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PubMed1983

Inhibition of sarcoplasmic reticulum calcium pump by cytosolic protein(s) endogenous to heart and slow skeletal muscle but not fast skeletal muscle.

Cytosol from rabbit heart and slow and fast skeletal muscles was fractionated using (NH4)2SO4 to yield three cytosolic protein fractions, viz., CPF-I (protein precipitated at 30% saturation), CPF-II (protein precipitated between 30 and 60% saturation), and cytosol supernatant (protein soluble at 60% saturation). The protein fractions were dialysed and tested for their effects on ATP-dependent, oxalate-supported Ca2+ uptake by sarcoplasmic reticulum from heart and slow and fast skeletal muscles. CPF-I from heart and slow muscle, but not from fast muscle, caused marked inhibition (up to 95%) of Ca2+ uptake by sarcoplasmic reticulum from heart and from slow and fast muscles. Neither unfractionated cytosol nor CPF-II or cytosol supernatant from any of the muscles altered the Ca2+ uptake activity of sarcoplasmic reticulum. Studies on the characteristics of inhibition of sarcoplasmic reticulum Ca2+ uptake by CPF-I (from heart and slow muscle) revealed the following: (a) Inhibition was concentration- and temperature-dependent (50% inhibition with approx. 80 to 100 micrograms CPF-I; seen only at temperatures above 20 degrees C). (b) The inhibitor reduced the velocity of Ca2+ uptake without appreciably influencing the apparent affinity of the transport system for Ca2+. (c) Inhibition was uncompetitive with respect to ATP. (d) Sarcoplasmic reticulum washed following exposure to CPF-I showed reduced rates of Ca2+ uptake, indicating that inhibition results from an interaction of the inhibitor with the sarcoplasmic reticulum membrane. (e) Concomitant with the inhibition of Ca2+ uptake, CPF-I also inhibited the Ca2+-ATPase activity of sarcoplasmic reticulum. (f) Heat-treatment of CPF-I led to loss of inhibitor activity, whereas exposure to trypsin appeared to enhance its inhibitory effect. (g) Addition of CPF-I to Ca2+-preloaded sarcoplasmic reticulum vesicles did not promote Ca2+ release from the vesicles. These results demonstrate the presence of a soluble protein inhibitor of sarcoplasmic reticulum Ca2+ pump in heart and slow skeletal muscle but not in fast skeletal muscle. The characteristics of the inhibitor and its apparently selective distribution suggest a potentially important role for it in the in vivo regulation of sarcoplasmic reticulum Ca2+ pump, and therefore in determining the duration of Ca2+ signal in slow-contracting muscle fibers.

Animals↗
PubMed1984

Kinetic studies of Ca2+ release from sarcoplasmic reticulum of normal and malignant hyperthermia susceptible pig muscles.

The time-course of Ca2+ release from sarcoplasmic reticulum isolated from muscles of normal pigs and those of pigs susceptible to malignant hyperthermia were investigated using stopped-flow spectrophotometry and arsenazo III as a Ca2+ indicator. Several methods were used to trigger Ca2+ release: (a) addition of halothane (e.g., 0.2 mM); (b) an increase of extravesicular Ca2+ concentration ([Ca2+0]); (c) a combination of (a) and (b), and (d) replacement of ions (potassium gluconate with choline chloride) to produce membrane depolarization. The initial rates of Ca2+ release induced by either halothane or Ca2+ alone, or both, are at least 70% higher in malignant hyperthermic sarcoplasmic reticulum than in normal. The amount of Ca2+ released by halothane at low [Ca2+0] in malignant hyperthermic sarcoplasmic reticulum is about twice as large as in normal sarcoplasmic reticulum. Membrane depolarization led to biphasic Ca2+ release in both malignant hyperthermic and normal sarcoplasmic reticulum, the rate constant of the rapid phase of Ca2+ release induced by membrane depolarization being significantly higher in malignant hyperthermic sarcoplasmic reticulum (k = 83 s-1) than in normal (k = 37 s-1). Thus, all types of Ca2+ release investigated (a, b, c and d) have higher rates in malignant hyperthermic sarcoplasmic reticulum than normal sarcoplasmic reticulum. These results suggest that the putative Ca2+ release channels located in the sarcoplasmic reticulum are altered in malignant hyperthermic sarcoplasmic reticulum.

