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.

Characterization of a cDNA-clone encoding Nc-p43, a major Neospora caninum tachyzoite surface protein

Parasitology ◽  
1997 ◽  
Vol 115 (6) ◽  
pp. 581-590 ◽  
Author(s):  
A. HEMPHILL ◽  
R. FELLEISEN ◽  
B. CONNOLLY ◽  
B. GOTTSTEIN ◽  
B. HENTRICH ◽  
...  
Keyword(s):  
Host Cell ◽  
Neospora Caninum ◽  
Surface Protein ◽  
Cell Types ◽  
Cdna Expression Library ◽  
Expression Library ◽  
Cdna Insert ◽  
Veterinary Importance ◽  
Major Surface

Neospora caninum is an apicomplexan parasite of veterinary importance which invades many different cell types and tissues. N. caninum tachyzoites proliferate intracellularly by endodyogeny. Eventually the massive proliferation of tachyzoites leads to host cell lysis and the newly formed parasites are released and invade neighbouring cells. Tachyzoite cell surface molecules could serve as ligands, mediating host cell adhesion and invasion. Nc-p43 is a recently identified N. caninum tachyzoite surface protein which is functionally involved in the processes leading to host cell invasion in vitro. Affinity-purified antibodies directed against Nc-p43 were used to screen a lambda gt22A-cDNA expression library constructed from N. caninum tachyzoites. The cDNA insert of one immunoreactive clone was subcloned and expressed in E. coli as a poly-histidine fusion protein. The identity of the resulting recombinant antigen termed recNc-p43 was confirmed by immunoblotting, immunofluorescence and electron microscopy using affinity-purified antibodies. The sequence of the cDNA insert encoding recNc-p43 was determined. Analysis of the deduced amino acid sequence revealed that Nc-p43 exhibited similarity to SAG1 (p30) and SAG3 (p43), 2 major surface antigens of Toxoplasma gondii tachyzoites. These similarities were not reflected on the immunochemical level, since no cross-antigenicity between SAG1, SAG3 and Nc-p43 was observed.

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 Conversion of secretory proteins into membrane proteins by fusing with a glycosylphosphatidylinositol anchor signal of alkaline phosphatase

1994 ◽  
Vol 301 (2) ◽  
pp. 577-583 ◽  
Author(s):  
K Oda ◽  
J Cheng ◽  
T Saku ◽  
N Takami ◽  
M Sohda ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Endoplasmic Reticulum ◽  
Aspartic Acid ◽  
Chimeric Protein ◽  
Attachment Site ◽  
In Vitro Translation ◽  
Placental Alkaline Phosphatase ◽  
Wild Type ◽  
Aspartic Acid Residue

Placental alkaline phosphatase (PLAP) is initially synthesized as a precursor (proPLAP) with a C-terminal extension. We constructed a recombinant cDNA which encodes a chimeric protein (alpha GL-PLAP) comprising rat alpha 2u-globulin (alpha GL) and the C-terminal extension of PLAP. Two molecular species (25 kDa and 22 kDa) were expressed in the COS-1 cell transfected with the cDNA for alpha GL-PLAP. Only the 22 kDa form was labelled with both [3H]stearic acid and [3H]ethanolamine. Upon digestion with phosphatidylinositol-specific phospholipase C the 22 kDa form was released into the medium, indicating that this form is anchored on the cell surface via glycosylphosphatidylinositol (GPI). A specific IgG raised against a C-terminal nonapeptide of proPLAP precipitated the 25 kDa form but not the 22 kDa form, suggesting that the 25 kDa form is a precursor retaining the C-terminal propeptide. When a mutant alpha GL-PLAP, in which the aspartic acid residue is replaced with tryptophan at a putative cleavage/attachment site, was expressed in COS-1 cells, the 25 kDa precursor was the only form found inside the cell and retained in the endoplasmic reticulum, as judged by immunofluorescence microscopy. In vitro translation programmed with mRNAs coding for the wild-type and mutant forms of alpha GL-PLAP demonstrated that the C-terminal propeptide was cleaved from the wild-type chimeric protein, but not from the mutant one. This gave rise to the 22 kDa form attached with a GPI anchor, suggesting that GPI is covalently linked to the aspartic acid residue (Asp159) of alpha GL-PLAP. Taken together, these results indicate that the C-terminal propeptide of PLAP functions as a signal to render alpha GL a GPI-linked membrane protein in vitro and in vivo in cultured cells, and that the chimeric protein constructed in this study may be useful for elucidating the mechanism underlying the cleavage of the propeptide and attachment of GPI, which occur in the endoplasmic reticulum.

