Chlorophyll Biosynthetic Reactions during Senescence of Excised Barley (Hordeum vulgare L. cv IB 65) Leaves

The inhibitor sensitivity of the endoplasmic reticulum (ER) and plasma membrane (PM) calcium pumps of red beet (Beta vulgaris L.) were studied by measuring the ATP-driven accumulation of 45Ca2+ into isolated membrane vesicles. Both transporters were strongly inhibited by 50 [mu]mol m-3 erythrosin B, but only by 50% in the presence of 100 mmol m-3 vanadate. A number of inhibitors considered to be specific for the sarcoplasmic reticulum (SR)/ER-type calcium pump in animal cells were used to further characterize the PM and ER Ca2+-ATPases in red beet and were compared with their effect on the transport and hydrolytic activities of the PM and tonoplast H+-ATPases. The hydroquinones 2,5-di(tert-butyl)-1,4-benzohydroquinone and 2,5-di(tert-amyl)-1,4-benzohydroquinone produced around 20 and 40% inhibition of activity, respectively, of the PM and ER calcium pumps and the PM H+-ATPase when present at concentrations of 30 mmol m-3. In contrast, the vacuolar proton pump displayed a much higher sensitivity to these two compounds. Nonylphenol appeared to have a general inhibitory effect on all four membrane transport proteins and gave almost complete inhibition when present at a concentration of 100 mmol m-3. Thapsigargin and the structurally related compound trilobolide produced 50% inhibition of both the ER and PM calcium pumps at concentrations of 12.5 and 24 mmol m-3, respectively. The PM and tonoplast proton pumps were also sensitive to these compounds. The ER and PM calcium pumps were almost completely insensitive to cyclopiazonic acid (CPA) up to a concentration of 20 mmol m-3. When present at 100 mmol m-3 CPA caused 30% inhibition of the transport properties of all four ATPases. The high concentrations of all of the inhibitors of the SR/ER Ca-ATPase required to inhibit the red beet ER calcium pump, together with the similar effects on the PM calcium pump and the PM and tonoplast proton pumps, suggests that these hydrophobic compounds have a general nonselective action in red beet, possibly through disruption of membrane lipid-protein interactions.     

Basal and physiological Ca(2+) leak from the endoplasmic reticulum of pancreatic acinar cells. Second messenger-activated channels and translocons

We have studied the Ca(2+) leak pathways in the endoplasmic reticulum of pancreatic acinar cells by directly measuring Ca(2+) in the endoplasmic reticulum ([Ca(2+)](ER)). Cytosolic Ca(2+) ([Ca(2+)](C)) was clamped to the resting level by a BAPTA-Ca(2+) mixture. Administration of cholecystokinin within the physiological concentration range caused a graded decrease of [Ca(2+)](ER), and the rate of Ca(2+) release generated by 10 pm cholecystokinin is at least 3x as fast as the basal Ca(2+) leak revealed by inhibition of the endoplasmic reticulum Ca(2+)-ATPase. Acetylcholine also evokes a dose-dependent decrease of [Ca(2+)](ER), with an EC(50) of 0.98 +/- 0.06 microm. Inhibition of receptors for inositol 1,4,5-trisphosphate (IP(3)) by heparin or flunarizine blocks the effect of acetylcholine but only partly blocks the effect of cholecystokinin. 8-NH(2) cyclic ADP-ribose (20 microm) inhibits the action of cholecystokinin, but not of acetylcholine(.) The basal Ca(2+) leak from the endoplasmic reticulum is not blocked by antagonists of the IP(3) receptor, the ryanodine receptor, or the receptor for nicotinic acid adenine dinucleotide phosphate. However, treatment with puromycin (0.1-1 mm) to remove nascent polypeptides from ribosomes increases Ca(2+) leak from the endoplasmic reticulum by a mechanism independent of the endoplasmic reticulum Ca(2+) pumps and of the receptors for IP(3) or ryanodine.     

