Time-dependent alteration in endoplasmic reticulum membrane profiles during in vitro incubation of Krebs II ascites cells

1983 ◽  
Vol 56 (2) ◽  
pp. 131-136 ◽  
Author(s):  
Anders Fjose ◽  
Ian F. Pryme
Keyword(s):  
Endoplasmic Reticulum ◽  
Time Dependent ◽  
Endoplasmic Reticulum Membrane ◽  
In Vitro Incubation

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.

Effect of creatine kinase-MM subtype composition on a CK-MB immunoinhibition assay.

1988 ◽  
Vol 34 (3) ◽  
pp. 535-538 ◽  
Author(s):  
I M Morison ◽  
K J Clayson ◽  
J S Fine
Keyword(s):  
Myocardial Infarction ◽  
Creatine Kinase ◽  
Human Serum ◽  
Time Dependent ◽  
Clinical Specimens ◽  
In Vitro Incubation ◽  
Dependent Change ◽  
Time Dependent Change

Abstract Immunoinhibition (INH) by use of polyclonal anti-human CK-M antibody may be used to measure CK-MB in serum. Previous studies have shown that inhibiting antibodies prepared against purified muscle extracts may inhibit CK-MM by greater than 99%. Using patients' sera and muscle homogenates incubated with human serum, we studied the effect of CK-MM subtype composition on an INH assay. We found that with increasing time from the CK-releasing event, e.g., myocardial infarction, or with longer in vitro incubation, the proportion of CK-MM1 increased and the proportion of uninhibited CK-MM increased from 0.2% to 0.7-0.8%. As a consequence, CK-MB activity may be overestimated by as much as 1.6% of total CK when uncorrected INH results are used. Inhibition was maximal in samples containing 100% CK-MM3, the tissue subtype. Because of the time-dependent change in CK-MM subtypes, published results for INH from studies in which CK-MM purified from muscle was used may not be directly applicable to clinical specimens.

scholarly journals TheSaccharomyces cerevisiaePrenylcysteine Carboxyl Methyltransferase Ste14p Is in the Endoplasmic Reticulum Membrane

1998 ◽  
Vol 9 (8) ◽  
pp. 2231-2247 ◽  
Author(s):  
Julia D. Romano ◽  
Walter K. Schmidt ◽  
Susan Michaelis
Keyword(s):  
Endoplasmic Reticulum ◽  
Subcellular Fractionation ◽  
Endoplasmic Reticulum Membrane ◽  
Carboxyl Methylation ◽  
C Terminus ◽  
Eukaryotic Proteins ◽  
Internal Site ◽  
Membrane Spanning ◽  
Er Membrane

Eukaryotic proteins containing a C-terminal CAAX motif undergo a series of posttranslational CAAX-processing events that include isoprenylation, C-terminal proteolytic cleavage, and carboxyl methylation. We demonstrated previously that the STE14gene product of Saccharomyces cerevisiae mediates the carboxyl methylation step of CAAX processing in yeast. In this study, we have investigated the subcellular localization of Ste14p, a predicted membrane-spanning protein, using a polyclonal antibody generated against the C terminus of Ste14p and an in vitro methyltransferase assay. We demonstrate by immunofluorescence and subcellular fractionation that Ste14p and its associated activity are localized to the endoplasmic reticulum (ER) membrane of yeast. In addition, other studies from our laboratory have shown that the CAAX proteases are also ER membrane proteins. Together these results indicate that the intracellular site of CAAX protein processing is the ER membrane, presumably on its cytosolic face. Interestingly, the insertion of a hemagglutinin epitope tag at the N terminus, at the C terminus, or at an internal site disrupts the ER localization of Ste14p and results in its mislocalization, apparently to the Golgi. We have also expressed the Ste14p homologue from Schizosaccharomyces pombe, mam4p, in S. cerevisiae and have shown that mam4p complements a Δste14 mutant. This finding, plus additional recent examples of cross-species complementation, indicates that the CAAX methyltransferase family consists of functional homologues.

scholarly journals p180 Is Involved in the Interaction between the Endoplasmic Reticulum and Microtubules through a Novel Microtubule-binding and Bundling Domain

