scholarly journals Molecular cloning, characterization, subcellular localization and dynamics of p23, the mammalian KDEL receptor.

1993 ◽  
Vol 120 (2) ◽  
pp. 325-338 ◽  
Author(s):  
B L Tang ◽  
S H Wong ◽  
X L Qi ◽  
S H Low ◽  
W Hong
Keyword(s):  
Golgi Apparatus ◽  
Brefeldin A ◽  
Cell Types ◽  
In Vitro Translation ◽  
Tubular Structures ◽  
Human Sequence ◽  
Intermediate Compartment ◽  
Membrane Spanning ◽  
Membrane Spanning Domains

We have isolated a cDNA clone (mERD2) for the mammalian (bovine) homologue of the yeast ERD2 gene, which codes for the yeast HDEL receptor. The deduced amino acid sequence bears extensive homology to its yeast counterpart and is almost identical to a previously described human sequence. The sequence predicts a very hydrophobic protein with multiple membrane spanning domains, as confirmed by analysis of the in vitro translation product. The protein encoded by mERD2 (p23) has widespread occurrence, being present in all the cell types examined. p23 was localized to the cis-side of the Golgi apparatus and to a spotty intermediate compartment which mediates ER to Golgi transport. A majority of the intracellular staining could be accumulated in the intermediate compartment by a low temperature (15 degrees C) or brefeldin A. During recovery from these treatments, the spotty intermediate compartment staining of p23 was shifted to the perinuclear staining of the Golgi apparatus and tubular structures marked by p23 were observed. These tubular structures may serve to mediate transport between the intermediate compartment and the Golgi apparatus.

Na+/H+Exchanger NHE3 Has 11 Membrane Spanning Domains and a Cleaved Signal Peptide:  Topology Analysis Using In Vitro Transcription/Translation†

Biochemistry ◽  
2000 ◽  
Vol 39 (27) ◽  
pp. 8102-8112 ◽  
Author(s):  
Mirza Zizak ◽  
Megan E. Cavet ◽  
Denis Bayle ◽  
Chung-Ming Tse ◽  
Stefan Hallen ◽  
...  
Keyword(s):  
Signal Peptide ◽  
In Vitro Transcription ◽  
Topology Analysis ◽  
Membrane Spanning ◽  
Membrane Spanning Domains

scholarly journals Dihydroceramide:sphinganine C-4-hydroxylation requires Des2 hydroxylase and the membrane form of cytochrome b5

2006 ◽  
Vol 397 (2) ◽  
pp. 289-295 ◽  
Author(s):  
Ayako Enomoto ◽  
Fumio Omae ◽  
Masao Miyazaki ◽  
Yasunori Kozutsumi ◽  
Toshitsugu Yubisui ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Erythrocyte Membrane ◽  
Cytochrome B5 ◽  
Soluble Form ◽  
Affinity Column ◽  
Vitro Assay ◽  
Membrane Spanning ◽  
Triton X ◽  
Membrane Spanning Domains

Des2 (degenerative spermatocyte 2) is a bifunctional enzyme that produces phytoceramide and ceramide from dihydroceramide. The molecular mechanism involved in C-4-hydroxylation has not been studied in detail. In the present paper, we report that C-4-hydroxylation requires an electron-transfer system that includes cytochrome b5 and that the hydroxylase activity is reconstituted in an in vitro assay with purified recombinant Des2. FLAG-tagged mouse Des2 was expressed in insect Sf9 cells and was purified by solubilization with digitonin and anti-FLAG antibody affinity column chromatography. The activity of dihydroceramide:sphinganine C-4-hydroxylase was reconstituted with the purified FLAG–Des2, mb5 (the membrane form of cytochrome b5) and bovine erythrocyte membrane. The apparent Km and Vmax of Des2 for the substrate N-octanoylsphinganine were 35 μM and 40 nmol·h−1·mg of protein−1 respectively. The Km of the hydroxylase for mb5 was 0.8 μM. Interestingly, mb5 was not replaced with the soluble form of cytochrome b5, which lacks the C-terminal membrane-spanning domain. The erythrocyte membrane was separated into Triton X-100-soluble and -insoluble fractions, and the detergent-soluble fraction was replaced by the soluble or membrane form of b5R (NADH-cytochrome b5 reductase). The Triton-X-100-insoluble fraction contained trypsin-resistant factors. The Des2 protein is found in the endoplasmic reticulum and is assumed to have three membrane-spanning domains. The findings of the present study indicate that the hydroxylation requires complex formation between Des2 and mb5 via their membrane-spanning domains and electron transfer from NADH to the substrate via the reduction of mb5 by b5R.

scholarly journals The Golgi apparatus remains associated with microtubule organizing centers during myogenesis.