Animals↗
PubMed1986

Functional characterization of junctional terminal cisternae from mammalian fast skeletal muscle sarcoplasmic reticulum.

Junctional terminal cisternae are a recently isolated sarcoplasmic reticulum fraction containing two types of membranes, the junctional face membrane with morphologically intact "feet" structures and the calcium pump membrane [Saito, A., Seiler, S., Chu, A., & Fleischer, S. (1984) J. Cell Biol. 99, 875-885]. In this study, the Ca2+ fluxes of junctional terminal cisternae are characterized and compared with three other well-defined fractions derived from the sarcotubular system of fast-twitch skeletal muscle, including light and heavy sarcoplasmic reticulum, corresponding to longitudinal and terminal cisternae regions of the sarcoplasmic reticulum, and isolated triads. Functionally, junctional terminal cisternae have low net energized Ca2+ transport measured in the presence or absence of a Ca2+-trapping anion, as compared to light and heavy sarcoplasmic reticulum and triads. Ca2+ transport and Ca2+ pumping efficiency can be restored to values similar to those of light sarcoplasmic reticulum with ruthenium red or high [Mg2+]. In contrast to junctional terminal cisternae, heavy sarcoplasmic reticulum and triads have higher Ca2+ transport and are stimulated less by ruthenium red. Heavy sarcoplasmic reticulum appears to be derived from the nonjunctional portion of the terminal cisternae. Our studies indicate that the decreased Ca2+ transport is referable to the enhanced permeability to Ca2+, reflecting the predominant localization of Ca2+ release channels in junctional terminal cisternae. This conclusion is based on the following observations: The Ca2+, -Mg2+ -dependent ATPase activity of junctional terminal cisternae in the presence of a Ca2+ ionophore is comparable to that of light sarcoplasmic reticulum when normalized for the calcium pump protein content; i.e., the enhanced Ca2+ transport cannot be explained by a faster turnover of the pump. Ruthenium red or elevated [Mg2+] enhances energized Ca2+ transport and Ca2+ pumping efficiency in junctional terminal cisternae so that values approaching those of light sarcoplasmic reticulum are obtained. Rapid Ca2+ efflux in junctional terminal cisternae can be directly measured and is blocked by ruthenium red or high [Mg2+]. Ryanodine at pharmacologically significant concentrations blocks the ruthenium red stimulation of Ca2+ loading. Ryanodine binding in junctional terminal cisternae, which appears to titrate Ca2+ release channels, is 2 orders of magnitude lower than the concentration of the calcium pump protein. By contrast, light sarcoplasmic reticulum has a high Ca2+ loading rate and slow Ca2+ efflux that are not modulated by ruthenium red, ryanodine, or Mg2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed2003

A class of mutant CHO cells resistant to cholera toxin rapidly degrades the catalytic polypeptide of cholera toxin and exhibits increased endoplasmic reticulum-associated degradation.

After binding to the eukaryotic cell surface, cholera toxin undergoes retrograde transport to the endoplasmic reticulum. The catalytic A1 polypeptide of cholera toxin (CTA1) then crosses the endoplasmic reticulum membrane and enters the cytosol in a process that may involve the quality control mechanism known as endoplasmic reticulum-associated degradation. Other toxins such as Pseudomonas exotoxin A and ricin are also thought to exploit endoplasmic reticulum-associated degradation for entry into the cytosol. To test this model, we mutagenized Chinese hamster ovary cells and selected clones that survived a prolonged coincubation with Pseudomonas exotoxin A and ricin. These lethal endoplasmic reticulum-translocating toxins bind different surface receptors and target different cytosolic substrates, so resistance to both would likely result from disruption of a shared trafficking or translocation event. Here we characterize two Pseudomonas exotoxin A/ricin-resistant clones that exhibited increased endoplasmic reticulum-associated degradation. Both clones acquired the following unselected traits: (i) resistance to cholera toxin; (ii) increased degradation of an endoplasmic reticulum-localized CTA1 construct; (iii) increased degradation of an established endoplasmic reticulum-associated degradation substrate, the Z variant of alpha1-antitrypsin (alpha1AT-Z); and (iv) reduced secretion of both alpha1AT-Z and the transport-competent protein alpha1AT-M. Proteosome inhibition partially rescued the alpha1AT-M secretion deficiencies. However, the mutant clones did not exhibit increased proteosomal activity against cytosolic proteins, including a second CTA1 construct that was expressed in the cytosol rather than in the endoplasmic reticulum. These results suggested that accelerated endoplasmic reticulum-associated degradation in the mutant clones produced a cholera toxin/Pseudomonas exotoxin A/ricin-resistant phenotype by increasing the coupling efficiency between toxin translocation and toxin degradation.

