Targeting and retention of type 1 ryanodine receptors to the endoplasmic reticulum

Most ryanodine receptors and their relatives, inositol 1,4,5-trisphosphate receptors, are expressed in the sarcoplasmic or endoplasmic reticulum (ER), where they mediate Ca(2+) release. We expressed fragments of ryanodine receptor type 1 (RyR1) in COS cells alone or fused to intercellular adhesion molecule-1 (ICAM-1), each tagged with yellow fluorescent protein, and used confocal imaging and glycoprotein analysis to identify the determinants of ER targeting and retention. Single transmembrane domains (TMD) of RyR1 taken from the first (TMD1-TMD2) or last (TMD5-TMD6) pair were expressed in the ER membrane. TMD3-TMD4 was expressed in the outer mitochondrial membrane. The TMD outer pairs (TMD1-TMD2 and TMD5-TMD6) retained ICAM-1, a plasma membrane-targeted protein, within the ER membrane. TMD1 alone provided a strong ER retention signal and TMD6 a weaker signal, but the other single TMD were unable to retain ICAM-1 in the ER. We conclude that TMD1 provides the first and sufficient signal for ER targeting of RyR1. The TMD outer pairs include redundant ER retention signals, with TMD1 providing the strongest signal.     

The role of cholesterol in the association of endoplasmic reticulum membranes with mitochondria

The unique endoplasmic reticulum (ER) subdomain termed the mitochondria-associated ER membrane (MAM) engages the physical connection between the ER and the mitochondrial outer membrane and plays a role in regulating IP(3) receptor-mediated Ca(2+) influx and the phospholipid transport between the two organelles. The MAM contains certain signaling and membrane-tethering proteins but also lipids including cholesterol. The biophysical role of lipids at the MAM, specifically in the physical interaction between the MAM of the ER and mitochondria, remains not totally clarified. Here we employed the in vitro membrane association assay to investigate the role of cholesterol in the association between MAMs and mitochondria. The purified MAMs and mitochondria were mixed in vitro in a test tube and then the physical association of the two subcellular organelles was quantified indirectly by measuring the presence of the MAM-specific protein sigma-1 receptors in the mitochondria fraction. Purified MAMs contained free cholesterol approximately 7 times higher than that in microsomes. We found that depletion of cholesterol in MAMs with methyl-β-cyclodextrin (MβC) significantly increases the association between MAMs and mitochondria, whereas MβC saturated with cholesterol does not change the association. (14)C-Serine pulse-labeling demonstrated that the treatment of living cells with MβC decreases the level of de novo synthesized (14)C-phosphatidylserine (PtSer) and concomitantly increases greatly the synthesis of (14)C-phosphatidylethanolamine (PtEt). Apparently, cholesterol depletion increased the PtSer transport from MAMs to mitochondria. Our findings suggest that cholesterol is an important substrate in regulating the association between MAMs of the ER and mitochondria.     

Calreticulin differentially modulates calcium uptake and release in the endoplasmic reticulum and mitochondria

To study the role of calreticulin in Ca(2+) homeostasis and apoptosis, we generated cells inducible for full-length or truncated calreticulin and measured Ca(2+) signals within the cytosol, the endoplasmic reticulum (ER), and mitochondria with "cameleon" indicators. Induction of calreticulin increased the free Ca(2+) concentration within the ER lumen, [Ca(2+)](ER), from 306 +/- 31 to 595 +/- 53 microm, and doubled the rate of ER refilling. [Ca(2+)](ER) remained elevated in the presence of thapsigargin, an inhibitor of SERCA-type Ca(2+) ATPases. Under these conditions, store-operated Ca(2+) influx appeared inhibited but could be reactivated by decreasing [Ca(2+)](ER) with the low affinity Ca(2+) chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine. In contrast, [Ca(2+)](ER) decreased much faster during stimulation with carbachol. The larger ER release was associated with a larger cytosolic Ca(2+) response and, surprisingly, with a shorter mitochondrial Ca(2+) response. The reduced mitochondrial signal was not associated with visible morphological alterations of mitochondria or with disruption of the contacts between mitochondria and the ER but correlated with a reduced mitochondrial membrane potential. Altered ER and mitochondrial Ca(2+) responses were also observed in cells expressing an N-truncated calreticulin but not in cells overexpressing calnexin, a P-domain containing chaperone, indicating that the effects were mediated by the unique C-domain of calreticulin. In conclusion, calreticulin overexpression increases Ca(2+) fluxes across the ER but decreases mitochondrial Ca(2+) and membrane potential. The increased Ca(2+) turnover between the two organelles might damage mitochondria, accounting for the increased susceptibility of cells expressing high levels of calreticulin to apoptotic stimuli.     

