共查询到20条相似文献,搜索用时 62 毫秒
1.
Hirooki Matsui Yasukazu Hozumi Toshiaki Tanaka Masashi Okada Tomoyuki Nakano Yusuke Suzuki Ken Iseki Seiji Kakehata Matthew K. Topham Kaoru Goto 《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2014,1841(10):1440-1450
The endoplasmic reticulum (ER), comprised of an interconnected membrane network, is a site of phospholipid and protein synthesis. The diacylglycerol kinase (DGK) enzyme family catalyzes phosphorylation of diacylglycerol to phosphatidic acid. Both of these lipids are known not only to serve as second messengers but also to represent intermediate precursors of lipids of various kinds. The DGK family is targeted to distinct subcellular sites in cDNA-transfected and native cells. Of DGKs, DGKε localizes primarily to the ER, suggesting that this isozyme plays a role in this organelle. Using experiments with various deletion and substitution mutants, this study examined the molecular mechanism of how DGKε is targeted to the ER. Results demonstrate that the N-terminal hydrophobic sequence 20–40 plays a necessary role in targeting of DGKε to the ER. This hydrophobic amino acid segment is predicted to adopt an α-helix structure, in which Leu22, L25, and L29 are present in mutual proximity, forming a hydrophobic patch. When these hydrophobic Leu residues were replaced with hydrophilic amino acid Gln, the mutant fragment designated DGKε (20–40/L22Q,L25Q,L29Q) exhibits diffuse distribution in the cytoplasm. Moreover, full-length DGKε containing these substitutions, DGKε (L22Q,L25Q,L29Q), is shown to distribute diffusely in the cytoplasm. These results indicate that the N-terminal hydrophobic residues play a key role in DGKε targeting to the ER membrane. Functionally, knockdown or deletion of DGKε affects the unfolding protein response pathways, thereby rendering the cells susceptible to apoptosis, to some degree, under ER stress conditions. 相似文献
2.
Recent data from studies of peroxisome assembly and the subcellular sorting of peroxisomal matrix and membrane proteins have led to an expansion of the 'growth and division' and 'endoplasmic reticulum-vesiculation' models of peroxisome biogenesis into a more flexible, unified model. Within this context, we discuss the proposed role for the endoplasmic reticulum in the formation of preperoxisomes and the potential for 15 Arabidopsis peroxin homologs to function in the biogenesis of peroxisomes in plant cells. 相似文献
3.
The endoplasmic reticulum (ER) is a major site of protein synthesis and its inside, or lumen, is a major site of protein folding. The lumen of the ER contains many folding factors and molecular chaperones, which facilitate protein folding by increasing both the rate and the efficiency of this process. Amongst the many ER folding factors, there are three components that specifically modulate the folding glycoproteins bearing N-linked carbohydrate side chains. These components are calnexin, calreticulin and ERp57, and this review focuses on the molecular basis for their capacity to influence glycoprotein folding. 相似文献
4.
Calcium, a signaling molecule in the endoplasmic reticulum? 总被引:10,自引:0,他引:10
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. 相似文献
5.
Summary Dilated cisternae (DC) of endoplasmic reticulum were found to be a typical component of stem-, hypocotyledon-, and root vascular parenchyma as well as root cap cells inBrassicaceae (14 species screened) andCapparaceae (7 species). In general, DC are very long, utricular organelles containing proteins and bound by a unit membrane which is studded with ribosomes. In aerial parts of the plants tested the proteins are tubular structures arranged parallel to the DC axis, in roots there ar filamentous proteins. In someBrassicaceae (e.g., Lunaria, Ptilotrichum, Schivereckia) DC are irregularly shaped and contain granular protein material. These forms are discussed to be leading to protein-containing vacuoles as found in companion cells ofTovaria. Since DC do occur in taxa which contain glucosinolates, an EM-cytochemical test for -thioglucosidases (as first performed byIversen) was applied to some of the species. The results, however, did not prove a specific location of the enzyme inside DC. In addition to questions on the reliability of the method it is discussed whether it is likely that DC are a site of -thioglucosidases while it is known thate.g., the glucosinolate-containingResedaceae do not contain this organelle. Also,Erythroxylum (Erythroxylaceae) andJacaranda (Bignoniaceae) gave the same precipitations when subjected to the cytochemical test (see note added in proof). 相似文献
6.
