Bcl-2 and Ca2+ homeostasis in the endoplasmic reticulum

Recent data have revealed an unexpected role of Bcl-2 in modulating the steady-state levels and agonist-dependent fluxes of Ca(2+) ions. Direct monitoring of endoplasmic reticulum (ER) Ca(2+) concentration with recombinant probes reveals a lower state of filling in Bcl-2-overexpressing cells and a higher leak rate from the organelle. The broader set of indirect data using cytosolic probes reveals a more complex scenario, as in many cases no difference was detected in the Ca(2+) content of the intracellular pools. At the same time, Ca(2+) signals have been shown to affect important checkpoints of the apoptotic process, such as mitochondria, thus tuning the sensitivity of cells to various challenges. In this contribution, we will review (i) the data on the effect of Bcl-2 on [Ca(2+)](er), (ii) the functional significance of the Ca(2+)-signalling alteration and (iii) the current insight into the possible mechanisms of this effect.     

Chaperone-mediated coupling of endoplasmic reticulum and mitochondrial Ca2+ channels

The voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane mediates metabolic flow, Ca(2+), and cell death signaling between the endoplasmic reticulum (ER) and mitochondrial networks. We demonstrate that VDAC1 is physically linked to the endoplasmic reticulum Ca(2+)-release channel inositol 1,4,5-trisphosphate receptor (IP(3)R) through the molecular chaperone glucose-regulated protein 75 (grp75). Functional interaction between the channels was shown by the recombinant expression of the ligand-binding domain of the IP(3)R on the ER or mitochondrial surface, which directly enhanced Ca(2+) accumulation in mitochondria. Knockdown of grp75 abolished the stimulatory effect, highlighting chaperone-mediated conformational coupling between the IP(3)R and the mitochondrial Ca(2+) uptake machinery. Because organelle Ca(2+) homeostasis influences fundamentally cellular functions and death signaling, the central location of grp75 may represent an important control point of cell fate and pathogenesis.     

Endoplasmic reticulum Ca2+ dysregulation and endoplasmic reticulum stress following in vitro neuronal ischemia: role of Na+-K+-Cl- cotransporter

We investigated the role of Na(+)-K(+)-Cl(-) cotransporter (NKCC1) in conjunction with Na(+)/Ca(2+) exchanger (NCX) in disruption of endoplasmic reticulum (ER) Ca(2+) homeostasis and ER stress development in primary cortical neurons following in vitro ischemia. Oxygen-glucose deprivation (OGD) and reoxygenation (REOX) caused a rise in [Na(+)](cyt) which was accompanied by an elevation in [Ca(2+)](cyt). Inhibition of NKCC1 with its potent inhibitor bumetanide abolished the OGD/REOX-induced rise in [Na(+)](cyt) and [Ca(2+)](cyt). Moreover, OGD significantly increased Ca(2+)(ER) accumulation. Following REOX, a biphasic change in Ca(2+)(ER) occurred with an initial release of Ca(2+)(ER) which was sensitive to inositol 1,4,5-trisphosphate receptor (IP(3)R) inhibition and a subsequent refilling of Ca(2+)(ER) stores. Inhibition of NKCC1 activity with its inhibitor or genetic ablation prevented the release of Ca(2+)(ER). A similar result was obtained with inhibition of reversed mode operation of NCX (NCX(rev)). OGD/REOX also triggered a transient increase of glucose regulated protein 78 (GRP78), phospho-form of the alpha subunit of eukaryotic initiation factor 2 (p-eIF2alpha), and cleaved caspase 12 proteins. Pre-treatment of neurons with NKCC1 inhibitor bumetanide inhibited upregulation of GRP78 and attenuated the level of cleaved caspase 12 and p-eIF2alpha. Inhibition of NKCC1 reduced cytochrome C release and neuronal death. Taken together, these results suggest that NKCC1 and NCX(rev) may be involved in ischemic cell damage in part via disrupting ER Ca(2+) homeostasis and ER function.     