Animals↗
PubMed2005

Multiple signals induce endoplasmic reticulum stress in both primary and immortalized chondrocytes resulting in loss of differentiation, impaired cell growth, and apoptosis.

The endoplasmic reticulum is the site of synthesis and folding of secretory proteins and is sensitive to changes in the internal and external environment of the cell. Both physiological and pathological conditions may perturb the function of the endoplasmic reticulum, resulting in endoplasmic reticulum stress. The chondrocyte is the only resident cell found in cartilage and is responsible for synthesis and turnover of the abundant extracellular matrix and may be sensitive to endoplasmic reticulum stress. Here we report that glucose withdrawal, tunicamycin, and thapsigargin induce up-regulation of GADD153 and caspase-12, two markers of endoplasmic reticulum stress, in both primary chondrocytes and a chondrocyte cell line. Other agents such as interleukin-1beta or tumor necrosis factor alpha induced a minimal or no induction of GADD153, respectively. The endoplasmic reticulum stress resulted in decreased chondrocyte growth based on cell counts, up-regulation of p21, and decreased PCNA expression. In addition, perturbation of endoplasmic reticulum function resulted in decreased accumulation of an Alcian Blue positive matrix by chondrocytes and decreased expression of type II collagen at the protein level. Further, quantitative real-time PCR was used to demonstrate a down-regulation of steady state mRNA levels coding for aggrecan, collagen II, and link protein in chondrocytes exposed to endoplasmic reticulum stress-inducing conditions. Ultimately, endoplasmic reticulum stress resulted in chondrocyte apoptosis, as evidenced by DNA fragmentation and annexin V staining. These findings have potentially important implications regarding consequences of endoplasmic reticulum stress in cartilage biology.

Animals↗
PubMed1984

Mechanisms of fatty acid effects on sarcoplasmic reticulum. II. Structural changes induced by oleic and palmitic acids.

The interaction of micromolar concentrations of palmitic and oleic acids with the sarcoplasmic reticulum membrane was studied by electron microscopic techniques in an attempt to define their different effects on ATP-induced calcium sequestration in sarcoplasmic reticulum vesicles. Oleic acid had a concentration-dependent effect on the morphology of sarcoplasmic reticulum vesicles, promoting vesicle fusion and eventual solubilization. Palmitic acid did not alter the morphology of sarcoplasmic reticulum, but its probable site(s) of interaction could be determined. In the presence of palmitic acid, large lamellar structures that formed external to sarcoplasmic reticulum vesicles are probably composed of pure palmitic acid and/or palmitic acid/phospholipid mixed "micelles," but internalization of palmitic acid into sarcoplasmic reticulum vesicles was not detected. Palmitic acid reduced the phospholipid content of sarcoplasmic reticulum membranes with a preservation of the average interparticle protein spacing as observed in freeze-fracture electron micrographs. Thus, palmitic acid appears to be incorporated into the sarcoplasmic reticulum lipid bi-layer. Oleic acid inhibition of ATP-induced calcium sequestration by sarcoplasmic reticulum vesicles is probably caused by net permeability changes of the membrane. A structural mechanism for palmitic acid stimulation of ATP-induced calcium sequestration is proposed in light of the probable insertion of palmitic acid into the sarcoplasmic reticulum lipid bilayer.

Adenosine Triphosphate↗
PubMed1992

Two kinds of calcium-induced release of calcium from the sarcoplasmic reticulum of skinned cardiac cells.

