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PubMed2000

Calcium, a signaling molecule in the endoplasmic reticulum?

For many years now, it has been known that Ca2+ is an important signaling molecule in the cytosol of the cell, but emerging evidence suggests that Ca2+ might also play a signaling role in the endoplasmic reticulum. For example, agonist-induced fluctuations in free Ca2+ concentration in the endoplasmic reticulum can affect many functions of the endoplasmic reticulum, including protein synthesis and modification, and interchaperone interactions.

Amino Acid Sequence↗
PubMed2002

Non-depolarizing cardioplegia activates Ca2+-ATPase in sarcoplasmic reticulum after reperfusion.

OBJECTIVES: Depolarizing cardioplegia is the most common method for myocardial preservation in cardiac operations. However, depolarizing cardioplegia causes depolarization of the membrane potential by extracellular hyperkalemia, resulting in depletion of energy stores and calcium overload. This study examined the hypothesis that non-depolarizing cardioplegia would provide superior protection compared with depolarizing cardioplegia. METHODS: In an isolated rat heart Langendorff model, hearts were perfused for 10 min with St. Thomas' Hospital cardioplegic solution (Group I: n=20), St. Thomas' Hospital cardioplegic solution+Lidocaine 1 mM (Group II: n=20) or non-depolarizing cardioplegia (Group III: n=20). The hearts then were subjected to 60 min of normothermic global ischemia, after which they were perfused with Krebs-Henseleit buffer at 37 degrees C for 30 min. The percent recovery of functional data, myocardial cyclic AMP contents, and myocardial cyclic GMP contents were recorded at each time point (base, after the administration of cardioplegia, after global ischemia, and after 30 min of reperfusion). Ca(2+)-ATPase in sarcoplasmic reticulum was measured at pre-ischemia and 30 min of reperfusion. RESULTS: The percent recovery of developed pressure and +/-dp/dt were significantly higher in Group III than in other groups. Myocardial cyclic AMP and GMP contents were elevated after reperfusion in all groups. However, in Group III, myocardial cyclic AMP contents after 30 min of reperfusion were significantly higher than in other groups (Group III: 14.7+/-1.6 vs. Group I: 8.7+/-1.0, Group II: 8.3+/-0.2 pmol/mg dry weight, P=0.05) but not cGMP. The sarcoplasmic reticulum Ca(2+)-ATPase activities at 30 min of reperfusion significantly increased in Group III compared with Groups II and I (Group III: 70.3+/-3.6 vs. Group I: 46.8+/-3.4, Group II: 53.9+/-6.1 micromol Pi/mg per h, P=0.025 and P=0.030). CONCLUSIONS: Non-depolarizing cardioplegia induced the activity of Ca(2+)-ATPase in sarcoplasmic reticulum after reperfusion. The activity would be increased by the cyclic AMP pathway. These findings suggested that non-depolarizing cardioplegia prevented calcium overload after reperfusion, especially decreased cytosolic calcium during the diastolic phase.

Animals↗
PubMed1995

Identification of a 44 kDa protein localized within the endoplasmic reticulum of Trypanosoma brucei brucei.

Immunoaffinity chromatography and gel electrophoresis were used to isolate a 44 kDa protein that was bound to a 72 kDa chaperone in Trypanosoma brucei brucei. A polyclonal antiserum to the 44 kDa protein was raised in rats and employed in conjunction with chromatography using DEAE-cellulose, Sephacryl S-300, and hydroxyapatite to purify the protein from membranes of bloodstream forms of the trypanosomes. Immunoblot analysis using this antiserum revealed a protein doublet of 44/45 kDa in T. b. brucei and a single protein band of 53 kDa in almost equivalent amounts throughout the life-cycle stages of T. congolense. Indirect immunofluorescence using affinity-purified antibodies specific for the 44 kDa protein showed labelling of the perinuclear area and reticular system extending throughout the parasites, suggesting that this protein was located in the endoplasmic reticulum. Localization of the 44 kDa molecule in the endoplasmic reticulum was confirmed by immunoelectron microscopy. Protease protection experiments demonstrated that the epitopes bound by antibody were buried within the membrane or towards the lumenal face of the endoplasmic reticulum. Ruthenium Red overlay of nitrocellulose blots containing the 44/45 kDa doublet suggested that the molecules have the potential to bind calcium. The N-terminal amino acid sequence of the 44 kDa protein showed no sequence similarity to any proteins in the database.