BI-1 regulates endoplasmic reticulum Ca2+ homeostasis downstream of Bcl-2 family proteins

BI-1 (Bax inhibitor-1) is an evolutionarily conserved multitransmembrane protein that resides in the endoplasmic reticulum (ER) and that has documented cytoprotective functions in both animals and plants. Recent studies indicate that BI-1 shares in common with Bcl-2/Bax family proteins the ability to regulate the amounts of Ca(2+) that can be released from the ER by agents, such as the ER-Ca(2+)-ATPase (SERCA) inhibitor thapsigargin (TG). Using an ER-targeted, Ca(2+) indicator (cameleon), with characteristics optimized for measuring ER Ca(2+) ([Ca(2+)](er)), we studied the effects of BI-1 on [Ca(2+)](er) in resting and TG-treated cells. Similar to cells overexpressing antiapoptotic Bcl-2 or Bcl-X(L), overexpression of BI-1 resulted in lower resting [Ca(2+)](er), with concomitantly less Ca(2+) released into the cytosol upon stimulation by TG and with a higher rate of Ca(2+) leakage from the ER. Co-expression of SERCA restored levels of [Ca(2+)](er) to normal, showing opposing actions of the ER-Ca(2+)ATPase and BI-1 on ER Ca(2+) homeostasis. Conversely, cells with deficient BI-1 have increased [Ca(2+)](er), and release more Ca(2+) into the cytosol when challenged with TG. In BI-1-deficient cells, Bcl-X(L) fails to reduce [Ca(2+)](er), indicating that BI-1 functions downstream of Bcl-X(L). In bax(-/-)bak(-/-) double knock-out cells, both BI-1 and Bcl-X(L) retained their ability to reduce [Ca(2+)](er), suggesting that BI-1 and Bcl-X(L) operate downstream of or parallel to Bax/Bak. The findings reveal a hierarchy of functional interactions of BI-1 with Bcl-2/Bax family proteins in regulating ER Ca(2+) homeostasis.     

Altered Ca2+ homeostasis and impaired mitochondrial function in cardiomyopathy

This work investigates whether purine metabolism and release is related to cardioprotection with hyperkalemia and hypothermia. Langendorff guinea-pig hearts were used to either monitor metabolism during ischemia or to measure functional recovery, myocardial injury and release of purine during reperfusion. Hearts underwent 30 min ischemia using one of the following protocols: control (normothermic buffer), hyperkalaemia (high-potassium buffer), hypothermia (20°C) and hyperkalemia + hypothermia. At the end of 30 min ischemia, hyperkalemia was associated with similar metabolic changes (rise in purine and lactate and fall in adenine nucleotides) to control group. Accumulation of purine was due to a rise in inosine, xanthine and hypoxanthine and was largely prevented by hypothermia and hyperkalemia + hypothermia. Upon reperfusion, there was a time-dependent release of all purine, lactate and AMP. A fast (peak in less than 20 sec) release of inosine, xanthine, hypoxanthine and lactate was highest in control followed by hyperkalemia then hypothermia and little release in hyperkalemia + hypothermia. Adenosine and AMP release was slow (peak at 3 min), only significant in control and was likely to be due to sarcolemmal disruption as the profile followed lactate dehydrogenase release. Recovery (left ventricular developed pressure) was 63% control, 82% hyperkalemia, 77% hypothermia and 98% for hyperkalemia + hypothermia. The loss of purine during reperfusion but not their production during ischemia is related to cardioprotection with hyperkalemia. The possibility that the consequences of hyperkalemia modulate a sodium-dependent purine efflux, is discussed. The reduced loss of purine in hypothermia or in hyperkalemia + hypothermia is likely to be due to a lower metabolic activity during ischemia.     

STIM2 is a feedback regulator that stabilizes basal cytosolic and endoplasmic reticulum Ca2+ levels.

Deviations in basal Ca2+ levels interfere with receptor-mediated Ca2+ signaling as well as endoplasmic reticulum (ER) and mitochondrial function. While defective basal Ca2+ regulation has been linked to various diseases, the regulatory mechanism that controls basal Ca2+ is poorly understood. Here we performed an siRNA screen of the human signaling proteome to identify regulators of basal Ca2+ concentration and found STIM2 as the strongest positive regulator. In contrast to STIM1, a recently discovered signal transducer that triggers Ca2+ influx in response to receptor-mediated depletion of ER Ca2+ stores, STIM2 activated Ca2+ influx upon smaller decreases in ER Ca2+. STIM2, like STIM1, caused Ca2+ influx via activation of the plasma membrane Ca2+ channel Orai1. Our study places STIM2 at the center of a feedback module that keeps basal cytosolic and ER Ca2+ concentrations within tight limits.     