This article compares the Ca(2+)-induced release of Ca2+ that is triggered by a rapid increase of free Ca2+ concentration at the outer surface of the sarcoplasmic reticulum of a skinned cardiac cell to the spontaneous release of Ca2+ that is produced by a steady-state high free Ca2+ concentration which overloads the sarcoplasmic reticulum with Ca2+ in a skinned cardiac cell. The first process, that is triggered by a rapid increase of free Ca2+ concentration at the outer surface of the sarcoplasmic reticulum, has a time- and Ca(2+)-dependent activation and inactivation, does not require any preload of the sarcoplasmic reticulum with Ca2+, and is not affected by the addition of inositol (1,4,5)-trisphosphate. The second process, i.e. the spontaneous release of Ca2+ from the sarcoplasmic reticulum, is not inactivated by a high free Ca2+ concentration, requires an overload of the sarcoplasmic reticulum with Ca2+ and is enhanced by inositol(1,4,5)-trisphosphate. The filling inside the sarcoplasmic reticulum with Ca2+ is critical for the triggering of the spontaneous release of Ca2+. On the other hand, the spontaneous release of Ca2+ has many similarities to the "Ca(2+)-induced release of Ca2+" that is observed for isolated sarcoplasmic reticulum vesicles incorporated into a lipid bilayer which is triggered by the increase of free Ca2+ concentration at the outer surface of the sarcoplasmic reticulum. Although the Ca(2+)-induced release of Ca2+ with time- and Ca(2+)-dependent activation and inactivation and the spontaneous release of Ca2+ are regulated by difference mechanisms, they are both inhibited by ryanodine, which suggests that they may take place through the same channel and may even have some partial common pathway.

Animals↗
PubMed1979

The smooth endoplasmic reticulum in neurohypophysial axons of the rat: possible involvement in transport, storage and release of neurosecretory material.

The intra-axonal organization of the smooth endoplasmic reticulum was studied in the neurohypophysis of rats during and after water deprivation. Parallel to conventional electron microscopy, the material was treated with a double impregnation staining technique specifically designed to contrast the intracellular membranous system. In conventionally stained ultrathin sections from severely dehydrated rats most axons appeared to be free of membranous organelles, whereas corresponding axons treated with the double-impregnation technique generally exhibited a highly developed system of smooth endoplasmic reticulum. In axonal endings, both techniques revealed a profusion of microvesicles in intimate relationship with tubular elements of the smooth endoplasmic reticulum. In short-term (12h) rehydrated rats, a similarly developed system of smooth endoplasmic reticulum was stilll observed at all axonal levels with both procedures. After 24 to 48 h or rehydration the tubules of the smooth endoplasmic reticulum exhibited, in double impregnated material, numerous dilatations which resembled the adjacent neurosecretory granules. In conventionally stained ultrathin sections, an accumulation of electron dense material occurred within tubules of the smooth endoplasmic reticulum in the more proximal axonal segments, while in the more terminal segments, which contained numerous elongated granules, membrane continuity was frequently observed between newly formed granules and the smooth endoplasmic reticulum. After 7 days of rehydration the general pattern of the axonal smooth endoplasmic reticulum was comparable to that in untreated rats. These results are discussed in the light of a suggested involvement of the axonal smooth endoplasmic reticulum in the non-granlular transport of neurosecretory material in connection with (1) storage in distally formed granules, and (2) release via microvesicles.

Animals↗
PubMed2000

Signaling pathways between the plasma membrane and endoplasmic reticulum calcium stores.

This review discusses multiple ways in which the endoplasmic reticulum participates in and is influenced by signal transduction pathways. The endoplasmic reticulum provides a Ca2+ store that can be mobilized either by calcium-induced calcium release or by the diffusible messenger inositol 1,4,5-trisphosphate. Depletion of endoplasmic reticulum Ca2+ stores provides a signal that activates surface membrane Ca2+ channels, a process known as capacitative calcium entry. Depletion of endoplasmic reticulum stores can also signal long-term cellular responses such as gene expression and programmed cell death or apoptosis. In addition to serving as a source of cellular signals, the endoplasmic reticulum is also functionally and structurally modified by the Ca2+ and protein kinase C pathways. Elevated cytoplasmic Ca2+ causes a rearrangement and fragmentation of endoplasmic reticulum membranes. Protein kinase C activation reduces the storage capacity of the endoplasmic reticulum Ca2+ pool. In some cell types, protein kinase C inhibits capacitative calcium entry. Protein kinase C activation also protects the endoplasmic reticulum from the structural effects of high cytoplasmic Ca2+. The emerging view is one of a complex network of pathways through which the endoplasmic reticulum and the Ca2+ and protein kinase C signaling pathways interact at various levels regulating cellular structure and function.