Amino Acid Sequence↗
PubMed1991

Orientation of cytochromes P450 in the endoplasmic reticulum.

The orientation of eukaryotic cytochromes P450, with respect to the membrane of the endoplasmic reticulum, has been investigated. There is now good evidence that the tertiary structure of these proteins is essentially the same as that of the soluble bacterial isoenzyme cytochrome P450CI, with the exception of an extension at the N-terminus which is thought to form a membrane-anchoring sequence. The remainder of the molecule protrudes from the cytosolic face of the membrane so that it can interact with substrates and electron-donating proteins. Two models based on this structure have been considered, in which the plane of the heme of cytochrome P450 is oriented either parallel with or perpendicular to the plane of the membrane of the endoplasmic reticulum. The validity of these models has been assessed from the results of studies involving the binding of antipeptide antibodies directed toward known regions of cytochromes P450, modeling of the interaction of cytochrome P450 with cytochrome b5, proposed intramolecular movements of cytochrome P450 during its catalytic cycle, and the partitioning of substrates for cytochrome P450 between the cytosol and membrane. It is concluded that cytochrome P450 is most likely oriented such that the heme is not fixed horizontal to the plane of the membrane of the endoplasmic reticulum and may well lie with the heme perpendicular to the membrane.

Amino Acid Sequence↗
PubMed1984

Cross-linking agents induce rapid calcium release from skeletal muscle sarcoplasmic reticulum.

The passive permeability of skeletal muscle sarcoplasmic reticulum vesicles to Ca2+ ions is drastically increased upon addition of the oxidizing agent cupric phenanthroline. The permeability change, which occurs very rapidly, is partially reversed by reducing agents and cannot be explained by a direct effect of cupric phenanthroline on the lipid moiety of the membranes. The rapid efflux phenomenon is due to protein cross-linking induced by the cupric phenanthroline catalyzed oxidation of SH groups to disulfide bridges. Similar effects are also induced by cross-linking sarcoplasmic reticulum proteins with dithiodipropionic acid disuccinimido ester. The rapid Ca2+ efflux is inhibited by micromolar concentrations of lanthanum and by labeling the Ca2+-ATPase with dicyclohexylcarbodiimide. These observations suggest that Ca2+ channels are formed by chemical modification of the ATPase. The Ca2+ permeability rate of sarcoplasmic reticulum obtained after cross-linking is compatible with the requirements of Ca2+ release in vivo. The possibility that Ca2+-ATPase oligomers might mediate the release process is discussed.

Animals↗
PubMed1987

Phospholipid fatty acyl chain asymmetry in the membrane bilayer of isolated skeletal muscle sarcoplasmic reticulum.

We previously showed [Herbette, L. G., Blasie, J. K., DeFoor, P., Fleischer, S., Bick, R. J., Van Winkle, W. B., Tate, C. A., & Entman, M. L. (1984) Arch. Biochem. Biophys. 234, 235-242; Herbette, L. G., DeFoor, P., Fleischer, S., Pascolini, D., Scarpa, A., & Blasie, J. K. (1985) Biochim. Biophys. Acta 817, 103-122] that the phospholipid head-group distribution in the membrane bilayer of isolated sarcoplasmic reticulum is asymmetric. From these studies, both the total number of phospholipid head groups and the total lipid, as well as the head-group species for these lipids, were found to be different for each monolayer of the membrane bilayer. In this paper, we demonstrate for the first time that there is significant asymmetry in the distribution of unsaturated fatty acids between the two monolayers; i.e., the outer monolayer of the sarcoplasmic reticulum contained more unsaturated and polyunsaturated chains when compared to the inner monolayer. X-ray diffraction measurements demonstrated that the time-averaged fatty acyl chain extension for the outer monolayer was approximately 20% less than for the inner monolayer. This is consistent with the concept that the greater degree of unsaturation in the outer monolayer may provide for a decreased average fatty acyl chain extension for that layer. This architecture for the bilayer may be related to both the "resting" state mass distribution of the calcium pump protein within the membrane bilayer and possible "conformational" states of the calcium pump protein during calcium transport by the sarcoplasmic reticulum.