Stable interactions between mitochondria and endoplasmic reticulum allow rapid accumulation of calcium in a subpopulation of mitochondria

To better understand the functional role of the mitochondrial network in shaping the Ca2+ signals in living cells, we took advantage both of the newest genetically engineered green fluorescent protein-based Ca2+ sensors ("Cameleons," "Camgaroos," and "Pericams") and of the classical Ca(2+)-sensitive photoprotein aequorin, all targeted to the mitochondrial matrix. The properties of the green fluorescent protein-based probes in terms of subcellular localization, photosensitivity, and Ca2+ affinity have been analyzed in detail. It is concluded that the ratiometric pericam is, at present, the most reliable mitochondrial Ca2+ probe for single cell studies, although this probe too is not devoid of problems. The results obtained with ratiometric pericam in single cells, combined with those obtained at the population level with aequorin, provide strong evidence demonstrating that the close vicinity of mitochondria to the Ca2+ release channels (and thus responsible for the fast uptake of Ca2+ by mitochondria upon receptor activation) are highly stable in time, suggesting the existence of specific interactions between mitochondria and the endoplasmic reticulum.     

Bile acids induce Ca2+ release from both the endoplasmic reticulum and acidic intracellular calcium stores through activation of inositol trisphosphate receptors and ryanodine receptors

Gallstones can cause acute pancreatitis, an often fatal disease in which the pancreas digests itself. This is probably because of biliary reflux into the pancreatic duct and subsequent bile acid action on the acinar cells. Because Ca(2+) toxicity is important for the cellular damage in pancreatitis, we have studied the mechanisms by which the bile acid taurolithocholic acid 3-sulfate (TLC-S) liberates Ca(2+). Using two-photon plasma membrane permeabilization and measurement of [Ca(2+)] inside intracellular stores at the cell base (dominated by ER) and near the apex (dominated by secretory granules), we have characterized the Ca(2+) release pathways. Inhibition of inositol trisphosphate receptors (IP(3)Rs), by caffeine and 2-APB, reduced Ca(2+) release from both the ER and an acidic pool in the granular area. Inhibition of ryanodine receptors (RyRs) by ruthenium red (RR) also reduced TLC-S induced liberation from both stores. Combined inhibition of IP(3)Rs and RyRs abolished Ca(2+) release. RyR activation depends on receptors for nicotinic acid adenine dinucleotide phosphate (NAADP), because inactivation by a high NAADP concentration inhibited release from both stores, whereas a cyclic ADPR-ribose antagonist had no effect. Bile acid-elicited intracellular Ca(2+) liberation from both the ER and the apical acidic stores depends on both RyRs and IP(3)Rs.     

Calcium wave propagation in pancreatic acinar cells: functional interaction of inositol 1,4,5-trisphosphate receptors, ryanodine receptors, and mitochondria

In pancreatic acinar cells, inositol 1,4,5-trisphosphate (InsP(3))-dependent cytosolic calcium ([Ca(2+)](i)) increases resulting from agonist stimulation are initiated in an apical "trigger zone," where the vast majority of InsP(3) receptors (InsP(3)R) are localized. At threshold stimulation, [Ca(2+)](i) signals are confined to this region, whereas at concentrations of agonists that optimally evoke secretion, a global Ca(2+) wave results. Simple diffusion of Ca(2+) from the trigger zone is unlikely to account for a global [Ca(2+)](i) elevation. Furthermore, mitochondrial import has been reported to limit Ca(2+) diffusion from the trigger zone. As such, there is no consensus as to how local [Ca(2+)](i) signals become global responses. This study therefore investigated the mechanism responsible for these events. Agonist-evoked [Ca(2+)](i) oscillations were converted to sustained [Ca(2+)](i) increases after inhibition of mitochondrial Ca(2+) import. These [Ca(2+)](i) increases were dependent on Ca(2+) release from the endoplasmic reticulum and were blocked by 100 microM ryanodine. Similarly, "uncaging" of physiological [Ca(2+)](i) levels in whole-cell patch-clamped cells resulted in rapid activation of a Ca(2+)-activated current, the recovery of which was prolonged by inhibition of mitochondrial import. This effect was also abolished by ryanodine receptor (RyR) blockade. Photolysis of d-myo InsP(3) P(4(5))-1-(2-nitrophenyl)-ethyl ester (caged InsP(3)) produced either apically localized or global [Ca(2+)](i) increases in a dose-dependent manner, as visualized by digital imaging. Mitochondrial inhibition permitted apically localized increases to propagate throughout the cell as a wave, but this propagation was inhibited by ryanodine and was not seen for minimal control responses resembling [Ca(2+)](i) puffs. Global [Ca(2+)](i) rises initiated by InsP(3) were also reduced by ryanodine, limiting the increase to a region slightly larger than the trigger zone. These data suggest that, while Ca(2+) release is initially triggered through InsP(3)R, release by RyRs is the dominant mechanism for propagating global waves. In addition, mitochondrial Ca(2+) import controls the spread of Ca(2+) throughout acinar cells by modulating RyR activation.     

