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1.
The eta isoform of protein kinase C is localized on rough endoplasmic reticulum. 总被引:3,自引:1,他引:2
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K Chida H Sagara Y Suzuki A Murakami S Osada S Ohno K Hirosawa T Kuroki 《Molecular and cellular biology》1994,14(6):3782-3790
The eta isoform of protein kinase C, isolated from a cDNA library of mouse skin, has unique tissue and cellular distributions. It is predominantly expressed in epithelia of the skin, digestive tract, and respiratory tract in close association with epithelial differentiation. We report here that this isoform is localized on the rough endoplasmic reticulum in transiently expressing COS1 cells and constitutively expressing keratinocytes. By the use of polyclonal antibodies raised against peptides of the diverse D1 and D2/D3 regions, we found that immunofluorescent signals were strongest in the cytoplasm around the nucleus and became weaker toward the peripheral cytoplasm. Under immunoelectron microscopic examination, electron-dense signals were located on the rough endoplasmic reticulum and on the outer nuclear membrane which is continuous with the endoplasmic reticulum membrane. However, no signals were detected in the nucleus, inner nuclear membrane, smooth endoplasmic reticulum, Golgi apparatus, mitochondria, or plasma membrane. Treatment of the cells in situ with detergents suggested association of the isoform of protein kinase C with intracellular structures. By immunoblotting, a distinct single band with an M(r) of 80,000 was detected in whole-cell lysate and in rough microsomal and crude nuclear fractions, all of which contain outer nuclear membrane and/or rough endoplasmic reticulum. We further demonstrated the absence of a nuclear localization signal in the pseudosubstrate sequence. The present observation is not consistent with the report of Greif et al. (H. Greif, J. Ben-Chaim, T. Shimon, E. Bechor, H. Eldar, and E. Livneh, Mol. Cell. Biol. 12:1304-1311, 1992). 相似文献
2.
Role of the nuclear envelope in synthesis, processing, and transport of membrane glycoproteins 总被引:7,自引:0,他引:7
The outer nuclear membrane is morphologically similar to rough endoplasmic reticulum. The presence of ribosomes bound to its cytoplasmic surface suggests that it could be a site of synthesis of membrane glycoproteins. We have examined the biogenesis of the vesicular stomatitis virus G protein in the nuclear envelope as a model for the biogenesis of membrane glycoproteins. G protein was present in nuclear membranes of infected Friend erythroleukemia cells immediately following synthesis and was transported out of nuclear membranes to cytoplasmic membranes with a time course similar to transport from rough endoplasmic reticulum (t 1/2 = 5-7 min). Temperature-sensitive mutations in viral membrane proteins which block transport of G protein from endoplasmic reticulum also blocked transport of G protein from the nuclear envelope. Friend erythroleukemia cells and NIH 3T3 cells differed in the fraction of newly synthesized G protein found in nuclear membranes, apparently reflecting the relative amount of nuclear membrane compared to endoplasmic reticulum available for glycoprotein synthesis. Nuclear membranes from erythroleukemia cells appeared to have the enzymatic activities necessary for cleavage of the signal sequence and core glycosylation of newly synthesized G protein. Signal peptidase activity was detected by the ability of detergent-solubilized membranes of isolated nuclei to correctly remove the signal sequence of human preplacental lactogen. RNA isolated from the nuclear envelope was highly enriched for G protein mRNA, suggesting that G protein was synthesized on the outer nuclear membrane rather than redistributing to nuclear membranes from endoplasmic reticulum before or during cell fractionation. These results suggest a mechanism for incorporation of membrane glycoproteins into the nuclear envelope and suggest that in some cell types the nuclear envelope is a major source of newly synthesized membrane glycoproteins. 相似文献
3.
Here we report a novel small protein that is highly conserved across vertebrates. The protein, which we have named TRIQK, has no homology to any previously reported proteins or functional domains, but all vertebrate homologs of this protein share a characteristic triple repeat of the sequence QXXK/R, as well as a hydrophobic C-terminal region. The Xenopus triqk gene (xTriqk) was isolated in an expression screen on the basis of its ability to cause dramatic changes in cell size and nuclear size and morphology in developing embryos. The Xenopus and mouse triqk genes are broadly expressed throughout embryogenesis, and mtriqk is also generally expressed in mouse adult tissues. TRIQK proteins are localized to the endoplasmic reticulum membrane. Depletion of endogenous xTRIQK protein in Xenopus embryos causes no detectable morphological or functional changes in tadpoles. 相似文献
4.