Animals↗
PubMed1984

Phospholipid asymmetry in the isolated sarcoplasmic reticulum membrane.

The total phospholipid content and distribution of phospholipid species between the outer and inner monolayers of the isolated sarcoplasmic reticulum membrane was measured by phospholipase A2 activities and neutron diffraction. Phospholipase measurements showed that specific phospholipid species were asymmetric in their distribution between the outer and inner monolayers of the sarcoplasmic reticulum lipid bilayer; phosphatidylcholine (PC) was distributed 48/52 +/- 2% between the outer and inner monolayer of the sarcoplasmic reticulum bilayer, 69% of the phosphatidyl-ethanolamine (PE) resided mainly in the outer monolayer of the bilayer, 85% of the phosphatidylserine (PS) and 88% of the phosphatidylinositol (PI) were localized predominantly in the inner monolayer. The total phospholipid distribution determined by these measurements was 48/52 +/- 2% for the outer/inner monolayer of the sarcoplasmic reticulum lipid bilayer. Sarcoplasmic reticulum phospholipids were biosynthetically deuterated and exchanged into isolated vesicles with both a specific lecithin and a general exchange protein. Neutron diffraction measurements directly provided lipid distribution profiles for both PC and the total lipid content in the intact sarcoplasmic reticulum membrane. The outer/inner monolayer distribution for PC was 47/53 +/- 1%, in agreement with phospholipase measurements, while that for the total lipid was 46/54 +/- 1%, similar to the phospholipase measurements. These neutron diffraction results regarding the sarcoplasmic reticulum membrane bilayer were used in model calculations for decomposing the electron-density profile structure (10 A resolution) of isolated sarcoplasmic reticulum previously determined by X-ray diffraction into structures for the separate membrane components. These structure studies showed that the protein profile structure within the membrane lipid bilayer was asymmetric, complementary to the asymmetric lipid structure. Thus, the total phospholipid asymmetry obtained by two independent methods was small but consistent with a complementary asymmetric protein structure, and may be related to the highly vectorial functional properties of the calcium pump ATPase protein in the sarcoplasmic reticulum membrane.

Animals↗
PubMed1985

The separate profile structures of the functional calcium pump protein and the phospholipid bilayer within isolated sarcoplasmic reticulum membranes determined by X-ray and neutron diffraction.

The detailed profile structure of the isolated sarcoplasmic reticulum membrane was studied utilizing a combination of X-ray and neutron diffraction. The water and lipid profile structures within the sarcoplasmic reticulum membrane were determined at 28 A resolution directly by neutron diffraction and selective deuteration of the water and lipid components. The previously determined electron density profile structure of the sarcoplasmic reticulum membrane at 12 A resolution was subjected to model refinement analysis constrained by the neutron diffraction results, thereby providing unique higher resolution calculated lipid and protein profile structures. It was found that the lipid bilayer profile structure of the isolated sarcoplasmic reticulum membrane is asymmetric, primarily the result of more lipid residing in the inner versus the outer monolayer of the sarcoplasmic reticulum lipid bilayer. The asymmetry in the lipid composition was necessarily coincident with a complimentary asymmetry in the protein mass distribution between the two monolayers in order to preserve the overall cross-sectional area of lipid and protein throughout the lipid bilayer region of the sarcoplasmic reticulum membrane profile structure. Approximately 50% of the mass of the total protein was found to be localized externally to the sarcoplasmic reticulum membrane lipid bilayer protruding from the outer lipid monolayer into the extravesicular medium. The structural features of the protein protrusion appear to be rather variable depending upon the environment of the sarcoplasmic reticulum membrane. This highly asymmetric structural organization of the sarcoplasmic reticulum membrane profile is consistent with its primary function of unidirectional calcium transport.