Endoplasmic reticulum Ca(2+) homeostasis and neuronal death

The endoplasmic reticulum (ER) is a universal signalling organelle, which regulates a wide range of neuronal functional responses. Calcium release from the ER underlies various forms of intracellular Ca²⁺ signalling by either amplifying Ca²⁺ entry through voltage-gated Ca²⁺ channels by Ca²⁺-induced Ca²⁺ release (CICR) or by producing local or global cytosolic calcium fluctuations following stimulation of metabotropic receptors through inositol-1,4,5-trisphosphate-induced Ca²⁺ release (IICR). The ER Ca²⁺ store emerges as a single interconnected pool, thus allowing for a long-range Ca²⁺ signalling via intra-ER tunnels. The fluctuations of intra-ER free Ca²⁺ concentration regulate the activity of numerous ER resident proteins responsible for post-translational protein folding and modification. Disruption of ER Ca²⁺ homeostasis results in the developing of ER stress response, which in turn controls neuronal survival. Altered ER Ca²⁺ handling may be involved in pathogenesis of various, neurodegenerative diseases including brain ischemia and Alzheimer dementia.     

Ca2+ shuttling between endoplasmic reticulum and mitochondria underlying Ca2+ oscillations

Although many cell functions are regulated by Ca(2+) oscillations induced by a cyclic release of Ca(2+) from intracellular Ca(2+) stores, the pacemaker mechanism of Ca(2+) oscillations remains to be explained. Using green fluorescent protein-based Ca(2+) indicators that are targeted to intracellular Ca(2+) stores, the endoplasmic reticulum (ER) and mitochondria, we found that Ca(2+) shuttles between the ER and mitochondria in phase with Ca(2+) oscillations. Following agonist stimulation, Ca(2+) release from the ER generated the first Ca(2+) oscillation and loaded mitochondria with Ca(2+). Before the second Ca(2+) oscillation, Ca(2+) release from the mitochondria by means of the Na(+)/Ca(2+) exchanger caused a gradual increase in cytoplasmic Ca(2+) concentration, inducing a regenerative ER Ca(2+) release, which generated the peak of Ca(2+) oscillation and partially reloaded the mitochondria. This sequence of events was repeated until mitochondrial Ca(2+) was depleted. Thus, Ca(2+) shuttling between the ER and mitochondria may have a pacemaker role in the generation of Ca(2+) oscillations.     

Herp, a new ubiquitin-like membrane protein induced by endoplasmic reticulum stress

Hyperhomocysteinemia, a risk factor for vascular disease, injures endothelial cells through undefined mechanisms. We previously identified several homocysteine-responsive genes in cultured human vascular endothelial cells, including the endoplasmic reticulum (ER)-resident molecular chaperone GRP78/BiP. Here, we demonstrate that homocysteine induces the ER stress response and leads to the expression of a novel protein, Herp, containing a ubiquitin-like domain at the N terminus. mRNA expression of Herp was strongly up-regulated by inducers of ER stress, including mercaptoethanol, tunicamycin, A23187, and thapsigargin. The ER stress-dependent induction of Herp was also observed at the protein level. Immunochemical analyses using Herp-specific antibodies indicated that Herp is a 54-kDa, membrane-associated ER protein. Herp is the first integral membrane protein regulated by the ER stress response pathway. Both the N and C termini face the cytoplasmic side of the ER; this membrane topology makes it unlikely that Herp acts as a molecular chaperone for proteins in the ER, in contrast to GRP78 and other ER stress-responsive proteins. Herp may, therefore, play an unknown role in the cellular survival response to stress.     

Myeloperoxidase-derived oxidants inhibit sarco/endoplasmic reticulum Ca2+-ATPase activity and perturb Ca2+ homeostasis in human coronary artery endothelial cells

The sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) plays a critical role in Ca(2+) homeostasis via sequestration of this ion in the sarco/endoplasmic reticulum. The activity of this pump is inhibited by oxidants and impaired in aging tissues and cardiovascular disease. We have shown previously that the myeloperoxidase (MPO)-derived oxidants HOCl and HOSCN target thiols and mediate cellular dysfunction. As SERCA contains Cys residues critical to ATPase activity, we hypothesized that HOCl and HOSCN might inhibit SERCA activity, via thiol oxidation, and increase cytosolic Ca(2+) levels in human coronary artery endothelial cells (HCAEC). Exposure of sarcoplasmic reticulum vesicles to preformed or enzymatically generated HOCl and HOSCN resulted in a concentration-dependent decrease in ATPase activity; this was also inhibited by the SERCA inhibitor thapsigargin. Decomposed HOSCN and incomplete MPO enzyme systems did not decrease activity. Loss of ATPase activity occurred concurrent with oxidation of SERCA Cys residues and protein modification. Exposure of HCAEC, with or without external Ca(2+), to HOSCN or HOCl resulted in a time- and concentration-dependent increase in intracellular Ca(2+) under conditions that did not result in immediate loss of cell viability. Thapsigargin, but not inhibitors of plasma membrane or mitochondrial Ca(2+) pumps/channels, completely attenuated the increase in intracellular Ca(2+) consistent with a critical role for SERCA in maintaining endothelial cell Ca(2+) homeostasis. Angiotensin II pretreatment potentiated the effect of HOSCN at low concentrations. MPO-mediated modulation of intracellular Ca(2+) levels may exacerbate endothelial dysfunction, a key early event in atherosclerosis, and be more marked in smokers because of their higher SCN(-) levels.     