Calcium homoeostasis modulator 1 (CALHM1) reduces the calcium content of the endoplasmic reticulum (ER) and triggers ER stress

CALHM1 (calcium homoeostasis modulator 1), a membrane protein with similarity to NMDA (N-methyl-D-aspartate) receptor channels that localizes in the plasma membrane and the ER (endoplasmic reticulum) of neurons, has been shown to generate a plasma-membrane Ca(2+) conductance and has been proposed to influence Alzheimer's disease risk. In the present study we have investigated the effects of CALHM1 on intracellular Ca(2+) handling in HEK-293T [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] cells by using targeted aequorins for selective monitorization of Ca(2+) transport by organelles. We find that CALHM1 increases Ca(2+) leak from the ER and, more importantly, reduces ER Ca(2+) uptake by decreasing both the transport capacity and the Ca(2+) affinity of SERCA (sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase). As a result, the Ca(2+) content of the ER is drastically decreased. This reduction in the Ca(2+) content of the ER triggered the UPR (unfolded protein response) with induction of several ER stress markers, such as CHOP [C/EBP (CCAAT/enhancer-binding protein)-homologous protein], ERdj4, GRP78 (glucose-regulated protein of 78 kDa) and XBP1 (X-box-binding protein 1). Thus CALHM1 might provide a relevant link between Ca(2+) homoeostasis disruption, ER stress and cell damage in the pathogenesis of neurodegenerative diseases.     

Overexpression of calsequestrin in L6 myoblasts: formation of endoplasmic reticulum subdomains and their evolution into discrete vacuoles where aggregates of the protein are specifically accumulated.

Calsequestrin (CSQ), the major low-affinity Ca(2+)-binding glycoprotein of striated muscle fibers, is concentrated to yield aggregates that occupy the lumen of the terminal cisternae of the sarcoplasmic reticulum (SR). When infected or transfected into L6 myoblast, the protein is also concentrated, however, in dense vacuoles apparently separate from the endoplasmic reticulum (ER). CSQ-rich cells appear otherwise normal; in particular, neither other proteins involved in Ca2+ homeostasis nor ER chaperones are increased. The CSQ dense vacuoles are shown herein to be specialized ER subdomains as demonstrated by 1) the endoglycosidase H sensitivity of their CSQ and 2) two markers, calreticulin and calnexin (but not others, protein disulfide isomerase and BiP), intermixed with the vacuole content. Their formation is shown to start with the aggregation of CSQ at discrete sites of the ER lumen. When cells were transfected with both CSQ and calreticulin, only the first gave rise to vacuoles; the second remained diffusely distributed within the ER lumen. The possibility that CSQ aggregation is an artifact of overexpression appears unlikely because 1) within dense vacuoles CSQ molecules are not disulfide cross-linked, 2) their turnover is relatively slow (t = 12 h), and 3) segregated CSQ is bound to large amounts of Ca2+. Transfection of a tagged CSQ into cells already overexpressing the protein revealed the continuous import of the newly synthesized protein into preassembled vacuoles. The tendency to aggregation appears, therefore, as a property contributing to the segregation of CSQ within the ER lumen and to its accumulation within specialized subdomains. The study of L6 cells expressing CSQ-rich vacuoles might thus ultimately help to unravel mechanisms by which the complexity of the sarcoplasmic reticulum is established in muscle fibers.     

Reverse mode of the sarcoplasmic reticulum calcium pump and load-dependent cytosolic calcium decline in voltage-clamped cardiac ventricular myocytes

We have characterized [Ca](i) decline in voltage-clamped rabbit ventricular myocytes with progressive increases in sarcoplasmic reticulum (SR) calcium load. "Backflux" through the SR calcium pump is a critical feature which allows realistically small values for SR calcium leak fluxes to be used. Total cytosolic calcium was calculated from the latter part of [Ca](i) decline using rate constants for cellular calcium buffers. Intra-SR calcium buffering characteristics were also deduced. We found that the net SR calcium pump flux and rate of [Ca](i) decline decreased as the SR free [Ca] rose, with pump parameters held constant. We have therefore characterized for the first time in intact myocytes both forward and reverse SR calcium pump kinetics as well as intra-SR calcium buffering and SR calcium leak. We conclude that the reverse flux through the SR calcium pump is an important factor in comprehensive understanding of dynamic SR calcium fluxes.     