Glycosylation site binding protein, a component of oligosaccharyl transferase, is highly similar to three other 57 kd luminal proteins of the ER 总被引:28,自引:0,他引:28
A 57 kd component of oligosaccharyl transferase, termed glycosylation site binding protein, specifically recognizes a photoaffinity probe containing the N-glycosylation site sequence Asn-Lys-Thr. It is present in the lumen of the ER (endoplasmic reticulum) and its release from this compartment results in a loss of N-glycosylation. Antibodies against this protein were used to identify cDNA clones from a lambda gt11 expression library. Analysis of its cDNA sequence reveals high sequence similarity to three other 57 kd luminal endoplasmic reticulum proteins: protein disulfide isomerase, the beta-subunit of prolyl hydroxylase, and thyroid hormone binding protein. This finding suggests that the capacity to recognize multiple polypeptide domains may reside in a single luminal protein that participates in co- and/or posttranslational modifications of newly synthesized proteins. 相似文献
5.
Haan GJ Baerends RJ Krikken AM Otzen M Veenhuis M van der Klei IJ 《FEMS yeast research》2006,6(2):186-194
The reassembly of peroxisomes in Hansenula polymorpha pex3 cells on reintroduction of Pex3p was examined. Using a Pex3-green fluorescent protein (Pex3-GFP) fusion protein, expressed under the control of an inducible promoter, it was observed that, initially on induction of Pex3-GFP synthesis, GFP fluorescence was localized to the endoplasmic reticulum and the nuclear envelope. Subsequently, a single organelle developed per cell that increased in size and multiplied by division. At these stages, GFP fluorescence was confined to peroxisomes. Fractionation experiments on homogenates of pex3 cells, in which the endoplasmic reticulum and nuclear envelope were marked with GFP, identified a small amount of GFP in peroxisomes present in the initial stage of peroxisome reassembly. Our data suggest a crucial role for the endoplasmic reticulum/nuclear envelope in peroxisome reintroduction on complementation of pex3 cells by the PEX3 gene. 相似文献
6.
We have investigated the behavior in in vivo cell fusion experiments of a transitional compartment lying between the endoplasmic reticulum and Golgi apparatus to determine if the compartment, as recognized by the antibody G1/93, might congregate in a similar manner to Golgi apparatus [W. C. Ho et al. (1990) Eur. J. Cell Biol. 52, 315-327]. The distributions of the transitional tubulovesicular compartment, endoplasmic reticulum, and Golgi apparatus in HeLa cells were assessed by immunofluorescent staining using mouse monoclonal antibody G1/93, mouse monoclonal antibody HP 24, and rabbit anti-galactosyltransferase, respectively. In agreement with previous results [W. C. Ho et al. (1990) Eur. J. Cell Biol. 52, 315-327], the Golgi apparatus was observed to congregate gradually over a 3- to 6-h period, forming a large, extended, central Golgi complex in uv-inactivated Sindbis virus-fused HeLa cells. Concomitant with this was a marked congregation of the transitional tubulovesicular compartment. Congregation of the tubulovesicular compartment was not affected by cycloheximide. The endoplasmic reticulum retained its web-like distribution throughout the syncytoplasm and rimmed the nuclear periphery. Treatment of HeLa cells with nocodazole prior to fusion followed by incubation of the syncytia in drug-containing media blocked congregation of the G1/93-positive compartment. With this long-term nocodazole treatment, Golgi apparatus was dispersed into scattered Golgi elements and the G1/93 distribution was endoplasmic reticulum-like. These results suggest that the transitional tubulovesicular compartment recognized by G1/93 is normally structured on microtubules and microtubule organizing centers and may be considered to be a subcompartment of a greater, perinuclear, Golgi complex. 相似文献
7.