Animals↗
PubMed1993

Ceramide excluded from cell-free vesicular lipid transfer from endoplasmic reticulum to Golgi apparatus. Evidence for lipid sorting.

The distribution and cell-free transfer of ceramide and other lipids were compared using highly purified fractions of endoplasmic reticulum, transitional endoplasmic reticulum, transition vesicles and Golgi apparatus from rat liver. Ceramides were present in both endoplasmic reticulum and Golgi apparatus where they represented between 0.3 and 1% of the total lipids. Ceramides, however, were much reduced or absent (< 0.05%) from transition vesicles. Transition vesicles were induced to form from transitional endoplasmic reticulum by incubation with ATP and a cytosol fraction. When transfer of [14C]choline-labeled phosphatidylcholine from transitional endoplasmic reticulum to Golgi apparatus was followed, transition vesicles were more efficient in transfer than the transitional endoplasmic reticulum from which they were derived. This transfer was temperature- and ATP-dependent and inhibited by N-ethylmaleimide. When transfer of [3H]ceramide was followed, there was little or no transfer via transition vesicles and that transfer which occurred was temperature-, ATP- and N-ethylmaleimide independent. Transfer of ceramide in the cell-free system did occur from endoplasmic reticulum to Golgi apparatus but via a non-vesicular mechanism that was temperature-dependent but not dependent on ATP or cytosol, alone, or in combination, nor was it inhibited by N-ethylmaleimide. A component of phosphatidylcholine transfer exhibited similar characteristics. The results provide evidence for two distinct mechanisms for cell-free transfer of lipids from endoplasmic reticulum to Golgi apparatus. The first is via 50 to 70 nm transition vesicles which is temperature- and ATP-dependent, inhibited by N-ethylmaleimide and from which ceramides are excluded. The second is non-vesicular, temperature-dependent, and neither ATP- nor cytosol-dependent. It accounts for the bulk of the ceramide transfer. As a result during cell-free lipid transfer from endoplasmic reticulum to Golgi apparatus, lipid sorting occurs such that ceramides are largely absent from the transition vesicles and, apparently are delivered to the Golgi apparatus by another mechanism.

Animals↗
PubMed1980

Evidence against the smooth endoplasmic reticulum as a continuous channel for the retrograde axonal transport of horseradish peroxidase.

The involvement of the axonal smooth endoplasmic reticulum as a channel for the retrograde axonal transport of horseradish peroxidase (HRP) has been tested by analysing serial sections of 52 HRP-positive organelles in chick optic nerves. The enzyme marker was injected in the posterior, contralateral optic tectum 10 h before fixation of young chicks. The two optic nerves, retinas and optic tecta were incubated for electron microscopic demonstration of HRP. Thin sections of the retinas and tecta and serial thin sections of the optic nerves were studied in some cases with the aid of a goniometer. Of the 52 organelles, 42% had a tubular shape, 46% were oval and 12% were multivesicular bodies. None of the organelles was found to have continuities with other membranous structures, including tubules or cisternae of the smooth endoplasmic reticulum. In 10 cases, the smooth endoplasmic reticulum was followed in serial sections over a length of up to 4 micrometer. In every case, the reticulum appeared to form a continuous system although some tubular extensions apparently ended blindly near other organelles. In neither the 10 series of serial sections nor in any other individual micrographs did any recognizable profile of the smooth endoplasmic reticulum contain HRP. Measurements of the thickness of the membranes of HRP-containing organelles, of the smooth endoplasmic reticulum and of plasmalemma were made, since these membranes have been distinguished on the basis of their thickness in other cells. The plasmalemma in the axons was about 20% thicker than that of the smooth endoplasmic reticulum, and about 9% thicker than that of HRP-labeled organelles. The membrane of the smooth endoplasmic reticulum and HRP-organelles could also be distinguished by this means. It is concluded that in chick retinal ganglion cell axons, HRP is not transported in a retrograde direction via a continuous channel of smooth endoplasmic reticulum.

Animals↗
PubMed2006

Nitric oxide-induced endoplasmic reticulum stress activates the expression of cargo receptor proteins and alters the glycoprotein transport to the Golgi complex.