scholarly journals EDEM2 stably disulfide-bonded to TXNDC11 catalyzes the first mannose trimming step in mammalian glycoprotein ERAD

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Ginto George ◽  
Satoshi Ninagawa ◽  
Hirokazu Yagi ◽  
Taiki Saito ◽  
Tokiro Ishikawa ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Gene Knockout ◽  
Covalent Bonding ◽  
Cxxc Motif ◽  
Endoplasmic Reticulum Associated Degradation ◽  
Endoplasmic Reticulum Protein ◽  
Hct116 Cells

Sequential mannose trimming of N-glycan (Man9GlcNAc2 -> Man8GlcNAc2 -> Man7GlcNAc2) facilitates endoplasmic reticulum-associated degradation of misfolded glycoproteins (gpERAD). Our gene knockout experiments in human HCT116 cells have revealed that EDEM2 is required for the first step. However, it was previously shown that purified EDEM2 exhibited no α1,2-mannosidase activity toward Man9GlcNAc2 in vitro. Here, we found that EDEM2 was stably disulfide-bonded to TXNDC11, an endoplasmic reticulum protein containing five thioredoxin (Trx)-like domains. C558 present outside of the mannosidase homology domain of EDEM2 was linked to C692 in Trx5, which solely contains the CXXC motif in TXNDC11. This covalent bonding was essential for mannose trimming and subsequent gpERAD in HCT116 cells. Furthermore, EDEM2-TXNDC11 complex purified from transfected HCT116 cells converted Man9GlcNAc2 to Man8GlcNAc2(isomerB) in vitro. Our results establish the role of EDEM2 as an initiator of gpERAD, and represent the first clear demonstration of in vitro mannosidase activity of EDEM family proteins.

Characterization of the integral membrane polypeptides of rat liver peroxisomes isolated from untreated and clofibrate-treated rats

1991 ◽  
Vol 69 (8) ◽  
pp. 499-508 ◽  
Author(s):  
Andrea G. Bodnar ◽  
Richard A. Rachubinski
Keyword(s):  
Endoplasmic Reticulum ◽  
Immunoblot Analysis ◽  
In Vitro Translation ◽  
Peroxisome Proliferator ◽  
Peroxisomal Membrane ◽  
Molecular Masses ◽  
Fold Induction ◽  
Membrane Bound ◽  
Liver Peroxisomes

We have characterized the integral membrane polypeptides of liver peroxisomes from untreated rats and rats treated with clofibrate, a peroxisome proliferator. Membranes, prepared by treatment of purified peroxisomes with sodium carbonate, were used to raise an antiserum in rabbits. Immunoblot analysis demonstrated the reaction of this antiserum with six peroxisomal integral membrane polypeptides (molecular masses, 140, 69, 50, 36, 22, and 15 kDa). Treatment of rats with the hypolipidemic drug clofibrate caused a 4- to 10-fold induction in the 69-kDa integral membrane polypeptide, while the other integral membrane polypeptides remained unchanged or varied to a lesser extent. The anti-peroxisomal membrane serum reacted with two integral membrane polypeptides of the endoplasmic reticulum which co-migrated with the 50- and 36-kDa integral membrane polypeptides of the peroxisome. Biochemical and immunoblot analyses indicated that these integral membrane polypeptides were co-localized to peroxisomes and endoplasmic reticulum. Immunoprecipitation of in vitro translation products of RNA isolated from free and membrane-bound polysomes indicated that the 22-, 36-, and 69-kDa integral membrane polypeptides were synthesized on free polysomes, while the 50-kDa integral membrane polypeptide was predominantly synthesized on membrane-bound polysomes. The predominant synthesis of the 50-kDa integral membrane polypeptide on membrane-bound polysomes raises interesting possibilities concerning its biosynthesis.Key words: peroxisomes, integral membrane polypeptides, clofibrate, free polysomes, membrane-bound polysomes.

scholarly journals Biosynthesis and processing of ribophorins in the endoplasmic reticulum.

