Ca2+ (10-20 mM), glycerol, anti-oxidants, proteinase inhibitors and appropriate detergents. Among eight detergents tested octaethylene glycol dodecyl ether, polyoxyethylene glycol(10) lauryl alcohol and polydocanol were found to be promotive in long-term preservation of the enzyme activity. Under these conditions (Ca2+ + Mg2+)-ATPase of erythrocyte ghosts became highly stable and developed microcrystalline arrays after storage for 35 days. Electron micrographs of the negatively stained and thin sectioned material indicated that crystals of purified, detergent-solubilized, lipid-stabilized erythrocyte (Ca2+ + Mg2+)-ATPase differ from those of Ca2(+)-ATPase of detergent-solubilized sarcoplasmic reticulum microsomes.     

Altered contractility and [Ca2+]i homeostasis in phospholemman-deficient murine myocytes: role of Na+/Ca2+ exchange

Phospholemman (PLM) regulates contractility and Ca(2+) homeostasis in cardiac myocytes. We characterized excitation-contraction coupling in myocytes isolated from PLM-deficient mice backbred to a pure congenic C57BL/6 background. Cell length, cell width, and whole cell capacitance were not different between wild-type and PLM-null myocytes. Compared with wild-type myocytes, Western blots indicated total absence of PLM but no changes in Na(+)/Ca(2+) exchanger, sarcoplasmic reticulum (SR) Ca(2+)-ATPase, alpha(1)-subunit of Na(+)-K(+)-ATPase, and calsequestrin levels in PLM-null myocytes. At 5 mM extracellular Ca(2+) concentration ([Ca(2+)](o)), contraction and cytosolic [Ca(2+)] ([Ca(2+)](i)) transient amplitudes and SR Ca(2+) contents in PLM-null myocytes were significantly (P < 0.0004) higher than wild-type myocytes, whereas the converse was true at 0.6 mM [Ca(2+)](o). This pattern of contractile and [Ca(2+)](i) transient abnormalities in PLM-null myocytes mimics that observed in adult rat myocytes overexpressing the cardiac Na(+)/Ca(2+) exchanger. Indeed, we have previously reported that Na(+)/Ca(2+) exchange currents were higher in PLM-null myocytes. Activation of protein kinase A resulted in increased inotropy such that there were no longer any contractility differences between the stimulated wild-type and PLM-null myocytes. Protein kinase C stimulation resulted in decreased contractility in both wild-type and PLM-null myocytes. Resting membrane potential and action potential amplitudes were similar, but action potential duration was much prolonged (P < 0.04) in PLM-null myocytes. Whole cell Ca(2+) current densities were similar between wild-type and PLM-null myocytes, as were the fast- and slow-inactivation time constants. We conclude that a major function of PLM is regulation of cardiac contractility and Ca(2+) fluxes, likely by modulating Na(+)/Ca(2+) exchange activity.     

Yeast plasma membrane Ena1p ATPase alters alkali-cation homeostasis and confers increased salt tolerance in tobacco cultured cells

In plants, the plasma membrane Na(+)/H(+) antiporter is the only key enzyme that extrudes cytosolic Na(+) and contributes to salt tolerance. But in fungi, the plasma membrane Na(+)/H(+) antiporter and Na(+)-ATPase are known to be key enzymes for salt tolerance. Saccharomyces cerevisiae Ena1p ATPase encoded by the ENA1/PMR2A gene is primarily responsible for Na(+) and Li(+) efflux across the plasma membrane during salt stress and for K(+) efflux at high pH and high K(+). To test if the yeast ATPase would improve salt tolerance in plants, we expressed a triple hemagglutinin (HA)-tagged Ena1p (Ena1p-3HA) in cultured tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 (BY2) cells. The Ena1p-3HA proteins were correctly localized to the plasma membrane of transgenic BY2 cells and conferred increased NaCl and LiCl tolerance to the cells. Under moderate salt stress conditions, the Ena1p-3HA-expressing BY2 clones accumulated lower levels of Na(+) and Li(+) than nonexpressing BY2 clones. Moreover, the Ena1p-3HA expressing BY2 clones accumulated lower levels of K(+) than nonexpressing cells under no-stress conditions. These results suggest that the yeast Ena1p can also function as an alkali-cation (Na(+), Li(+), and K(+)) ATPase and alter alkali-cation homeostasis in plant cells. We conclude that, even with K(+)-ATPase activity, Na(+)-ATPase activity of the yeast Ena1p confers increased salt tolerance to plant cells during salt stress.