Mesencephalic astrocyte-derived neurotrophic factor protects the heart from ischemic damage and is selectively secreted upon sarco/endoplasmic reticulum calcium depletion

The endoplasmic reticulum (ER) stress protein mesencephalic astrocyte-derived neurotrophic factor (MANF) has been reported to protect cells from stress-induced cell death before and after its secretion; however, the conditions under which it is secreted are not known. Accordingly, we examined the mechanism of MANF release from cultured ventricular myocytes and HeLa cells, both of which secrete proteins via the constitutive pathway. Although the secretion of proteins via the constitutive pathway is not known to increase upon changes in intracellular calcium, MANF secretion was increased within 30 min of treating cells with compounds that deplete sarcoplasmic reticulum (SR)/ER calcium. In contrast, secretion of atrial natriuretic factor from ventricular myocytes was not increased by SR/ER calcium depletion, suggesting that not all secreted proteins exhibit the same characteristics as MANF. We postulated that SR/ER calcium depletion triggered MANF secretion by decreasing its retention. Consistent with this were co-immunoprecipitation and live cell, zero distance, photo affinity cross-linking, demonstrating that, in part, MANF was retained in the SR/ER via its calcium-dependent interaction with the SR/ER-resident protein, GRP78 (glucose-regulated protein 78 kDa). This unusual mechanism of regulating secretion from the constitutive secretory pathway provides a potentially missing link in the mechanism by which extracellular MANF protects cells from stresses that deplete SR/ER calcium. Consistent with this was our finding that administration of recombinant MANF to mice decreased tissue damage in an in vivo model of myocardial infarction, a condition during which ER calcium is known to be dysregulated, and MANF expression is induced.     

Expression of the sarco/endoplasmic Ca(2+)-ATPase, SERCA1a, in fibroblasts induces the formation of organelle membrane arrays

Members of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) family are transmembrane proteins that are essential for the function of intracellular Ca(2+) storage organelles. We found that overexpression of avian muscle SERCA1a in transfected mouse fibroblasts led to the appearance of tubular membrane bundles that we termed plaques. These structures were generated in transfected cells when SERCA1a protein expression approached the endogenous level measured in chicken skeletal muscle. Plaque membranes had associated ribosomes and contained endoplasmic reticulum (ER) proteins. Endogenous ER protein levels were not elevated in SERCA1a-expressing cells, indicating that plaques were not generalized proliferations of ER but rather a reorganization of existing organelle membrane. Plaque formation also was observed in cells expressing a green fluorescent protein-SERCA1a fusion protein (GFP-SERCA1a). GFP-SERCA1a molecules displayed extensive lateral mobility between plaques, suggesting the presence of membrane continuities between these structures. Plaques were induced in cells expressing cDNA encoding a catalytically silent SERCA1a mutant indicating that ER redistribution was driven by a structural feature of the enzyme. SERCA1a-induced plaque formation shares some characteristics of sarcoplasmic reticulum (SR) biogenesis during muscle differentiation, and high-level SERCA1a expression in vivo may contribute to the formation of SR from ER during embryonic myogenesis.     