1984 ◽  
Vol 99 (3) ◽  
pp. 1076-1082 ◽  
Author(s):  
M G Rosenfeld ◽  
E E Marcantonio ◽  
J Hakimi ◽  
V M Ort ◽  
P H Atkinson ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Posttranslational Modifications ◽  
Rat Hepatocytes ◽  
Direct Analysis ◽  
In Vitro Translation ◽  
Endoplasmic Reticulum Membrane ◽  
Pituitary Cells ◽  
Amino Terminal

Ribophorins are two transmembrane glycoproteins characteristic of the rough endoplasmic reticulum, which are thought to be involved in the binding of ribosomes. Their biosynthesis was studied in vivo using lines of cultured rat hepatocytes (clone 9) and pituitary cells (GH 3.1) and in cell-free synthesis experiments. In vitro translation of mRNA extracted from free and bound polysomes of clone 9 cells demonstrated that ribophorins are made exclusively on bound polysomes. The primary translation products of ribophorin messengers obtained from cultured hepatocytes or from regenerating livers co-migrated with the respective mature proteins, but had slightly higher apparent molecular weights (2,000) than the unglycosylated forms immunoprecipitated from cells treated with tunicamycin. This indicates that ribophorins, in contrast to all other endoplasmic reticulum membrane proteins previously studied, contain transient amino-terminal insertion signals which are removed co-translationally. Kinetic and pulse-chase experiments with [35S]methionine and [3H]mannose demonstrated that ribophorins are not subjected to electrophoretically detectable posttranslational modifications, such as proteolytic cleavage or trimming and terminal glycosylation of oligosaccharide side chain(s). Direct analysis of the oligosaccharides of ribophorin l showed that they do not contain the terminal sugars characteristic of complex oligosaccharides and that they range in composition from Man8GlcNAc to Man5GlcNAc. These findings, as well as the observation that the mature proteins are sensitive to endoglycosidase H and insensitive to endoglycosidase D, are consistent with the notion that the biosynthetic pathway of the ribophorins does not require a stage of passage through the Golgi apparatus.

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.

scholarly journals Molecular cloning and expression of flagellar radial spoke and dynein genes of Chlamydomonas.

1986 ◽  
Vol 103 (1) ◽  
pp. 1-11 ◽  
Author(s):  
B D Williams ◽  
D R Mitchell ◽  
J L Rosenbaum
Keyword(s):  
Heavy Chain ◽  
Polyclonal Antiserum ◽  
Cloning And Expression ◽  
In Vitro Translation ◽  
Temperature Sensitive ◽  
Expression Library ◽  
Recombinant Phage ◽  
Accumulation Pattern ◽  
Radial Spoke

Several flagellar dynein ATPase and radial spokehead genes have been isolated from a Chlamydomonas genomic expression library in lambda gt11. The library was probed with polyclonal and monoclonal antibodies raised against purified flagellar polypeptides, and recombinant phage giving positive signals were cloned. In vitro translation of mRNAs hybrid-selected by the cloned sequences from whole cell RNA provided confirmation of identity for three of the four clones. Evidence supporting the identification of the fourth, which encodes a dynein heavy chain, was provided by antibody selection; the fusion protein produced by this clone selected heavy chain-specific antibodies from a complex polyclonal antiserum recognizing many dynein determinants. One of the radial spoke sequences isolated here is of particular interest because it encodes the wild-type allele of a locus which was defined previously by temperature-sensitive paralyzed flagella mutation pf-26ts (Huang, B., G. Piperno, Z. Ramanis, and D. J. L. Luck, 1981, J. Cell Biol., 88:80-88). The cloned sequence was used to hybrid-select mRNA from mutant pf-26ts cells, and when translated in vitro, the selected mRNA produced a mutant spokehead polypeptide with an altered electrophoretic mobility. This confirms that the pf-26ts mutation alters the primary structure of a radial spokehead polypeptide. To quantify spokehead and dynein mRNAs during flagellar regeneration, all of the cloned sequences were used as hybridization probes in RNA dot experiments. Levels increased rapidly and coordinately after deflagellation, peaked 3-10-fold above nondeflagellated controls, and then returned to control values within 2 h. This accumulation pattern was similar to that of flagellar alpha-tubulin mRNA.

scholarly journals A novel 205-kilodalton testis-specific serine/threonine protein kinase associated with microtubules of the spermatid manchette.