High c-myc levels are linked to poor prognosis in medulloblastoma (MB), and it was the aim of the current study to search for c-myc-dependent proteins in the MB cell line D425Med. For this purpose D425Med cells and cells with knocked-down c-myc (by siRNA) were analysed by a gel-based differential proteomics study using mass spectrometry. Heterogeneous nuclear ribonucleoproteins C1/C2, heterogeneous nuclear ribonucleoprotein A/B, stathmin, endoplasmic reticulum protein ERp29 precursor and guanidinoacetate N-methyltransferase were c-myc dependently expressed. Signalling, the protein machinery, metabolism and endoplasmic reticulum function may be affected and these results enable studying tumour tissue for these proteins as potential dignity markers or pharmacological targets. 相似文献
8.
Riikka Jokinen Taina Lahtinen Paula Marttinen Maarit My?h?nen Pilvi Ruotsalainen Nicolas Yeung Antonina Shvetsova Alexander J. Kastaniotis J. Kalervo Hiltunen Tiina ?hman Tuula A. Nyman Hartmut Weiler Brendan J. Battersby 《Genetics》2015,200(1):221-235
Mammalian mitochondrial DNA (mtDNA) is a high-copy maternally inherited genome essential for aerobic energy metabolism. Mutations in mtDNA can lead to heteroplasmy, the co-occurence of two different mtDNA variants in the same cell, which can segregate in a tissue-specific manner affecting the onset and severity of mitochondrial dysfunction. To investigate mechanisms regulating mtDNA segregation we use a heteroplasmic mouse model with two polymorphic neutral mtDNA haplotypes (NZB and BALB) that displays tissue-specific and age-dependent selection for mtDNA haplotypes. In the hematopoietic compartment there is selection for the BALB mtDNA haplotype, a phenotype that can be modified by allelic variants of Gimap3. Gimap3 is a tail-anchored member of the GTPase of the immunity-associated protein (Gimap) family of protein scaffolds important for leukocyte development and survival. Here we show how the expression of two murine Gimap3 alleles from Mus musculus domesticus and M. m. castaneus differentially affect mtDNA segregation. The castaneus allele has incorporated a uORF (upstream open reading frame) in-frame with the Gimap3 mRNA that impairs translation and imparts a negative effect on the steady-state protein abundance. We found that quantitative changes in the expression of Gimap3 and the paralogue Gimap5, which encodes a lysosomal protein, affect mtDNA segregation in the mouse hematopoietic tissues. We also show that Gimap3 localizes to the endoplasmic reticulum and not mitochondria as previously reported. Collectively these data show that the abundance of protein scaffolds on the endoplasmic reticulum and lysosomes are important to the segregation of the mitochondrial genome in the mouse hematopoietic compartment. 相似文献
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Vladimir M. Korkhov 《Journal of cellular biochemistry》2009,107(1):86-95
Disruption of autophagy leads to accumulation of intracellular multilamellar inclusions morphologically similar to organised smooth endoplasmic reticulum (OSER) membranes. However, the relation of these membranous compartments to autophagy is unknown. The purpose of this study was to test whether OSER plays a role in the autophagic protein degradation pathway. Here, GFP‐LC3 is shown to localise to the OSER membranes induced by calnexin expression both in transiently transfected HEK293 cells and in mouse embryo fibroblasts. In contrast to GFP‐LC3, endogenous LC3 is excluded from these membranes under normal conditions as well as after cell starvation. Furthermore, YFP‐Atg5, a protein essential for autophagy and known to reside on autophagic membranes, is excluded from the calnexin‐positive inclusion structures. In cells devoid of Atg5, a protein essential for autophagy and known to reside on autophagic membranes, colocalisation of calnexin with GFP‐LC3 within the multilamellar bodies is preserved. I show that calnexin, a protein enriched in the OSER, is not subject to autophagic or lysosomal degradation. Finally, GFP‐LC3 targeting to these membranes is independent of its processing and insensitive to drugs modulating autophagic and lysosomal protein degradation. These observations are inconsistent with a role of autophagic/lysosomal degradation in clearance of multilamellar bodies comprising OSER. Furthermore, GFP‐LC3, a fusion protein widely used as a marker for autophagic vesicles and pre‐autophagic compartments, may be trapped in this compartment and this artefact must be taken into account if the construct is used to visualise autophagic membranes. J. Cell. Biochem. 107: 86–95, 2009. © 2009 Wiley‐Liss, Inc. 相似文献
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13.
The regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase requires a short-lived protein and occurs in the endoplasmic reticulum 总被引:12,自引:0,他引:12
A chimeric gene consisting of the coding sequence for the membrane domain of the endoplasmic reticulum protein, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, fused to the coding sequence for the soluble enzyme, beta-galactosidase of Escherichia coli, has been previously constructed. This fusion protein, HMGal, has been localized to the membrane of the endoplasmic reticulum of Chinese hamster ovary cells transfected with this chimeric gene, and its beta-galactosidase activity has declined in the presence of low density lipoprotein (Skalnik, D. G., Narita, H., Kent, C., and Simoni, R. D. (1988) J. Biol. Chem. 263, 6836-6841). In this report, we demonstrate that the loss of beta-galactosidase activity results from the accelerated degradation of the HMGal protein. Taking advantage of a fluorescence-activated cell sorter technique, we have selected transfected cells which express sufficient levels of HMGal to improve its immunodetection. Based on pulse-chase experiments, the half-life of HMGal is 6.0 h, and, in the presence of 20 mM mevalonate, the half-life declines 1.7-fold. Under these conditions, mevalonate accelerates the degradation of HMG-CoA reductase in these cells 1.6-fold, from 8.4 h to 5.3 h, most probably by the same mechanism. This mevalonate-regulated degradation of HMGal is not due to a heteromeric association of HMGal with reductase, since the same effect has been observed in cells lacking the reductase protein. In addition, we demonstrate that inhibition of protein synthesis with cycloheximide abolishes the mevalonate-dependent accelerated degradation of HMGal, in agreement with previous studies which have presented indirect evidence that a short-lived protein is essential for mediating the loss of HMG-CoA reductase activity. Finally, using brefeldin A, we show that the mevalonate-dependent accelerated degradation of HMGal may occur in the endoplasmic reticulum. 相似文献
14.
The cytochemical localization of endogenous peroxidase activity in sinus lining cells of mouse liver has been investigated. Kupffer cells, as identified by their exclusive ability to phagocytize large (0.8 micron) latex particles, exhibited strong peroxidase activity in nuclear envelope and endoplasmic reticulum. In addition, weak to moderate peroxidase activity was found in 57% of all endothelial cells. The enzyme in endothelial cells was also localized in nuclear envelope and endoplasmic reticulum, with a negative reaction in the Golgi apparatus. These observations indicate that peroxidase staining, as a marker for identification of Kupffer cells in mouse liver, is only of limited value and should be used in conjunction with other methods (e.g., latex phagocytosis). 相似文献
15.
Calreticulin: not just another calcium-binding protein 总被引:15,自引:0,他引:15
In this paper we review some of the rapidly expanding information about calreticulin, a Ca2+-binding/storage protein of the endoplasmic reticulum. The emphasis is placed on the structure and function of calreticulin. We believe that calreticulin is a multifunctional Ca2+-binding protein and that distinct functional properties of the protein may be localized to each of the three structural domains of calreticulin. Most evidence indicates that calreticulin is a resident endoplasmic reticulum protein. However, it can also be found outside of the endoplasmic reticulum compartment, i.e. in the nuclear envelope, in the nucleus, in the cytotoxic granules in T-lymphocytes and in acrosomal vesicles of sperm cells. The evidence reviewed here clearly suggests that calreticulin has other functions in addition to its role as a Ca2+ storage protein in the endoplasmic reticulum.Abbreviations SR
sarcoplasmic reticulum
- ER
endoplasmic reticulum 相似文献
16.