Presynaptic store-operated Ca2+ entry drives excitatory spontaneous neurotransmission and augments endoplasmic reticulum stress

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Mitochondrial Ca2+ uptake requires sustained Ca2+ release from the endoplasmic reticulum

We analyzed the role of inositol 1,4,5-trisphosphate-induced Ca(2+) release from the endoplasmic reticulum (ER) (i) in powering mitochondrial Ca(2+) uptake and (ii) in maintaining a sustained elevation of cytosolic Ca(2+) concentration ([Ca(2+)](c)). For this purpose, we expressed in HeLa cells aequorin-based Ca(2+)-sensitive probes targeted to different intracellular compartments and studied the effect of two agonists: histamine, acting on endogenous H(1) receptors, and glutamate, acting on co-transfected metabotropic glutamate receptor (mGluR1a), which rapidly inactivates through protein kinase C-dependent phosphorylation and thus causes transient inositol 1,4,5-trisphosphate production. Glutamate induced a transient [Ca(2+)](c) rise and drop in ER luminal [Ca(2+)] ([Ca(2+)](er)), and then the ER refilled with [Ca(2+)](c) at resting values. With histamine, [Ca(2+)](c) after the initial peak stabilized at a sustained plateau, and [Ca(2+)](er) decreased to a low steady-state value. In mitochondria, histamine evoked a much larger mitochondrial Ca(2+) response than glutamate ( approximately 15 versus approximately 65 microm). Protein kinase C inhibition, partly relieving mGluR1a desensitization, reestablished both the [Ca(2+)](c) plateau and the sustained ER Ca(2+) release and markedly increased the mitochondrial Ca(2+) response. Conversely, mitochondrial Ca(2+) uptake evoked by histamine was drastically reduced by very transient ( approximately 2-s) agonist applications. These data indicate that efficient mitochondrial Ca(2+) uptake depends on the preservation of high Ca(2+) microdomains at the mouth of ER Ca(2+) release sites close to mitochondria. This in turn depends on continuous Ca(2+) release balanced by Ca(2+) reuptake into the ER and maintained by Ca(2+) influx from the extracellular space.     

Ca2+ regulation of interactions between endoplasmic reticulum chaperones

Casade Blue (CB), a fluorescent dye, was used to investigate the dynamics of interactions between endoplasmic reticulum (ER) lumenal chaperones including calreticulin, protein disulfide isomerase (PDI), and ERp57. PDI and ERp57 were labeled with CB, and subsequently, we show that the fluorescence intensity of the CB-conjugated proteins changes upon exposure to microenvironments of a different polarity. CD analysis of the purified proteins revealed that changes in the fluorescence intensity of CB-ERp57 and CB-PDI correspond to conformational changes in the proteins. Using this technique we demonstrate that PDI interacts with calreticulin at low Ca2+ concentration (below 100 microM), whereas the protein complex dissociates at >400 microM Ca2+. These are the Ca2+ concentrations reminiscent of Ca2+ levels found in empty or full ER Ca2+ stores. The N-domain of calreticulin interacts with PDI, but Ca2+ binding to the C-domain of the protein is responsible for Ca2+ sensitivity of the interaction. ERp57 also interacts with calreticulin through the N-domain of the protein. Initial interaction between these proteins is Ca2+-independent, but it is modulated by Ca2+ binding to the C-domain of calreticulin. We conclude that changes in ER lumenal Ca2+ concentration may be responsible for the regulation of protein-protein interactions. Calreticulin may play a role of Ca2+ "sensor" for ER chaperones via regulation of Ca2+-dependent formation and maintenance of structural and functional complexes between different proteins involved in a variety of steps during protein synthesis, folding, and post-translational modification.     