1993 ◽  
Vol 13 (12) ◽  
pp. 7625-7635 ◽  
Author(s):  
P D Walden ◽  
N J Cowan
Keyword(s):  
Protein Complex ◽  
Catalytic Domain ◽  
Signal Transduction Pathway ◽  
Kinase Domain ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Expression Library ◽  
Serine Threonine Kinase ◽  
Expression Clone

To identify proteins which interact with and potentially modulate the function of microtubules during spermatogenesis, we prepared a total testis MAP (microtubule-associated protein) antiserum and used it to isolate cDNA clones from a mouse testis cDNA expression library. Antibodies affinity purified by using one expression clone recognized a 205-kDa protein, termed MAST205, which colocalizes with the spermatid manchette. Sequencing of full-length cDNA clones encoding MAST205 revealed it to be a novel serine/threonine kinase with a catalytic domain related to those of the A and C families. The testis-specific MAST205 RNA increases in abundance during prepuberal testis development, peaking at the spermatid stage. The microtubule-binding region of MAST205 occupies a central region of the molecule including the kinase domain and sequences C terminal to this domain. Binding of MAST205 to microtubules requires interaction with other MAPs, since it does not bind to MAP-free tubulin. A 75-kDa protein associated with immunoprecipitates of MAST205 from extracts of both whole testis and testis microtubules becomes phosphorylated in in vitro kinase assays. This 75-kDa substrate of the MAST205 kinase may form part of the MAST205 protein complex which binds microtubules. The MAST205 protein complex may function to link the signal transduction pathway with the organization of manchette microtubules.

scholarly journals Identification and molecular characterization of E-MAP-115, a novel microtubule-associated protein predominantly expressed in epithelial cells.

1993 ◽  
Vol 123 (2) ◽  
pp. 357-371 ◽  
Author(s):  
D Masson ◽  
T E Kreis
Keyword(s):  
Epithelial Cells ◽  
Molecular Characterization ◽  
Hela Cells ◽  
Binding Proteins ◽  
Cdna Expression Library ◽  
Expression Library ◽  
Microtubule Binding ◽  
Terminal Domain

A novel microtubule-associated protein (MAP) of M(r) 115,000 has been identified by screening of a HeLa cell cDNA expression library with an anti-serum raised against microtubule-binding proteins from HeLa cells. Monoclonal and affinity-purified polyclonal antibodies were generated for the further characterization of this MAP. It is different from the microtubule-binding proteins of similar molecular weights, characterized so far, by its nucleotide-insensitive binding to microtubules and different sedimentation behavior. Since it is predominantly expressed in cells of epithelial origin (Caco-2, HeLa, MDCK), and rare (human skin, A72) or not detectable (Vero) in fibroblastic cells, we name it E-MAP-115 (epithelial MAP of 115 kD). In HeLa cells, E-MAP-115 is preferentially associated with subdomains or subsets of perinuclear microtubules. In Caco-2 cells, labeling for E-MAP-115 increases when they polarize and form blisters. The molecular characterization of E-MAP-115 reveals that it is a novel protein with no significant homologies to other known proteins. The secondary structure predicted from its sequence indicates two domains connected by a putative hinge region rich in proline and alanine (PAPA region). E-MAP-115 has two highly charged regions with predicted alpha-helical structure, one basic with a pI of 10.9 in the NH2-terminal domain and one neutral with a pI of 7.6 immediately following the PAPA region in the acidic COOH-terminal half of the molecule. A novel microtubule-binding site has been localized to the basic alpha-helical region in the NH2-terminal domain using in vitro microtubule-binding assays and expression of mutant polypeptides in vivo. Overexpression of this domain of E-MAP-115 by transfection of fibroblasts lacking significant levels of this protein with its cDNA renders microtubules stable to nocodazole. We conclude that E-MAP-115 is a microtubule-stabilizing protein that may play an important role during reorganization of microtubules during polarization and differentiation of epithelial cells.

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