Calcium homeostasis in the thymocyte apoptosis II. Accumulation of calcium in mitochondria and endoplasmic reticulum

The activity of energy-dependent Ca2+-accumulation systems in rat thymocytes mitochondria and endoplasmic reticulum (ER) in control and at the early stage of X-irradiation or H2O2-induced apoptosis were determined in experiments using the model of digitonin-permeabilized cells with addition of thapsigargin and ruthenium red. The mitochondrial Ca2+-transporting system proved to be more sensitive to both apoptotic stimuli. The stationary level of Ca2+, accumulated in mitochondria and initial rate of Ca2+ accumulation in ER were reduced 15 min after H2O2 treatment. The parameters of mitochondrial Ca2+-accumulation system in irradiated cells were decreased 30 min after irradiation. Cyclosporin A almost completely inhibited DNA fragmentation in irradiated and partly--in peroxide-treated cells. The mitochondrial calcium homeostasis imbalance is suggested to be an early event in thymocytes apoptosis initiation.     

Deubiquitination, a new player in Golgi to endoplasmic reticulum retrograde transport

Modification by ubiquitin plays a major role in a broad array of cellular functions. Although reversal of this process, deubiquitination, likely represents an important regulatory step contributing to cellular homeostasis, functions of deubiquitination enzymes still remain poorly characterized. We have previously shown that the ubiquitin protease Ubp3p requires a co-factor, Bre5p, to specifically deubiquitinate the coat protein complex II (COPII) subunit Sec23p, which is involved in anterograde transport between endoplasmic reticulum and Golgi compartiments. In the present report, we show that disruption of BRE5 gene also led to a defect in the retrograde transport from the Golgi to the endoplasmic reticulum. Further analysis indicate that the COPI subunit beta'-COP represents another substrate of the Ubp3p.Bre5p complex. All together, our results indicate that the Ubp3p.Bre5p deubiquitination complex co-regulates anterograde and retrograde transports between endoplasmic reticulum and Golgi compartments.     

Diospyrin derivative, an anticancer quinonoid, regulates apoptosis at endoplasmic reticulum as well as mitochondria by modulating cytosolic calcium in human breast carcinoma cells

Diospyrin diethylether (D7), a bisnaphthoquinonoid derivative, exhibited an oxidative stress-dependent apoptosis in several human cancer cells and tumor models. The present study was aimed at evaluation of the increase in cytosolic calcium [Ca(2+)](c) leading to the apoptotic cell death triggered by D7 in MCF7 human breast carcinoma cells. A phosphotidylcholine-specific phospholipase C (PC-PLC) inhibitor, viz. U73122, and an antioxidant, viz. N-acetylcysteine, could significantly prevent the D7-induced rise in [Ca(2+)](c) and PC-PLC activity. Using an endoplasmic reticulum (ER)-Ca(2+) mobilizer (thapsigargin) and an ER-IP3R antagonist (heparin), results revealed ER as a major source of [Ca(2+)](c) which led to the activation of calpain and caspase12, and cleavage of fodrin. These effects including apoptosis were significantly inhibited by the pretreatment of Bapta-AM (a cell permeable Ca(2+)-specific chelator), or calpeptin (a calpain inhibitor). Furthermore, D7-induced [Ca(2+)](c) was found to alter mitochondrial membrane potential and induce cytochrome c release, which was inhibited by either Bapta-AM or ruthenium red (an inhibitor of mitochondrial Ca(2+) uniporter). Thus, these results provided a deeper insight into the D7-induced redox signaling which eventually integrated the calcium-dependent calpain/caspase12 activation and mitochondrial alterations to accentuate the induction of apoptotic cell death.     