The endoplasmic reticulum and calcium storage

Calcium storage is one of the functions commonly attributed to the endoplasmic reticulum (ER) in nonmuscle cells. Several recent studies have added support to this concept. Analysis of reticuloplasm, the luminal ER content, has shown that it contains several proteins (reticuloplasmins) which are prospective calcium storage proteins. One of these, calreticulin, is also present in the sarcoplasmic reticulum (SR). In sea urchin eggs, a calsequestrin-like protein has been clearly localised to the ER. The recent demonstration that the IP3 receptor, which has similarities with the calcium release channel in the SR is also localised in the ER membrane suggests that calcium stored in the ER is important for intracellular signalling. The alternative view, that the physiologically important calcium store is a specialised organelle, the calciosome, is not supported by these observations. Recent evidence also suggests that ER calcium might be important in ER structure and in the retention of the luminal ER proteins.     

Steady-state free Ca(2+) in the yeast endoplasmic reticulum reaches only 10 microM and is mainly controlled by the secretory pathway pump pmr1.

Over recent decades, diverse intracellular organelles have been recognized as key determinants of Ca(2+) signaling in eukaryotes. In yeast however, information on intra-organellar Ca(2+) concentrations is scarce, despite the demonstrated importance of Ca(2+) signals for this microorganism. Here, we directly monitored free Ca(2+) in the lumen of the endoplasmic reticulum (ER) of yeast cells, using a specifically targeted version of the Ca(2+)-sensitive photoprotein aequorin. Ca(2+) uptake into the yeast ER displayed characteristics distinctly different from the mammalian ER. At steady-state, the free Ca(2+) concentration in the ER lumen was limited to approximately 10 microM, and ER Ca(2+) sequestration was insensitive to thapsigargin, an inhibitor specific for mammalian ER Ca(2+) pumps. In pmr1 null mutants, free Ca(2+) in the ER was reduced by 50%. Our findings identify the secretory pathway pump Pmr1, predominantly localized in the Golgi, as a major component of ER Ca(2+) uptake activity in yeast.     

The sarcoplasmic reticulum Ca(2+)-ATPase, SERCA1a, contains endoplasmic reticulum targeting information.

The fast-twitch skeletal muscle Ca(2+)-ATPase isoenzyme, SERCA1a, is localized in chick skeletal myotubes to both the sarcoplasmic reticulum (SR) and to the nuclear envelope, an extension of the endoplasmic reticulum (ER). The ER labeling remained after cycloheximide treatment, indicating that it did not represent newly synthesized SERCA1a in transit to the SR. Expression of the cDNA encoding SERCA1a in cultured non-muscle cells led to the localization of the enzyme in the ER, as indicated by organelle morphology and the co-localization of SERCA1a with the endogenous ER luminal protein, BiP. Immunopurification analysis showed that SERCA1a was not bound to BiP, nor was any degradation apparent. Thus, the SR Ca(2+)-ATPase appears to contain ER targeting information.     

A new hypothesis for Ca2+ flows in skeletal muscle and its implications for other cell types

We offer a new hypothesis to explain calcium flows in skeletal muscle cells. Our model accounts for the uptake of Ca²⁺ from the extracellular fluid, and the release of Ca²⁺ from the sarcoplasmic reticulum (SR/ER) (the endoplasmic reticulum in muscle is named sarcoplasmic reticulum); this has engendered difficulty in reviews encompassing both muscle and nonmuscle cells. Here we will typically refer to the organelle as ER, except when specifically discussing muscle cells. The broad consideration of two major, still unexplained properties of skeletal muscle function, namely excitation contraction coupling and capacitative calcium entry are accounted for in a unitary hypothesis. This model allows a reinterpretation of existing data, and points to areas where new investigation may be fruitful. While primarily aimed at explaining Ca²⁺ flows in skeletal muscle, we consider findings of other systems to explore the implications of this hypothesis for other cell types.     

Functional comparisons between isoforms of the sarcoplasmic or endoplasmic reticulum family of calcium pumps.