Bcl-2 increases emptying of endoplasmic reticulum Ca2+ stores during photodynamic therapy-induced apoptosis.

Photodynamic therapy (PDT) is clinically approved for the treatment of several types of cancer as well as age-related macular degeneration, the leading cause of blindness in the elderly. PDT using the photosensitizer verteporfin has been previously shown to induce rapid apoptosis via a mitochondrial-caspase activation pathway. The impact of PDT on other cellular organelles such as the endoplasmic reticulum (ER) is undefined. The effect of PDT on intracellular Ca2+ ([Ca2+]i) in control and Bcl-2-overexpressing HeLa cells was assessed. A greater [Ca2+]i transient was observed for Bcl-2 overexpressing cells in response to PDT. The PDT-induced Ca2+ release was due to the emptying of Ca2+ from ER and possibly mitochondrial stores and was not due to an influx of Ca2+ from the medium. For Bcl-2-transfected cells, the release of Ca2+ was incomplete as determined by a further [Ca2+]i transient produced by the addition of the Ca2+ ionophore ionomycin after PDT. Furthermore, extrusion of Ca2+ was not hindered while ER-mediated sequestration of Ca2+ was impaired after PDT. Impairment of ER-mediated sequestration of Ca2+ may be due to the immediate caspase-independent depletion of sarco/endoplasmic reticulum Ca2+ ATPase-2 (SERCA2) that occurred in response to PDT in birth HeLa/Neo and Bcl-2 overexpressed HeLa cells. In summary, PDT induced the rapid degradation of SERCA2 and release of ER and mitochondrial Ca2+ stores. Although overexpression of Bcl-2 did not protect against SERCA2 degradation, it may influence the release of Ca2+ from ER and mitochondrial stores in PDT-treated cells.     

Calreticulin, Ca2+-binding chaperon of the endoplasmic reticulum

The endoplasmic reticulum (ER) plays a vital role in many cellular processes, including Ca2+ storage and release. Calreticulin is a Ca2+-binding chaperon residing in ER. The protein is a key component of the quality control pathways in ER. In the ER lumen, calreticulin performs two major functions, works as a chaperon and regulates Ca2+ homeostasis. In cardiac muscle, calreticulin plays an important role in cardiac development and pathology.     

Mitoregulin controls mitochondrial function and stress-adaptation response during early phase of endoplasmic reticulum stress in breast cancer cells

The proper regulation of mitochondrial function is important for cellular homeostasis. Especially, in cancer cells, dysregulation of mitochondria is associated with diverse cellular events such as metabolism, redox status, and stress responses. Mitoregulin (MTLN), a micro protein encoded by LINC00116, recently has been reported to control mitochondrial functions in skeletal muscle cells and adipocytes. However, the role of MTLN in cancer cells remains unclear. In the present study, we found that MTLN regulates membrane potential and reactive oxygen species (ROS) generation of mitochondria in breast cancer cells. Moreover, MTLN deficiency resulted in abnormal mitochondria-associated ER membranes (MAMs) formation, which is crucial for stress adaptation. Indeed, the MTLN-deficient breast cancer cells failed to successfully resolve ER (endoplasmic reticulum) stress, and cell vulnerability to ER-stress inducers was significantly enhanced by the downregulation of MTLN. In conclusion, MTLN controls stress-adaptation responses in breast cancer cells as a key regulator of mitochondria-ER harmonization, and thereby its expression level may serve as an indicator of the responsiveness of cancer cells to proteasome inhibitors.     

Taurine resumed neuronal differentiation in arsenite-treated N2a cells through reducing oxidative stress,endoplasmic reticulum stress,and mitochondrial dysfunction

The goal of the study is to investigate the preventive effect of taurine against arsenite-induced arrest of neuronal differentiation in N2a cells. Our results revealed that taurine reinstated the neurite outgrowth in arsenite-treated N2a cells. Meanwhile, arsenite-induced oxidative stress and mitochondrial dysfunction as well as degradation of mitochondria DNA (mtDNA) were also inhibited by co-treatment of taurine. Since oxidative stress and mitochondrial dysfunction is closely associated with endoplasmic reticulum (ER) stress, we further examined indicators of ER stress, 78 kDa glucose-regulated protein (GRP78), and C/EBP-homologous protein (CHOP) protein expression. The results demonstrated that taurine significantly reduced arsenite-induced ER stress in N2a cells. In the parallel experiment, arsenite-induced disruption of intracellular calcium homeostasis was also ameliorated by taurine. The proven bio-function of taurine preserved a preventive effect against deleteriously cross-talking between oxidative stress, mitochondria, and ER. Overall, the results of the study suggested that taurine reinstated neuronal differentiation by inhibiting oxidative stress, ER stress, and mitochondrial dysfunction in arsenite-treated N2a cells.     