1985 ◽  
Vol 101 (2) ◽  
pp. 630-638 ◽  
Author(s):  
A M Tassin ◽  
M Paintrand ◽  
E G Berger ◽  
M Bornens
Keyword(s):  
Golgi Apparatus ◽  
Nuclear Periphery ◽  
Spatial Relationship ◽  
Cell Types ◽  
Microtubule Depolymerization ◽  
Animal Cells ◽  
Microtubule Organizing Center ◽  
Microtubule Disruption ◽  
Tight Association

In vitro myogenesis involves a dramatic reorganization of the microtubular network, characterized principally by the relocalization of microtubule nucleating sites at the surface of the nuclei in myotubes, in marked contrast with the classical pericentriolar localization observed in myoblasts (Tassin, A. M., B. Maro, and M. Bornens, 1985, J. Cell Biol., 100:35-46). Since a spatial relationship between the Golgi apparatus and the centrosome is observed in most animal cells, we have decided to follow the fate of the Golgi apparatus during myogenesis by an immunocytochemical approach, using wheat germ agglutinin and an affinity-purified anti-galactosyltransferase. We show that Golgi apparatus in myotubes displays a perinuclear distribution which is strikingly different from the polarized juxtanuclear organization observed in myoblasts. As a result, the Golgi apparatus in myotubes is situated close to the microtubule organizing center (MTOC), the cis-side being situated at a fixed distance from the nuclear envelope, a situation which suggests the existence of a structural association between the Golgi apparatus and the nuclear periphery. This is supported by experiments of microtubule depolymerization by nocodazole, in which a minimal effect was observed on Golgi apparatus localization in myotubes in contrast with the dramatic scattering observed in myoblasts. In both cell types, electron microscopy reveals that microtubule disruption generates individual dictyosomes; this suggests that the connecting structures between dictyosomes are principally affected. This structural dependency of the Golgi apparatus upon microtubules is not apparently accompanied by a reverse dependency of MTOC structure or function upon Golgi apparatus activity. Golgi apparatus modification by monensin, as effective in myotubes as in myoblasts, is without apparent effect on MTOC localization or activity and on microtubule stability. The main result of our study is to show that in a cell type where the MTOC is dissociated from centrioles and where antero-posterior polarity has disappeared, the association between the Golgi apparatus and the MTOC is maintained. The significance of such a tight association is discussed.

scholarly journals Morphological and Functional Association of Sec22b/ERS-24 with the pre-Golgi Intermediate Compartment

1999 ◽  
Vol 10 (2) ◽  
pp. 435-453 ◽  
Author(s):  
Tao Zhang ◽  
Siew Heng Wong ◽  
Bor Luen Tang ◽  
Yue Xu ◽  
Wanjin Hong
Keyword(s):  
Golgi Apparatus ◽  
Mammalian Cells ◽  
Intact Cell ◽  
Brefeldin A ◽  
Cell System ◽  
Fusion Event ◽  
Homologous Proteins ◽  
Golgi Transport ◽  
Protein Receptor ◽  
Intermediate Compartment