ATP-dependent calcium pumps that reside in intracellular organelles are encoded by a family of structurally related enzymes, termed the sarcoplasmic or endoplasmic reticulum Ca(2+)-ATPases (SERCA), which each have a distinct pattern of tissue-specific and developmentally regulated expression. A COS-1 cell expression system was used to examine the biochemical properties of the isoforms: SERCA1 (fast-twitch skeletal muscle). SERCA2a (cardiac/slow-twitch skeletal muscle), SERCA2b (ubiquitous smooth- and non-muscle), and SERCA3 (non-muscle). Each isoform was expressed efficiently and appeared to be targeted to the endoplasmic reticulum. All isoforms displayed qualitatively similar enzymatic properties and were activated by calcium in a cooperative manner with a Hill coefficient of 2. The quantitative properties of SERCA1 and SERCA2a (the muscle isoforms) were identical in all respects. SERCA2b, however, appeared to have a lower turnover rate for both calcium transport and ATP hydrolysis. SERCA3 displayed a reduced apparent affinity for calcium, an increased apparent affinity for vanadate, and an altered pH dependence when compared with the other isoforms. These properties are consistent with an enzyme in which the equilibrium between the E1 and E2 conformations is shifted toward the E2 state.     

Calcium is required for folding of newly made subunits of the asialoglycoprotein receptor within the endoplasmic reticulum.

By resolving immunoprecipitates on nonreducing sodium dodecyl sulfate gels, we have detected several disulfide-bonded intermediates in folding within the endoplasmic reticulum of newly made H1 subunits of the asialoglycoprotein receptor. H1 in the endoplasmic reticulum (ER) can be partially unfolded by treatment of cells with dithiothreitol, but H1 in Golgi or post-Golgi organelles is resistant to such unfolding. This defines a late step in H1 folding that occurs just prior to exit from the ER. Depletion of calcium from the endoplasmic reticulum, either by treatment with A23187 or thapsigargin, has no effect on folding or secretion of newly made albumin, but totally blocks H1 maturation from the ER. No ER intermediates in H1 folding are formed in cells treated with A23187 or thapsigargin, indicating that at least an early step in H1 folding requires a high Ca2+ concentration in the ER lumen. As judged by cross-linking experiments, formation of H1 dimers and trimers occurs immediately after biosynthesis of the peptide chain, before monomer folding, and occurs normally in cells in which ER Ca2+ is reduced and where the monomer never folds properly. Calcium is essential for the asialoglycoprotein receptor to bind galactose, and our results suggest that Ca2+ is also essential for the receptor polypeptides to fold in the ER.     

Retrograde activation of store-operated calcium channel

Store-operated Ca2+ entry represents an important mechanism for refilling of a depleted intracellular-reticulum Ca2+ store following sustained activation of the IP3 receptor or ryanodine receptor RyR/Ca2+ release channel in the endoplasmic/sarcoplasmic reticulum (ER/SR). Recent studies have demonstrated the existence of store-operated Ca2+ channel (SOC) in muscle cells, whose activation process appears to be coupled to conformational changes of the RyR. Regulation of the plasma membrane (PM)-resided SOC by the SR-located RyR requires an integrity of the junctional membrane structure between SR and PM. Proteins that interact with RyR or influence the Ca2+ buffering capacity in the ER or SR lumen also participate in the activation process of SOC. Calsequestrin (CSQ) and calreticulin (CRT) are SR/ER-resident proteins, with highly negative charged regions at the carboxyl-terminal end that exhibit high buffering capacity for luminal Ca2+. CSQ and CRT not only modulate the intracellular Ca2+ release process but also might provide retrograde signals to regulate the function of SOC. The functional interplay between CSQ, RyR and SOC may serve essential roles of Ca2+ signaling in muscle contraction and development. A tight link between the expression of CRT and operation of SOC exist in certain cancer cells, where the reduced sensitivity to apoptosis may correlate with the altered function of SOC.     