R-Ras alters Ca2+ homeostasis by increasing the Ca2+ leak across the endoplasmic reticular membrane

Evidence in the literature implicating both Ras-like Ras (R-Ras) and intracellular Ca(2+) in programmed cell death and integrin-mediated adhesion prompted us to investigate the possibility that R-Ras alters cellular Ca(2+) handling. Chinese hamster ovary cells expressing the cholecystokinin (CCK)-A receptor were loaded with indo-1 to study the effects of constitutively active V38R-Ras and dominant negative N43R-Ras on the kinetics of the thapsigargin (Tg)- and CCK(8)-induced Ca(2+) rises using high speed confocal microscopy. In the absence of extracellular Ca(2+), both 1 microm Tg, a potent and selective inhibitor of the Ca(2+) pump of the intracellular Ca(2+) store, and 100 nm CCK(8) evoked a transient rise in Ca(2+), the size of which was decreased significantly after expression of V38R-Ras. At 0.1 nm, CCK(8) evoked periodic Ca(2+) rises. The frequency of these Ca(2+) oscillations was reduced significantly in V38R-Ras-expressing cells. In contrast to V38R-Ras, N43R-Ras did not alter the kinetics of the Tg- and CCK(8)-induced Ca(2+) rises. The present findings are compatible with the idea that V38R-Ras expression increases the passive leak of Ca(2+) of the store leading to a decrease in Ca(2+) content of this store, which, in turn, leads to a decrease in frequency of the CCK(8)-induced cytosolic Ca(2+) oscillations. The effect of V38R-Ras on the Ca(2+) content of the intracellular Ca(2+) store closely resembles that of the antiapoptotic protein Bcl-2 observed earlier. Together with reports on the role of dynamic Ca(2+) changes in integrin-mediated adhesion, this leads us to propose that the reduction in endoplasmic reticulum Ca(2+) content may underlie the antiapoptotic effect of R-Ras, whereas the decrease in frequency of stimulus-induced Ca(2+) oscillations may play a role in the inhibitory effect of R-Ras on stimulus-induced cell detachment and migration.     

Gibberellic-acid-stimulated Ca2+ accumulation in endoplasmic reticulum of barley aleurone: Ca2+ transport and steady-state levels

The steady-state levels of Ca²⁺ within the endoplasmic reticulum (ER) and the transport of ⁴⁵Ca²⁺ into isolated ER of barley (Hordeum vulgare L. cv. Himalaya) aleurone layers were studied. The Ca²⁺-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca²⁺ level in the ER was measured using the Ca²⁺-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca²⁺ level in the lumen of the ER was determined by the fluorescence-ratio method to be at least 3 M. Transport of ⁴⁵Ca²⁺ into the ER was studied in microsomal fractions isolated from aleurone layers incubated in the presence and absence of gibberellic acid (GA₃) and Ca²⁺. Isopycinic sucrose density gradient centrifugation of microsomal fractions isolated from aleurone layers or protoplasts separates ER from tonoplast and plasma membranes but not from the Golgi apparatus. Transport of ⁴⁵Ca²⁺ occurs primarily in the microsomal fraction enriched in ER and Golgi. Using monensin and heat-shock treatments to discriminate between uptake into the ER and Golgi, we established that ⁴⁵Ca²⁺ transport was into the ER. The sensitivity of ⁴⁵Ca²⁺ transport to inhibitors and the K_m of ⁴⁵Ca²⁺ uptake for ATP and Ca²⁺ transport in the microsomal fraction of barley aleurone cells. The rate of ⁴⁵Ca²⁺ transport is stimulated several-fold by treatment with GA₃. This effect of GA₃ is mediated principally by an effect on the activity of the Ca²⁺ transporter rather than on the amount of ER.Abbreviations CCR cytochrome-c reductase - DCCD dicyclohexylcarbodiimide - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - ER endoplasmic reticulum - FCCP carbonylcyanide p-trifluoromethoxyphenyl hydrazone - GA₃ gibberellic acid - IDPase inosine diphosphatase - Mon monensin