Animals↗
PubMed1980

Characterization of calmodulin effects on calcium transport in cardiac microsomes enriched in sarcoplasmic reticulum.

Calmodulin prepared from red cell hemolysates was found to significantly increase Ca2+ uptake into cardiac microsomal preparations enriched in sarcoplasic reticulum in a dose-dependent manner. The stimulation of calcium uptake by calmodulin was additive to that stimulation produced by maximal stimulatory concentrations of adenosine cyclic 3',5'-phosphate (cAMP) dependent protein kinase and cAMP, indicating separate mechanisms of action and potentially different modulatory roles for these two systems in the control of calcium transport. K+ significantly decreased calmodulin stimulation of calcium uptake, while in the absence of calmodulin, K+ increased Ca2+ uptake. In the absence of K+, calmodulin increased Ca2+ uptake to levels observed at maximal K+ concentrations without calmodulin present. Na+ produced effects similar to those of K+ in this preparation both in the presence and absence of calmodulin. The effect of calmodulin on the intermediate steps of the (Mg2+,Ca2+)ATPase in cardiac sarcoplasmic reticulum was also investigated. Calmodulin was found to reduce the steady-state level of the Ca2+-dependent phosphoprotein (ECaP) and increase the (Mg2+,Ca2+)ATPase activity of this preparation. Dephosphorylation of ECaP in the presence of Tris-ATP (0.5 mM) was significantly stimulated by calmodulin. These studies indicate that calmodulin stimulates Ca2+ transport in cardiac sarcoplasmic reticulum by increasing the turnover rate of the transport process.

Animals↗
PubMed2001

Localization of glucose-6-phosphate dehydrogenase activity on ribosomes of granular endoplasmic reticulum, in peroxisomes and peripheral cytoplasm of rat liver parenchymal cells.

Glucose-6-phosphate dehydrogenase activity has been localized ultrastructurally in fixed tissues. Activity was found in particular in association with ribosomes of granular endoplasmatic reticulum. Biochemical studies indicated that glucose-6-phosphate dehydrogenase activity is also present in the cytoplasm and in peroxisomes. Fixation may be held responsible for selective inactivation of part of glucose-6-phosphate dehydrogenase activity. In the present study, we applied the ferricyanide method for the demonstration of glucose-6-phosphate dehydrogenase activity in unfixed cryostat sections of rat liver in combination with the semipermeable membrane technique and in isolated rat liver parenchymal cells. Isolated liver parenchymal cells were permeabilized with 0.025% glutaraldehyde after NADP+ protection of the active site of glucose-6-phosphate dehydrogenase. This treatment resulted in only slight inactivation of glucose-6-phosphate dehydrogenase activity. The composition of the incubation medium was optimized on the basis of rapid light microscopical analysis of the formation of reddish-brown final reaction product in sections. With the optimized method, electron dense reaction product was observed in cryostat sections on granular endoplasmic reticulum, in mitochondria and at the cell border. However, the ultrastructural morphology was rather poor. In contrast, the morphology of incubated isolated cells was preserved much better. Electron dense precipitate was found on ribosomes of the granular endoplasmic reticulum, in peroxisomes and the cytoplasm, particularly at the periphery of cells. In conclusion, our ultrastructural study clearly demonstrates that it is essential to use mildly-fixed cells to allow detection of glucose-6-phosphate dehydrogenase activity in all cellular compartments where activity is present.