The endoplasmic reticulum Golgi intermediate compartment 53 protein recycles continuously between the endoplasmic reticulum and the Golgi complex and ensures the anterograde transport of specific glycoproteins with the assistance of the Multiple Clotting Factor Deficiency adaptor protein. Therefore, to analyze the effect of the endoplasmic reticulum stress on the secretory pathway beyond the endoplasmic reticulum, we analyzed the expression of both proteins in J774 macrophages incubated with the nitric oxide donor DETA NONOate or with thapsigargin. Both proteins accumulated progressively, by a transcriptional mechanism, in response to these inducers. Nitric oxide also induced a higher level of calreticulin and glucose regulated 78 protein, two endoplasmic reticulum proteins controlled by the unfolded protein response. Interestingly, nitric oxide induced the processing of the activating transcription factor 6alpha of the unfolded protein response, while thapsigargin also induced the activation of the transcription factor X-box Binding Protein 1. In addition, we showed that the accumulation of both transporters occurred simultaneously with the activation of endoplasmic reticulum-stress-dependent apoptosis, suggesting that these proteins may participate in the events that will eventually decide the fate of the cell. Induction of endoplasmic reticulum stress affected the rate of anterograde transport of a reporter glycoprotein, indicating that the endoplasmic reticulum to Golgi transport is remarkably impaired. Our results indicate that increased levels of cargo receptor proteins might have a function either in the quality control of protein folding in the endoplasmic reticulum or in the homeostasis of the intermediate compartment and Golgi complex during cell stress.

Activating Transcription Factor 6↗
PubMedDate not supplied

Ca2+ signaling and calcium binding chaperones of the endoplasmic reticulum.

The endoplasmic reticulum is a centrally located organelle which affects virtually every cellular function. Its unique luminal environment consists of Ca(2+) binding chaperones, which are involved in protein folding, post-translational modification, Ca(2+) storage and release, and lipid synthesis and metabolism. The environment within the lumen of the endoplasmic reticulum has profound effects on endoplasmic reticulum function and signaling, including apoptosis, stress responses, organogenesis, and transcriptional activity. Calreticulin, a major Ca(2+) binding (storage) chaperone in the endoplasmic reticulum, is a key component of the calreticulin/calnexin cycle which is responsible for the folding of newly synthesized proteins and glycoproteins and for quality control pathways in the endoplasmic reticulum. The function of calreticulin, calnexin and other endoplasmic reticulum proteins is affected by continuous fluctuations in the concentration of Ca(2+) in the endoplasmic reticulum. Thus, changes in Ca(2+) concentration may play a signaling role in the lumen of the endoplasmic reticulum as well as in the cytosol. Recent studies on calreticulin-deficient and transgenic mice have revealed that calreticulin and the endoplasmic reticulum may be upstream regulators in the Ca(2+)-dependent pathways that control cellular differentiation and/or organ development.

Animals↗
PubMed1992

The effects of volatile anesthetics on calcium regulation by malignant hyperthermia-susceptible sarcoplasmic reticulum.

To clarify the mechanism by which volatile anesthetics initiate malignant hyperthermia (MH), we examined the effect of halothane, isoflurane, and enflurane on Ca2+ uptake and release by sarcoplasmic reticulum vesicles isolated from MH-susceptible (MHS) and normal pig muscle. Clinical concentrations of these anesthetics (0.1-0.5 mM) stimulated sarcoplasmic reticulum ATP-dependent Ca2+ uptake (maximal at approximately 4 mM), whereas 10-20 times the clinical anesthetic concentration inhibited Ca2+ uptake. There was no significant difference between MHS and normal sarcoplasmic reticulum in any aspect of Ca2+ uptake. Ca2+ release from 45Ca(2+)-filled sarcoplasmic reticulum vesicles in a 10(-8) M Ca(2+)-containing medium (pH 7.0) was significantly stimulated at clinical concentrations of all three volatile anesthetics (anesthetic concentration for the 50% stimulation of Ca2+ release = 0.096-0.22 mM); however, the rate constant for Ca2+ release from MHS sarcoplasmic reticulum was in all cases significantly greater than that from normal sarcoplasmic reticulum. Furthermore, 0.5 mM halothane had no effect on Ca2+ release from normal sarcoplasmic reticulum at pH values less than 6.8, although it could still significantly stimulate Ca2+ release from MHS sarcoplasmic reticulum even at pH 6.4; similar results were obtained for isoflurane and enflurane. These studies thus demonstrate that the interaction of volatile anesthetics with the sarcoplasmic reticulum Ca(2+)-release channel is altered in MHS porcine muscle such that the channel may be activated even at a Ca2+ concentration or pH that would be expected to maintain the channel in the closed state.