The subcellular site of -amylase (EC 1.6.2.1) synthesis and transport was studied in barley aleurone layers incubated in the presence or absence of gibberellic acid (GA3). Using [35S]methionine as a marker, the site of amino-acid incorporation into organelles isolated from aleurone layers incubated with and without GA3 was determined following purification by isopycnic sucrose-density-gradient centrifugation. Incorporation of radioactivity into trichloroacetic-acid-insoluble proteins was greatest in those fractions exhibiting activity of an endoplasmic reticulum (ER) marker enzyme. Further fractionation of densitygradient fractions by sodium-dodecyl-sulfate polyacrylamide-gel electrophoresis showed that a major portion of the radioactivity in the ER fractions was present in a protein co-migrating with marker -amylase. This protein was identified as authentic -amylase by immunoadsorbent chromatography and affinity chromatography. The newly synthesized -amylase associated with the ER was shown to be sequenstered within the lumen of the ER by experiments which showed that the enzyme was resistant to proteolytic degradation. The labelled -amylase sequestered in the ER can be chased from this organelle when tissue is incubated in unlabelled methionine following a 1-h pulse of labelled methionine. The isoenzymic forms of -amylase found in tissue homogenates and incubation media of aleurone layers incubated with and without GA3 were characterized after chromatography on diethylaminoethyl cellulose. In homogenates of GA3-treated aleurone layers, five peaks of -amylase activity were detected, while in homogenates of aleurone layers incubated with-out GA3 only three peaks of activity were found. In incubation media, four isoenzymes were found after GA3 treatment and two were found after incubation without GA3. We conclude that at least five -amylase isoenzymes are synthesized by the ER of barley aleurone layers and that this membrane system is involved in the sequestration and transport of four of these isoenzymes.Abbreviations CHA
cyclohepataamylose
- DEAE-cellulose
diethylaminoethyl-cellulose
- ER
endoplasmic reticulum
- GA3
gibberellic acid
- SDS-PAGE
sodium-dodecyl-sulfate polyacrylamide-gel electrophoresis 相似文献
7.
Heterotypic tubular connections at the endoplasmic reticulum–Golgi complex interface 总被引:1,自引:1,他引:0
Vivero-Salmerón G Ballesta J Martínez-Menárguez JA 《Histochemistry and cell biology》2008,130(4):709-717
Electron microscopy and cryoimmunocytochemistry were used to characterize tubular connections in the secretory pathway using rat spermatids as model. Our results support the existence of a complex tubular network enriched in the Golgi matrix protein GM130 that transiently joins the cis-Golgi side and the endoplasmic reticulum. These tubules occasionally contain the endoplasmic reticulum resident protein PDI but not COPII complexes or KDEL receptor. At the lateral edges of the stacks tubules were seen to connect cisternae belonging to the same or adjacent stacks. These connections were observed in all cisternae but preferentially on the cis side. Giantin, Gos28 and Rab6 were detected in the tubules; importantly, we reported the presence of cis-trans heterotypic connections between cisternae. On the trans-Golgi side, we occasionally observed tubules highly immunoreactive for Rab6 connecting the stack with the forming acrosome. Together, our results support the existence of transient continuities throughout the secretory pathways. 相似文献
8.
9.
Braun M 《Plant physiology》2001,125(4):1611-1619
Spectrin-like epitopes were immunochemically detected and immunofluorescently localized in gravitropically tip-growing rhizoids and protonemata of characean algae. Antiserum against spectrin from chicken erythrocytes showed cross-reactivity with rhizoid proteins at molecular masses of about 170 and 195 kD. Confocal microscopy revealed a distinct spherical labeling of spectrin-like proteins in the apices of both cell types tightly associated with an apical actin array and a specific subdomain of endoplasmic reticulum (ER), the ER aggregate. The presence of spectrin-like epitopes, the ER aggregate, and the actin cytoskeleton are strictly correlated with active tip growth. Application of cytochalasin D and A23187 has shown that interfering with actin or with the calcium gradient, which cause the disintegration of the ER aggregate and abolish tip growth, inhibits labeling of spectrin-like proteins. At the beginning of the graviresponse in rhizoids the labeling of spectrin-like proteins remained in its symmetrical position at the cell tip, but was clearly displaced to the upper flank in gravistimulated protonemata. These findings support the hypothesis that a displacement of the Spitzenk?rper is required for the negative gravitropic response in protonemata, but not for the positive gravitropic response in rhizoids. It is evident that the actin/spectrin system plays a role in maintaining the organization of the ER aggregate and represents an essential part in the mechanism of gravitropic tip growth. 相似文献
10.