The ins and outs of calreticulin: from the ER lumen to the extracellular space

Calreticulin was first isolated 26 years ago. Since its discovery as a minor Ca(2+)-binding protein of the sarcoplasmic reticulum, the appreciation of its importance has grown, and it is now recognized to be a multifunctional protein, most abundant in the endoplasmic reticulum (ER). The protein has well-recognized physiological roles in the ER as a molecular chaperone and Ca(2+)-signalling molecule. However, it has also been found in other membrane-bound organelles, at the cell surface and in the extracellular environment, where it has recently been shown to exert a number of physiological and pathological effects. Here, we will focus on these less-well-characterized functions of calreticulin.     

内质网应激诱导自噬的分子机制

在真核细胞中,内质网对蛋白质的折叠和运输至关重要,多种病理因素对内质网稳态的扰乱,可导致内质网腔中未折叠或错误折叠蛋白蓄积,即内质网应激(ERS)。细胞为此通过激活一种叫做未折叠蛋白反应(UPR)的防御反应来恢复内质网稳态。自噬是一种被描述为"自我吞食"的细胞代谢过程,其通过批量清除和降解未折叠蛋白以及破损细胞器在ERS时作为一种重要的保护机制。近年的研究显示这两个系统动态互联,且ERS可以通过多种方式诱导自噬的发生。在本文中,我们将总结目前关于ERS尤其是UPR诱导自噬的分子机制的相关知识,以进一步指导关于ERS与自噬关系的的研究。     

Transmembrane redox sensor of ryanodine receptor complex

Inositol 1,4,5-trisphosphate receptors (IP(3)R) and ryanodine receptors (RyR) mediate the release of endoplasmic and sarcoplasmic reticulum (ER/SR) Ca(2+) stores and regulate Ca(2+) entry through voltage-dependent or ligand-gated channels of the plasma membrane. A prominent property of ER/SR Ca(2+) channels is exquisite sensitivity to sulfhydryl-modifying reagents. A plausible role for sulfhydryl chemistry in physiologic regulation of Ca(2+) release channels and the fidelity of Ca(2+) release from ER/SR is lacking. This study reveals the existence of a transmembrane redox sensor within the RyR1 channel complex that confers tight regulation of channel activity in response to changes in transmembrane redox potential produced by cytoplasmic and luminal glutathione. A transporter selective for glutathione is co-localized with RyR1 within the SR membrane to maintain local redox potential gradients consistent with redox regulation of ER/SR Ca(2+) release. Hyperreactive sulfhydryls previously shown to reside within the RyR1 complex (Liu, G., and Pessah, I. N. (1994) J. Biol. Chem. 269, 33028-33034) are an essential biochemical component of a transmembrane redox sensor. Transmembrane redox sensing may represent a fundamental mechanism by which ER/SR Ca(2+) channels respond to localized changes in transmembrane glutathione redox potential produced by physiologic and pathophysiologic modulators of Ca(2+) release from stores.     

Effects of cyclic nucleotide dependent protein kinases on the endoplasmic reticulum Ca2+ pump of bovine pulmonary artery

This paper describes the stimulation by cyclic nucleotide dependent protein kinases on the Ca2+ uptake by isolated endoplasmic reticulum (ER) vesicles from the bovine main pulmonary artery. This ER fraction has previously been shown to be highly enriched in phospholamban, a protein kinase substrate that has been well characterized in cardiac sarcoplasmic reticulum (SR), where its phosphorylation is accompanied by an increased rate of Ca2+ uptake. As previously observed for the phosphorylation of phospholamban, the stimulation of the rate of Ca uptake was as high with cGMP dependent protein kinase as with cAMP dependent protein kinase. The effect of phosphorylation of the ER membranes from smooth muscle on the Ca2+ uptake was smaller than that seen in cardiac SR, and it was only observed if albumin was included during the isolation of the membranes. This relatively small effect is probably not due to a lower ratio of phospholamban to Ca2(+)-transport enzyme in the ER membranes as compared to cardiac SR. Several alternative explanations are discussed.