Yeast Sec22p participates in both anterograde and retrograde vesicular transport between the endoplasmic reticulum (ER) and the Golgi apparatus by functioning as a v-SNARE (solubleN-ethylmaleimide-sensitive factor [NSF] attachment protein receptor) of transport vesicles. Three mammalian proteins homologous to Sec22p have been identified and are referred to as Sec22a, Sec22b/ERS-24, and Sec22c, respectively. The existence of three homologous proteins in mammalian cells calls for detailed cell biological and functional examinations of each individual protein. The epitope-tagged forms of all three proteins have been shown to be primarily associated with the ER, although functional examination has not been carefully performed for any one of them. In this study, using antibodies specific for Sec22b/ERS-24, it is revealed that endogenous Sec22b/ERS-24 is associated with vesicular structures in both the perinuclear Golgi and peripheral regions. Colabeling experiments for Sec22b/ERS-24 with Golgi mannosidase II, the KDEL receptor, and the envelope glycoprotein G (VSVG) of vesicular stomatitis virus (VSV) en route from the ER to the Golgi under normal, brefeldin A, or nocodazole-treated cells suggest that Sec22b/ERS-24 is enriched in the pre-Golgi intermediate compartment (IC). In a well-established semi-intact cell system that reconstitutes transport from the ER to the Golgi, transport of VSVG is inhibited by antibodies against Sec22b/ERS-24. EGTA is known to inhibit ER–Golgi transport at a stage after vesicle/transport intermediate docking but before the actual fusion event. Antibodies against Sec22b/ERS-24 inhibit ER–Golgi transport only when they are added before the EGTA-sensitive stage. Transport of VSVG accumulated in pre-Golgi IC by incubation at 15°C is also inhibited by Sec22b/ERS-24 antibodies. Morphologically, VSVG is transported from the ER to the Golgi apparatus via vesicular intermediates that scatter in the peripheral as well as the Golgi regions. In the presence of antibodies against Sec22b/ERS-24, VSVG is seen to accumulate in these intermediates, suggesting that Sec22b/ERS-24 functions at the level of the IC in ER–Golgi transport.

scholarly journals Expression of a dominant allele of human ARF1 inhibits membrane traffic in vivo

1994 ◽  
Vol 124 (3) ◽  
pp. 289-300 ◽  
Author(s):  
CJ Zhang ◽  
AG Rosenwald ◽  
MC Willingham ◽  
S Skuntz ◽  
J Clark ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Rat Kidney ◽  
Brefeldin A ◽  
Membrane Traffic ◽  
Immunoblot Analysis ◽  
In Vitro Assays ◽  
Wild Type ◽  
Discernible Effect

ADP-ribosylation factor (ARF) proteins and inhibitory peptides derived from ARFs have demonstrated activities in a number of in vitro assays that measure ER-to-Golgi and intra-Golgi transport and endosome fusion. To better understand the roles of ARF proteins in vivo, stable cell lines were obtained from normal rat kidney (NRK) cells transfected with either wild-type or a dominant activating allele ([Q71L]) of the human ARF1 gene under the control of the interferon-inducible mouse Mx1 promoter. Upon addition of interferon, expression of ARF1 proteins increased with a half-time of 7-8 h, as determined by immunoblot analysis. Induction of mutant ARF1, but not wild-type ARF1, led to an inhibition of protein secretion with kinetics similar to that observed for induction of protein expression. Examination of the Golgi apparatus and the ER by indirect immunofluorescence or transmission electron microscopy revealed that expression of low levels of mutant ARF1 protein correlated with a dramatic increase in vesiculation of the Golgi apparatus and expansion of the ER lumen, while expression of substantially higher levels of wild-type ARF1 had no discernible effect. Endocytosis was also inhibited by expression of mutant ARF1, but not by the wild-type protein. Finally, the expression of [Q71L]ARF1, but not wild-type ARF1, antagonized the actions of brefeldin A, as determined by the delayed loss of ARF and beta-COP from Golgi membranes and disruption of the Golgi apparatus. General models for the actions of ARF1 in membrane traffic events are discussed.

scholarly journals cis-Golgi proteins accumulate near the ER exit sites and act as the scaffold for Golgi regeneration after brefeldin A treatment in tobacco BY-2 cells

2012 ◽  
Vol 23 (16) ◽  
pp. 3203-3214 ◽  
Author(s):  
Yoko Ito ◽  
Tomohiro Uemura ◽  
Keiko Shoda ◽  
Masaru Fujimoto ◽  
Takashi Ueda ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Fluorescent Proteins ◽  
Plant Cells ◽  
Brefeldin A ◽  
Eukaryotic Cells ◽  
Er Exit Sites ◽  
Intermediate Compartment ◽  
Golgi Protein ◽  
And Function

The Golgi apparatus forms stacks of cisternae in many eukaryotic cells. However, little is known about how such a stacked structure is formed and maintained. To address this question, plant cells provide a system suitable for live-imaging approaches because individual Golgi stacks are well separated in the cytoplasm. We established tobacco BY-2 cell lines expressing multiple Golgi markers tagged by different fluorescent proteins and observed their responses to brefeldin A (BFA) treatment and BFA removal. BFA treatment disrupted cis, medial, and trans cisternae but caused distinct relocalization patterns depending on the proteins examined. Medial- and trans-Golgi proteins, as well as one cis-Golgi protein, were absorbed into the endoplasmic reticulum (ER), but two other cis-Golgi proteins formed small punctate structures. After BFA removal, these puncta coalesced first, and then the Golgi stacks regenerated from them in the cis-to-trans order. We suggest that these structures have a property similar to the ER-Golgi intermediate compartment and function as the scaffold of Golgi regeneration.