Animals↗
PubMed1998

Mutants of the yeast Yarrowia lipolytica defective in protein exit from the endoplasmic reticulum are also defective in peroxisome biogenesis.

Mutations in the SEC238 and SRP54 genes of the yeast Yarrowia lipolytica not only cause temperature-sensitive defects in the exit of the precursor form of alkaline extracellular protease and of other secretory proteins from the endoplasmic reticulum and in protein secretion but also lead to temperature-sensitive growth in oleic acid-containing medium, the metabolism of which requires the assembly of functionally intact peroxisomes. The sec238A and srp54KO mutations at the restrictive temperature significantly reduce the size and number of peroxisomes, affect the import of peroxisomal matrix and membrane proteins into the organelle, and significantly delay, but do not prevent, the exit of two peroxisomal membrane proteins, Pex2p and Pex16p, from the endoplasmic reticulum en route to the peroxisomal membrane. Mutations in the PEX1 and PEX6 genes, which encode members of the AAA family of N-ethylmaleimide-sensitive fusion protein-like ATPases, not only affect the exit of precursor forms of secretory proteins from the endoplasmic reticulum but also prevent the exit of the peroxisomal membrane proteins Pex2p and Pex16p from the endoplasmic reticulum and cause the accumulation of an extensive network of endoplasmic reticulum membranes. None of the peroxisomal matrix proteins tested associated with the endoplasmic reticulum in sec238A, srp54KO, pex1-1, and pex6KO mutant cells. Our data provide evidence that the endoplasmic reticulum is required for peroxisome biogenesis and suggest that in Y. lipolytica, the trafficking of some membrane proteins, but not matrix proteins, to the peroxisome occurs via the endoplasmic reticulum, results in their glycosylation within the lumen of the endoplasmic reticulum, does not involve transport through the Golgi, and requires the products encoded by the SEC238, SRP54, PEX1, and PEX6 genes.

ATPases Associated with Diverse Cellular Activitie↗
PubMedDate not supplied

Interrelationships of endoplasmic reticulum, mitochondria, intermediate filaments, and microtubules--a quadruple fluorescence labeling study.

To study the interrelationships of endoplasmic reticulum, mitochondria, intermediate filaments, and microtubules, we have developed a quadruple fluorescence labeling procedure to visualize all four structures in the same cell. We applied this approach to study cellular organization in control cells and in cells treated with the microtubule drugs vinblastine or taxol. Endoplasmic reticulum was visualized by staining glutaraldehyde-fixed cells with the dye 3,3'-dihexyloxacarbocyanine iodide. After detergent permeabilization, triple immunofluorescence was carried out to specifically visualize mitochondria, vimentin intermediate filaments, and microtubules. Mitochondria in human fibroblasts were found to be highly elongated tubular structures (lengths up to greater than 50 microns), which in many cases were apparently fused to each other. Mitochondria were always observed to be associated with endoplasmic reticulum, although endoplasmic reticulum also existed independently. Intermediate filament distribution could not completely account for endoplasmic reticulum or mitochondrial distributions. Microtubules, however, always codistributed with these organelles. Microtubule depolymerization in vinblastine treated cells resulted in coaggregation of endoplasmic reticulum and mitochondria, and in the collapse of intermediate filaments. The spatial distributions of organelles compared with intermediate filaments were not identical, indicating that attachment of organelles to intermediate filaments was not responsible for organelle aggregation. Mitochondrial associations with endoplasmic reticulum, on the other hand, were retained, indicating this association was stable regardless of endoplasmic reticulum form or microtubules. In taxol-treated cells, endoplasmic reticulum, mitochondria, and intermediate filaments were all associated with taxol-stabilized microtubule bundles.

Animals↗