《The Journal of cell biology》1996,133(2):257-268
We have identified and characterized Cab45, a novel 45-kD protein from mouse 3T3-L1 adipocytes. Cab45 is ubiquitously expressed, contains an NH2-terminal signal sequence but no membrane-anchor sequences, and binds Ca2+ due to the presence of six EF-hand motifs. Within the superfamily of calcium-binding proteins, it belongs to a recently identified group of proteins consisting of Reticulocalbin (Ozawa, M., and T. Muramatsu. 1993. J. Biol. Chem. 268:699-705) and ERC 55 (Weis, K., G. Griffiths, and A.I. Lamond. 1994. J. Biol Chem. 269:19142- 19150), both of which share significant sequence homology with Cab45 outside the EF-hand motifs. In contrast to reticulocalbin and ERC-55 which are soluble components of the endoplasmic reticulum, Cab45 is a soluble protein localized to the Golgi. Cab45 is the first calcium- binding protein localized to the lumenal portion of a post-ER compartment; Cab45 is also the first known soluble protein resident in the Golgi lumen. Cab45 can serve as a model protein to determine the mechanism of retention of soluble proteins in the Golgi compartment. 相似文献
17.
The cytochrome P450 2C1 N-terminal signal anchor sequence mediates direct retention of the protein in the endoplasmic reticulum and consists of a hydrophobic transmembrane domain, residues 3-20, followed by a hydrophilic linker, residues 21-28. Fusions of the N-terminal 21 or 28 amino acids of P450 2C1 to green fluorescent protein resulted in endoplasmic reticulum localization of the chimera in transfected cells. Disruption of microtubules by nocodazole treatment resulted in redistribution into a punctate pattern for the 1-21, but not for the 1-28, chimera indicating that the linker was preventing transport from the endoplasmic reticulum but was not required for retrieval to the endoplasmic reticulum from the pre-Golgi compartment. In the 1-28 chimera, mutations of residues 21-23 (KQS) in the linker resulted in redistribution of the chimera after nocodazole treatment. Mutations in the transmembrane domain affected both direct retention in the endoplasmic reticulum and retrieval from the pre-Golgi compartment, and although structural requirements for each process are distinct, in both cases the arrangement of amino acids and distribution of hydrophobicity are critical. In contrast, the linker region exhibits a sequence-specific requirement for direct retention in the endoplasmic reticulum. 相似文献
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There is an increasing need for tissue-specific gene expression regulatory elements to study normal and disease development in the mouse. However, the cloning and characterization of these elements are time-consuming and costly. Thus, there is a particular need to be able to identify gene expression patterns without having to clone the promoter elements. Gene-trap strategies identify expression patterns assigned for endogenous genes using reporters, such as LacZ (Gossler et al., 1989; Skarnes, 1990) or green fluorescent protein (GFP) (Ishida and Leder, 1999; Zheng and Hughes, 1999). The gene-trap vector randomly inserts into the genome and "steals" regulatory elements for the reporter. Here we describe an improved gene-trap strategy, which allows an efficient Cre recombinase-mediated insertion of any transgene into the trapped loci as a post-integrational modification and links the expression of the transgene to that of the reporter. 相似文献
20.
Intracytoplasmic vacuoles were produced within keratinocytes of the epidermis by hypertonic solutions of sodium chloride, dextrose, and human albumin injected into the dermis of guinea pigs. They arose from a canalicular system which unfolded at higher osmolarities; the degree of unfolding was related to the degree of hyperosmolarity chosen. Tracers (horseradish peroxidase and colloidal silver) incorporated into the test solutions freely permeated the cisternal system and the vacuoles, demonstrating their continuity with the intercellular space. The membranes lining this cisternal system were not stained by ruthenium red and exhibited no nucleosidetriphosphatase activity which indicated that they did not represent infoldings of the plasma membrane; they were impregnated by the osmium soaking technique (which stains endoplasmic reticulum) and their dimensions were identical with those of the outer nuclear membrane and endoplasmic reticulum. It is concluded that a continuity exists between endoplasmic reticulum of keratinocytes and extracellular compartment which unfolds under hyperosmolar conditions. Since endoplasmic reticulum and perinuclear space are continuous the continuity of plasma membrane and endoplasmic reticulum, as demonstrated in this paper, suggest the existence of a potential path for the exchange of substances and information between the nuclear membrane and the extracellular compartment. 相似文献