11.
12.
Erdmann F Schäuble N Lang S Jung M Honigmann A Ahmad M Dudek J Benedix J Harsman A Kopp A Helms V Cavalié A Wagner R Zimmermann R 《The EMBO journal》2011,30(1):17-31
In eukaryotes, protein transport into the endoplasmic reticulum (ER) is facilitated by a protein-conducting channel, the Sec61 complex. The presence of large, water-filled pores with uncontrolled ion permeability, as formed by Sec61 complexes in the ER membrane, would seriously interfere with the regulated release of calcium from the ER lumen into the cytosol, an essential mechanism for intracellular signalling. We identified a calmodulin (CaM)-binding motif in the cytosolic N-terminus of mammalian Sec61α that bound CaM but not Ca2+-free apocalmodulin with nanomolar affinity and sequence specificity. In single-channel measurements, CaM potently mediated Sec61-channel closure in Ca2+-dependent manner. At the cellular level, two different CaM antagonists stimulated calcium release from the ER through Sec61 channels. However, protein transport into microsomes was not modulated by Ca2+-CaM. Molecular modelling of the ribosome/Sec61/CaM complexes supports the view that simultaneous ribosome and CaM binding to the Sec61 complex may be possible. Overall, CaM is involved in limiting Ca2+ leakage from the ER. 相似文献
13.
In pancreatic β-cells, the endoplasmic reticulum (ER) is the crucial site for insulin biosynthesis, as this is where the protein-folding machinery for secretory proteins is localized. Perturbations to ER function of the β-cell, such as those caused by high levels of free fatty acid and insulin resistance, can lead to an imbalance in protein homeostasis and ER stress, which has been recognized as an important mechanism for type 2 diabetes. Macroautophagy (hereafter referred to as autophagy) is activated as a novel signaling pathway in response to ER stress. In this review, we outline the mechanism of ER stress-mediated β-cell death and focus on the role of autophagy in ameliorating ER stress. The development of drugs to take advantage of the potential protective effect of autophagy in ER stress, such as glucagon like peptide-1, will be a promising avenue of investigation. 相似文献
14.
15.
Oxidative protein folding in the luminal compartment of the endoplasmic reticulum is thought to be mediated by a proteinaceous electron relay system composed by PDI and ER oxidoreductin 1 (Ero1), transferring electrons from the cysteinyl residues of substrate proteins to oxygen. However, recent observations revealed that Ero1 isoforms are dispensable. Endoplasmic reticulum is known as a generator and accumulator of low molecular weight oxidants; some of them have already been shown to promote oxidative folding. On the basis of these observations a new theory of oxidative folding is proposed where the oxidative power is provided by the stochastic contribution of prooxidants. 相似文献
16.
Kimura S Maruyama J Watanabe T Ito Y Arioka M Kitamoto K 《Fungal genetics and biology : FG & B》2010,47(12):1044-1054
Properly folded proteins destined for secretion exit through a specific subdomain of the endoplasmic reticulum (ER) known as transitional ER (tER) sites or ER exit sites (ERES). While such proteins in filamentous fungi localize at the hyphal tips overlapping the Spitzenk?rper, the distribution of misfolded proteins remains unknown. In the present study, we analyzed the distribution of mutant protein as well as ER and tER sites visualized by expression of AoClxA and AoSec13 fused with fluorescent protein, respectively, in the filamentous fungus Aspergillus oryzae. Discrete tER subdomains were visualized as the punctate dots of AoSec13 overlapping or associated with AoClxA distribution. Both ER and tER sites were concentrated near hyphal tips and formed apical gradients. Interestingly, while the expression of wild-type α-amylase fusion protein (AmyB-mDsRed) showed its localization coinciding with the Spitzenk?rper, a disulfide bond-deletion in AmyB causing its misfolding resulted in its accumulation in the subapical and basal ER, creating a reciprocal gradient to the tER sites. Furthermore, the reciprocal gradient enabled a clear distinction between the tER sites and the mutant AmyB accumulation sites near the apex. Based on these findings, we conclude that A. oryzae accumulates aberrant proteins toward basal hyphae while maintaining polarized tER sites for secretion of properly folded proteins at the hyphal tip. 相似文献
17.