scholarly journals Cloning of human Ca2+ homoeostasis endoplasmic reticulum protein (CHERP): regulated expression of antisense cDNA depletes CHERP, inhibits intracellular Ca2+ mobilization and decreases cell proliferation

2000 ◽  
Vol 348 (1) ◽  
pp. 189-199 ◽  
Author(s):  
Janice M. LAPLANTE ◽  
Flavia O'ROURKE ◽  
Xinghua LU ◽  
Alan FEIN ◽  
Anne OLSEN ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Types ◽  
In Vitro Translation ◽  
Cdna Expression Library ◽  
Expression Library ◽  
Regulated Expression ◽  
Endoplasmic Reticulum Protein ◽  
Hel Cells ◽  
Expressed Sequence

A monoclonal antibody which blocks InsP3-induced Ca2+ release from isolated endoplasmic reticulum was used to isolate a novel 4.0 kb cDNA from a human erythroleukaemia (HEL) cell cDNA expression library. A corresponding mRNA transcript of approx. 4.2 kb was present in all human cell lines and tissues examined, but cardiac and skeletal muscle had an additional transcript of 6.4 kb. The identification in GenBank® of homologous expressed sequence tags from many tissues and organisms suggests that the gene is ubiquitously expressed in higher eukaryotes. The gene was mapped to human chromosome 19p13.1. The cDNA predicts a 100 kDa protein, designated Ca2+ homoeostasis endoplasmic reticulum protein (CHERP), with two putative transmembrane domains, multiple consensus phosphorylation sites, a polyglutamine tract of 12 repeats and regions of imperfect tryptophan and histadine octa- and nona-peptide repeats. In vitro translation of the full-length cDNA produced proteins of Mr 128000 and 100000, corresponding to protein bands detected by Western blotting of many cell types. CHERP was co-localized in HEL cells with the InsP3 receptor by two-colour immunofluorescence. Transfection of HEL cells with antisense cDNA led to an 80% decline in CHERP within 5 days of antisense induction, with markedly decreased intracellular Ca2+ mobilization by thrombin, decreased DNA synthesis and growth arrest, indicating that the protein has an important function in Ca2+ homoeostasis, growth and proliferation.

NSP-encoded reticulons, neuroendocrine proteins of a novel gene family associated with membranes of the endoplasmic reticulum

1994 ◽  
Vol 107 (9) ◽  
pp. 2403-2416 ◽  
Author(s):  
H.J. van de Velde ◽  
A.J. Roebroek ◽  
N.H. Senden ◽  
F.C. Ramaekers ◽  
W.J. Van de Ven
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Gene Family ◽  
Biochemical Characterization ◽  
Smooth Endoplasmic Reticulum ◽  
Dendritic Tree ◽  
Cell Types ◽  
In Vitro Translation ◽  
Amino Terminal

The novel NSP gene was previously shown to encode, among a variety of neuroendocrine cell types, two 3′-overlapping transcripts, a 3.4 kb one for NSP-A (776 amino acids) and a 1.8 kb one for NSP-C (208 amino acids). The deduced proteins, which were predicted to possess distinct amino-terminal regions, appeared to exhibit some architectural resemblance to known neuroendocrine proteins. In this paper the biochemical characterization and subcellular localization of the two proteins is addressed. In vitro translation of NSP-A and -C RNA produced proteins of about 135 and 23 kDa, respectively. Proteins of similar molecular mass were also detected in immunoprecipitation and western blot analyses of neural and endocrine cells using specific anti-NSP-A or -C antisera; some heterogeneity of NSP-A was observed. NSP-A, but not NSP-C, appeared to be highly phosphorylated and preferentially on serine residues. In immunocytochemical studies, we demonstrated that NSP-A and -C are associated with the endoplasmic reticulum; NSP-A was found to co-localize with SERCA2b, a membrane-associated Ca(2+)-ATPase of the endoplasmic reticulum. In Purkinje cells, we found NSP-immunostaining in the perikaryon, the extensive dendritic tree and the axon, also suggesting association with the smooth endoplasmic reticulum. Biochemical studies of NSP-A provided evidence that NSP-A is strongly associated with microsomal membranes and analysis of deletion mutants of NSP-A revealed that the hydrophobic carboxy-terminal portion of the protein, which is also present in NSP-C, is critical for membrane binding. Through database searches, finally, we found two different NSP-related sequences, one in a sequenced region of human chromosome 19, and the second in a human, pancreatic islet-derived partial cDNA, suggesting that the NSP gene is the prototype of a larger gene family. The results of our studies seem to indicate that the NSP-encoded proteins are novel, membrane-anchored components of the endoplasmic reticulum for which we propose the name reticulons.