Animals↗
PubMed2002

Ascorbyl free radical and dehydroascorbate formation in rat liver endoplasmic reticulum.

The mechanism of ascorbate oxidation was studied in rat liver microsomes. A continuous consumption of the added ascorbate was observed, which was accompanied with a prompt appearance of ascorbyl free radical and dehydroascorbate. Microsomes sustained steady-state level of ascorbyl free radical and dehydroascorbate till ascorbate was present in the medium. Ascorbyl free radical formation was diminished when microsomes had been pretreated with heat or trypsine. It was also decreased by addition of quercetin, econazole or metal chelators, including the copper specific neocuproine. Enzymatic (superoxide dismutase, catalase) and nonenzymatic (dimethyl sulfoxide, mannitol) antioxidants did not modify the microsomal production of ascorbyl free radical. Investigation of the subcellular distribution of ascorbate oxidation showed that the microsomal fraction of liver had the highest activity. The decrease of ascorbate oxidation after protease treatment and the negligible increase upon permeabilization of microsomal vesicles showed that a membrane protein is responsible for the activity, which is exposed to the outer surface of the endoplasmic reticulum. The results indicate the presence of a primary enzymatic ascorbate oxidation in rat liver endoplasmic reticulum which is able to generate dehydroascorbate, an important source of the oxidizing environment in the endoplasmic reticulum.

Animals↗
PubMed2002

Function of Ca-pump in sarcoplasmic reticulum of rat myocardium during adaptation to electromagnetic field.

We studied the effect of adaptation to electromagnetic field on functional state of Ca-pump in sarcoplasmic reticulum, intensity of lipid peroxidation, and energy metabolism in rat myocardium. Membrane viscosity was assessed in crude preparation of myocardial sarcoplasmic reticulum. Adaptation to electromagnetic field did not change the rate of endogenous respiration, including ouabain-sensitive respiration, but potentiated coupling between catalytic activity and Ca-transporting capacity of Ca-pump. It is hypothesized that modification of structural and functional properties of the membrane due to inhibition of free radical processes increases functional activity of Ca-pump in sarcoplasmic reticulum.

Adaptation, Physiological↗
PubMed2001

The mammalian guanine nucleotide exchange factor mSec12 is essential for activation of the Sar1 GTPase directing endoplasmic reticulum export.

The Sar1 GTPase is an essential component of COPII vesicle coats involved in export of cargo from the endoplasmic reticulum of mammalian cells. To begin to elucidate its mechanism of action, we now report the identity of the mammalian homolog to the yeast Sec12 guanine nucleotide exchange factor (18% identity) that promotes Sar1 activation. Mammalian Sec12 (mSec12) is a type II transmembrane protein with a large cytosolic domain, a fragment of which has previously been reported as the transcription factor prolactin regulatory element binding protein (PREB). mSec12 promotes efficient guanine nucleotide exchange on Sar1, but not Arf1 or Rab GTPases. mSec12 is localized to the endoplasmic reticulum and an antibody to the cytosolic domain of mSec12 potently inhibits Sar1 recruitment and the formation of COPII vesicles in vitro. The dominant negative GDP-restricted mutant Sar1[T39N] is shown to be a potent inhibitor of mSec12 activity, consistent with its role in preventing COPII vesicle formation in vitro and during transient expression in vivo. We propose that mSec12 is an evolutionarily distant guanine nucleotide exchange factor directing Sar1 GTPase activation in mammalian cells. Its divergence from yeast Sec12p may reflect the specialized needs of the mammalian endoplasmic reticulum involving the formation of Sar1-dependent transitional elements (Aridor M, et al. J Cell Biol 2001;152:213-229) and selection of cargo into prebudding complexes.