Mutations in a tRNA import signal define distinct receptors at the two membranes of Leishmania mitochondria

Nucleus-encoded tRNAs are selectively imported into the mitochondrion of Leishmania, a kinetoplastid protozoan. An oligoribonucleotide constituting the D stem-loop import signal of tRNA(Tyr)(GUA) was efficiently transported into the mitochondrial matrix in organello as well as in vivo. Transfer through the inner membrane could be uncoupled from that through the outer membrane and was resistant to antibody against the outer membrane receptor TAB. A number of mutations in the import signal had differential effects on outer and inner membrane transfer. Some mutants which efficiently traversed the outer membrane were unable to enter the matrix. Conversely, restoration of the loop-closing GC pair in reverse resulted in reversion of transfer through the inner, but not the outer, membrane, and binding of the RNA to the inner membrane was restored. These experiments indicate the presence at the two membranes of receptors with distinct specificities which mediate stepwise transfer into the mitochondrial matrix. The combination of oligonucleotide mutagenesis and biochemical fractionation may provide a general tool for the identification of tRNA transport factors.     

Mechanism of the calcium pump in the endoplasmic reticulum of liver: phosphoproteins as reaction intermediates

Microsomal fractions, highly enriched with endoplasmic reticulum of rat and human liver exhibit Ca2+ uptake catalyzed by a Ca2+-pumping ATPase. The mechanism of Ca2+-translocation involves: (i) reversible Ca2+-dependent formation of an acyl-phosphoenzyme intermediate (Mr 116,000 to 118,000) with bound Ca2+, which in the reversed reaction can transphosphorylate its Pi to ADP to re-synthesize ATP; (ii) reversible transition of the ADP-reactive phosphoenzyme into an isomer without bound Ca2+, not further reactive to ADP; (iii) hydrolytic cleavage, stimulated by Mg2+, K+, and ATP of the ADP-unreactive phosphoenzyme with liberation of Pi. By analogy to a mechanism proposed for the Ca2+ pump of sarcoplasmic reticulum, the translocation of Ca2+ to and dissociation from the inner side of the membrane is suggested to occur by a conformational change, coupled with a decrease in Ca2+-affinity of the phosphoenzyme during its transition into the ADP-unreactive isomer. With CaATP as the effective substrate the reactions proceed normally but at a considerably slower rate.     

Lipids and proteolipids in membranes of the smooth endoplasmic reticulum of rat enterocytes. Vitamin D-dependent calcium binding

The lipid and proteolipid composition of endoplasmic reticulum smooth membranes from rat enterocytes in normalcy and under D-hypovitaminosis is studied to show the Ca2+-binding with these components. These membranes in vitamin D-deficiency are established to differ in the cholesterol content. Proteolipids 1 and 2 are isolated from the lipid fraction of membranes. Their amino acid composition is determined. The vitamin D-dependent binding of Ca by the proteolipid fraction is shown.     

The endoplasmic reticulum network in the ascidian egg: localization and calcium content

In contrast with most other eggs, where the endoplasmic reticulum is mixed with many other organelles, in ascidians, continuous sheets and tubes of endoplasmic reticulum constitute the only prominent organelle in the immediate layer (0.5-1μm) beneath the plasma membrane, and occupies 16–20% of the cortical volume. We took advantage of this unusual stratification of the organelles in the ascidian egg, to carry X-ray microanalysis. Our measurements provide the first estimate of the calcium content of the endoplasmic reticulum network in an egg, and show it is the main calcium store.     

The effects of inducers of the endoplasmic reticulum, peroxisomes and mitochondria on the amounts and synthesis of ubiquinone in rat liver subcellular membranes

Rats were treated with inducers of peroxisomes, mitochondria and the endoplasmic reticulum, as well as receiving diets and drug known to influence the mevalonate pathway. Treatment with clofibrate and 2-diethylhexylphthalate (DEHP) increased microsomal and mitochondrial ubiquinone contents, but a decrease was observed in lysosomes. In vivo labeling of this lipid with [3H]mevalonate was also elevated. The amount of cholesterol did not change upon exposure to these inducers of peroxisomes and mitochondria, but its rate of labeling was decreased. The concentration of dolichol increased only after treatment with DEHP and only in lysosomes. The inducers of the endoplasmic reticulum phenobarbital, 3-methylcholanthrene and N-nitrosodiethylamine enhanced the rate of ubiquinone synthesis and exposure to the latter two substances also elevated the amount of this lipid in microsomes. A cholesterol-rich diet increased the labeling of ubiquinone and decreased cholesterol labeling, while cholestyramine treatment had opposite effects on lipid labeling in both microsomes and mitochondria. The results demonstrate that the ubiquinone contents of the various membranes of hepatocytes change in a characteristic manner under the influence of inducers and dietary factors. Clearly, the level of ubiquinone and its biosynthesis are regulated separately from those of the other products of the mevalonate pathway, cholesterol and dolichol.