For insulin synthesis, the proinsulin precursor is translated at the endoplasmic reticulum (ER), folds to include its three native disulfide bonds, and is exported to secretory granules for processing and secretion. Protein disulfide isomerase (PDI) has long been assumed to assist proinsulin in this process. Herein we have examined the effect of PDI knockdown (PDI-KD) in β-cells. The data establish that upon PDI-KD, oxidation of proinsulin to form native disulfide bonds is unimpaired and in fact enhanced. This is accompanied by improved proinsulin exit from the ER and increased total insulin secretion, with no evidence of ER stress. We provide evidence for direct physical interaction between PDI and proinsulin in the ER of pancreatic β-cells, in a manner requiring the catalytic activity of PDI. In β-cells after PDI-KD, enhanced export is selective for proinsulin over other secretory proteins, but the same effect is observed for recombinant proinsulin trafficking upon PDI-KD in heterologous cells. We hypothesize that PDI exhibits unfoldase activity for proinsulin, increasing retention of proinsulin within the ER of pancreatic β-cells. 相似文献
18.
STIM1 is a core component of the store‐operated Ca2+‐entry channel involved in Ca2+‐signaling with an important role in the activation of immune cells and many other cell types. In response to cell activation, STIM1 protein senses low Ca2+ concentration in the lumen of the endoplasmic reticulum (ER) and activates the channel protein Orai1 in the plasma membrane by direct physical contact. The related protein STIM2 functions similar but its physiological role is less well defined. We found that STIM2, but not STIM1, contains a di‐lysine ER‐retention signal. This restricts the function of STIM2 as Ca2+ sensor to the ER while STIM1 can reach the plasma membrane. The intracellular distribution of STIM1 is regulated in a cell‐cycle‐dependent manner with cell surface expression of STIM1 during mitosis. Efficient retention of STIM1 in the ER during interphase depends on its lysine‐rich domain and a di‐arginine ER retention signal. Store‐operated Ca2+‐entry enhanced ER retention, suggesting that trafficking of STIM1 is regulated and this regulation contributes to STIM1s role as multifunctional component in Ca2+‐signaling. 相似文献
19.
Hereditary tyrosinemia type 1 (HT1) is the most severe metabolic disease associated with tyrosine catabolism. An accumulation of toxic metabolites seems responsible for the pathology of HT1. The metabolite fumarylacetoacetate, accumulating due to a deficiency in fumarylacetoacetate hydrolase, displays apoptogenic, mutagenic, aneugenic and mitogenic activities. These effects may underlie the tumorigenic phenomenon observed in HT1. Fumarylacetoacetate in addition to causing disturbances in Ca2+ homeostasis, may induce endoplasmic reticulum stress. 相似文献
20.
《Biochimica et Biophysica Acta - Proteins and Proteomics》2019,1867(4):382-395
Sphingolipids are diverse lipids with essential, and occasionally opposing, functions in the cell and therefore tight control over biosynthesis is vital. Mechanisms governing this regulation are not understood. Initial steps in sphingolipid biosynthesis take place on the cytosolic face of the endoplasmic reticulum (ER). Serine palmitoyltransferase (SPT) is an ER-resident enzyme catalyzing the first-committed step in sphingolipid biosynthesis. Not surprisingly, SPT activity is tightly regulated. ORMDLs are ER-resident proteins recently identified as regulators of SPT activity. ORMDL proteins interact directly with SPT but the nature of this interaction is unknown. ORMDL protein sequences contain hydrophobic regions, yet algorithm-based predictions of transmembrane segments are highly ambiguous, making topology of this key regulator unclear. Here we report use of substituted cysteine accessibility to analyze topology of mammalian ORMDLs. We constructed multiple mutant ORMDLs, each containing a single cysteine strategically placed along the protein length. Combined use of selective membrane permeabilization with an impermeant cysteine modification reagent allowed us to assign transmembrane and cytosolic segments of ORMDL. We confirmed that mammalian ORMDL proteins transit the membrane four times, with amino- and carboxy termini facing the cytosol along with a large cytosolic loop. This model will allow us to determine details of the ORMDL-SPT interaction and identify regions acting as the “lipid sensor” to detect changes in cellular sphingolipid levels. We also observe that SPT and ORMDL are substantially resistant to extraction from membranes with non-ionic detergent, indirectly suggesting that both proteins reside in a specialized subdomain of the ER. 相似文献