ADP-Ribosylation Factor 1↗
PubMed2003

Effects of HIV-1 Nef on retrograde transport from the plasma membrane to the endoplasmic reticulum.

HIV-1 Nef protein down-regulates several important immunoreceptors through interactions with components of the intracellular sorting machinery. Nef expression is also known to induce modifications of the endocytic pathway. Here, we analyzed the effects of Nef on retrograde transport, from the plasma membrane to the endoplasmic reticulum using Shiga toxin B-subunit (STxB). Nef expression inhibited access of STxB to the endoplasmic reticulum, but did not modify the surface expression level of STxB receptor, Gb3, nor its internalization rate as measured with a newly developed assay. Mutation of the myristoylation site or of a di-leucine motif of Nef involved in the interaction with the clathrin adaptor complexes AP1 and AP2 abolished the inhibition of retrograde transport. In contrast, mutations of Nef motifs known to interact with PACS-1, beta COP or a subunit of the v-ATPase did not modify the inhibitory activity of Nef on retrograde transport. Ultrastructural analysis revealed that Nef was present in clusters located on endosomal or Golgi membranes together with internalized STxB. Furthermore, in strongly Nef-expressing cells, STxB accumulated in endosomal structures that labeled with AP1. Our observations show that Nef perturbs retrograde transport between the early endosome and the endoplasmic reticulum. The potential transport steps targeted by Nef are discussed.

Cell Membrane↗
PubMed1989

Three-dimensional architecture of the calcium channel/foot structure of sarcoplasmic reticulum.

The calcium channel responsible for the release of Ca2+ from the sarcoplasmic reticulum of skeletal muscle during excitation-contraction coupling has recently been identified and purified. The isolated calcium channel has been identified morphologically with the 'foot' structures which are associated with the junctional face membrane of the terminal cisternae of sarcoplasmic reticulum. In situ, the foot structure extends across the gap of the triad junction from the terminal cisternae of the reticulum to the transverse tubule. We describe here the three-dimensional architecture (3.7 nm resolution) of the calcium channel/foot structure from fast-twitch rabbit skeletal muscle, which we determined from electron micrographs of isolated, non-crystalline structures that had been tilted in the electron microscope. The reconstruction reveals two different faces and an internal structure in which stain accumulates at several interconnected locations, which could empty into the junctional gap of the triad junction. The detailed architecture of the channel complex is relevant to understanding both the physical path followed by calcium ions during excitation-contraction coupling and the association of the terminal cisternae and the transverse tubules in the triad junction.

Animals↗
PubMed1998

Structure of the calcium pump from sarcoplasmic reticulum at 8-A resolution.

The calcium pump from sarcoplasmic reticulum (Ca2+-ATPase) is typical of the large family of P-type cation pumps. These couple ATP hydrolysis with cation transport, generating cation gradients across membranes. Ca2+-ATPase specifically maintains the low cytoplasmic calcium concentration of resting muscle by pumping calcium into the sarcoplasmic reticulum; subsequent release is used to initiate contraction. No high-resolution structure of a P-type pump has yet been determined, although a 14-A structure of Ca2+-ATPase, obtained by electron microscopy of frozen-hydrated, tubular crystals, showed a large cytoplasmic head connected to the transmembrane domain by a narrow stalk. We have now improved the resolution to 8A and can discern ten transmembrane alpha-helices, four of which continue into the stalk On the basis of constraints from transmembrane topology, site-directed mutagenesis and disulphide crosslinking, we have made tentative assignments for these alpha-helices within the amino-acid sequence. A distinct cavity leads to the putative calcium-binding site, providing a plausible path for calcium release to the lumen of the sarcoplasmic reticulum.

Amino Acid Sequence↗
PubMed1989

Nature and site of phospholamban regulation of the Ca2+ pump of sarcoplasmic reticulum.