scholarly journals PtK1 cells contain a nondiffusible, dominant factor that makes the Golgi apparatus resistant to brefeldin A.

1991 ◽  
Vol 113 (5) ◽  
pp. 1009-1023 ◽  
Author(s):  
N T Ktistakis ◽  
M G Roth ◽  
G S Bloom
Keyword(s):  
Golgi Apparatus ◽  
G Protein ◽  
Golgi Complex ◽  
Brefeldin A ◽  
Cell Types ◽  
Live Cells ◽  
Dominant Factor ◽  
Glycoprotein G ◽  
Numerous Cell ◽  
Vesicular Stomatitis

Brefeldin A (BFA) was shown in earlier studies of numerous cell types to inhibit secretion, induce enzymes of the Golgi stacks to redistribute into the ER, and to cause the Golgi cisternae to disappear. Here, we demonstrate that the PtK1 line of rat kangaroo kidney cells is resistant to BFA. The drug did not disrupt the morphology of the Golgi complex in PtK1 cells, as judged by immunofluorescence using antibodies to 58- (58K) and 110-kD (beta-COP) Golgi proteins, and by fluorescence microscopy of live cells labeled with C6-NBD-ceramide. In addition, BFA did not inhibit protein secretion, not alter the kinetics or extent of glycosylation of the vesicular stomatitis virus (VSV) glycoprotein (G-protein) in VSV-infected PtK1 cells. To explore the mechanism of resistance to BFA, PtK1 cells were fused with BFA-sensitive CV-1 cells that had been infected with a recombinant SV-40 strain containing the gene for VSV G-protein and, at various times following fusion, the cultures were exposed to BFA. Shortly after cell fusion, heterokaryons contained one Golgi complex associated with each nucleus. Golgi membranes derived from CV-1 cells were sensitive to BFA, whereas those of PtK1 origin were BFA resistant. A few hours after fusion, most heterokaryons contained a single, large Golgi apparatus that was resistant to BFA and contained CV-1 galactosyltransferase. In unfused cells that had been perforated using nitrocellulose filters, retention of beta-COP on the Golgi was optimal in the presence of cytosol, ATP, and GTP. In perforated cell models of the BFA-sensitive MA104 line, BFA caused beta-COP to be released from the Golgi complex in the presence of nucleotides, and either MA104 or PtK1 cytosol. In contrast, when perforated PtK1 cells were incubated with BFA, nucleotides, and cytosol from either cell type, beta-COP remained bound to the Golgi complex. We conclude that PtK1 cells contain a nondiffusible factor, which is located on or very close to the Golgi complex, and confers a dominant resistance to BFA. It is possible that this factor is homologous to the target of BFA in cells that are sensitive to the drug.

In vitro translation in a hybrid cell free lysate with exogenous cellular ribosomes

2015 ◽  
Vol 467 (3) ◽  
pp. 387-398 ◽  
Author(s):  
Baptiste Panthu ◽  
Didier Décimo ◽  
Laurent Balvay ◽  
Théophile Ohlmann
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Hybrid System ◽  
Hybrid Cell ◽  
Cell Types ◽  
In Vitro Translation ◽  
Translation System ◽  
Different Origins ◽  
Cell Free Protein Synthesis

Cell free protein synthesis systems (CFPS) have been widely used to express proteins and to explore the pathways of gene expression. In the present manuscript, we describe the design of a novel adaptable hybrid in vitro translation system which is assembled with ribosomes isolated from many different origins. We first show that this hybrid system exhibits all important features such as efficiency, sensitivity, reproducibility and the ability to translate specialized mRNAs in less than 1 h. In addition, the unique design of this cell free assay makes it highly adaptable to utilize ribosomes isolated from many different organs, tissues or cell types.

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