The rapid removal of Ca2+ ions from the cytosol, necessary for the efficient relaxation of cardiac muscle cells, is performed by the Ca2+-pumping ATPase of the sarcoplasmic reticulum. The calcium pump is activated by cyclic AMP- and calmodulin-dependent phosphorylation of phospholamban, an integral membrane protein of the sarcoplasmic reticulum. Using a heterobifunctional crosslinking agent which can be cleaved and photoactivated, we provide evidence for a direct interaction between the two proteins. Only the non-phosphorylated form of phospholamban interacts with the ATPase, demonstrating that phospholamban is an endogenous inhibitor that is removed from the ATPase by phosphorylation. Non-phosphorylated phospholamban interacts only with the calcium-free conformation of the ATPase and is released when it is converted to the calcium-bound state. We localized the site of interaction to a single peptide isolated after cyanogen bromide cleavage of the ATPase. The peptide derives from a domain just C-terminal to the aspartyl phosphate of the active site. This domain is unique to ATPases of the sarcoplasmic reticulum in that it has no homology with any other phosphorylation-type ion pump. The domain occurs in both slow- and fast-twitch isoforms of the ATPase, even though phospholamban is not expressed in fast-twitch muscles.

Adenosine Triphosphatases↗
PubMed1990

Voltage-dependent InsP3-insensitive calcium channels in membranes of pancreatic endoplasmic reticulum vesicles.

Stimulus-secretion coupling in exocrine glands involves Ca2+ release from intracellular stores. In endoplasmic reticulum vesicle preparations from rat exocrine pancreas, an inositol 1,4,5-trisphosphate(InsP3)-sensitive, as well as an InsP3-insensitive, Ca2+ pool has been characterized. But Ca2+ channels in the endoplasmic reticulum of rat exocrine pancreas have not been demonstrated at the level of single-channel current. We have now used the patch-clamp technique on endoplasmic reticulum vesicles fused by means of the dehydration-rehydration method. In excised patches, single Ba2(+)- and Ca2(+)-selective channels were recorded. The channel activity was markedly voltage-dependent. Caffeine increased channel open-state probability, whereas ruthenium red and Cd2+ blocked single-channel currents. Ryanodine, nifedipine and heparin had no effect on channel activity. The channel activity was not dependent on the free Ca2+ concentration, the presence of InsP3, or pH. We conclude that this calcium channel mediates Ca2+ release from an intracellular store through an InsP3-insensitive mechanism.

Animals↗
PubMed1991

Characterization of the rough endoplasmic reticulum ribosome-binding activity.

The rough endoplasmic reticulum membranes of mammalian cells contain specific ribosome-binding sites. A purification to apparent homogeneity of a negatively charged protein (ERp180) of relative molecular mass 180,000 (180 K) was reported which was proposed to function as a rough endoplasmic reticulum ribosome receptor. We report here that ribosome-binding site activity quantitatively solubilized from rough endoplasmic reticulum membranes does not cofractionate with ERp180. By contrast, ribosome-binding site activity fractionates as a much smaller, positively charged protein.

Animals↗
PubMed2006

A complex of Yos9p and the HRD ligase integrates endoplasmic reticulum quality control into the degradation machinery.

A quality-control system surveys the lumen of the endoplasmic reticulum for terminally misfolded proteins. Polypeptides singled out by this system are ultimately degraded by the cytosolic ubiquitin-proteasome pathway. Key components of both the endoplasmic reticulum quality-control system and the degradation machinery have been identified, but a connection between the two systems has remained elusive. Here, we report an association between the endoplasmic reticulum quality-control lectin Yos9p and Hrd3p, a component of the ubiquitin-proteasome system that links these pathways. We identify designated regions in the luminal domain of Hrd3p that interact with Yos9p and the ubiquitin ligase Hrd1p. Binding of misfolded proteins occurs through Hrd3p, suggesting that Hrd3p recognises proteins that deviate from their native conformation, whereas Yos9p ensures that only terminally misfolded polypeptides are degraded.

Blotting, Western↗