2007 ◽  
Vol 18 (10) ◽  
pp. 3741-3751 ◽  
Author(s):  
Kiyoko Ogawa-Goto ◽  
Keiko Tanaka ◽  
Tomonori Ueno ◽  
Keisuke Tanaka ◽  
Takeshi Kurata ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Specific Interaction ◽  
Endoplasmic Reticulum Membrane ◽  
Dependent Manner ◽  
Small Interfering Rnas ◽  
Animal Cells ◽  
Microtubule Binding

p180 was originally reported as a ribosome-binding protein on the rough endoplasmic reticulum membrane, although its precise role in animal cells has not yet been elucidated. Here, we characterized a new function of human p180 as a microtubule-binding and -modulating protein. Overexpression of p180 in mammalian cells induced an elongated morphology and enhanced acetylated microtubules. Consistently, electron microscopic analysis clearly revealed microtubule bundles in p180-overexpressing cells. Targeted depletion of endogenous p180 by small interfering RNAs led to aberrant patterns of microtubules and endoplasmic reticulum in mammalian cells, suggesting a specific interaction between p180 and microtubules. In vitro sedimentation assays using recombinant polypeptides revealed that p180 bound to microtubules directly and possessed a novel microtubule-binding domain (designated MTB-1). MTB-1 consists of a predicted coiled-coil region and repeat domain, and strongly promoted bundle formation both in vitro and in vivo when expressed alone. Overexpression of p180 induced acetylated microtubules in cultured cells in an MTB-1-dependent manner. Thus, our data suggest that p180 mediates interactions between the endoplasmic reticulum and microtubules mainly through the novel microtubule-binding and -bundling domain MTB-1.

scholarly journals The influenza hemagglutinin insertion signal is not cleaved and does not halt translocation when presented to the endoplasmic reticulum membrane as part of a translocating polypeptide.

1987 ◽  
Vol 104 (6) ◽  
pp. 1705-1714 ◽  
Author(s):  
J Finidori ◽  
L Rizzolo ◽  
A Gonzalez ◽  
G Kreibich ◽  
M Adesnik ◽  
...  
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Signal Sequence ◽  
In Vitro Translation ◽  
Signal Peptidase ◽  
Endoplasmic Reticulum Membrane ◽  
Hydrophobic Region ◽  
Amino Terminal ◽  
Influenza Hemagglutinin

The co-translational insertion of polypeptides into endoplasmic reticulum membranes may be initiated by cleavable amino-terminal insertion signals, as well as by permanent insertion signals located at the amino-terminus or in the interior of a polypeptide. To determine whether the location of an insertion signal within a polypeptide affects its function, possibly by affecting its capacity to achieve a loop disposition during its insertion into the membrane, we have investigated the functional properties of relocated insertion signals within chimeric polypeptides. An artificial gene encoding a polypeptide (THA-HA), consisting of the luminal domain of the influenza hemagglutinin preceded by its amino-terminal signal sequence and linked at its carboxy-terminus to an intact prehemagglutinin polypeptide, was constructed and expressed in in vitro translation systems containing microsomal membranes. As expected, the amino-terminal signal initiated co-translational insertion of the hybrid polypeptide into the membranes. The second, identical, interiorized signal, however, was not recognized by the signal peptidase and was translocated across the membrane. The failure of the interiorized signal to be cleaved may be attributed to the fact that it enters the membrane as part of a translocating polypeptide and therefore cannot achieve the loop configuration that is thought to be adopted by signals that initiate insertion. The finding that the interiorized signal did not halt translocation of downstream sequences, even though it contains a hydrophobic region and must enter the membrane in the same configuration as natural stop-transfer signals, indicates that the HA insertion signal lacks essential elements of halt transfer signals that makes the latter effective membrane-anchoring domains. When the amino-terminal insertion signal of the THA-HA chimera was deleted, the interior signal was incapable of mediating insertion, probably because of steric hindrance by the folded preceding portions of the chimera. Several chimeras were constructed in which the interiorized signal was preceded by polypeptide segments of various lengths. A signal preceded by a segment of 111 amino acids was also incapable of initiating insertion, but insertion took place normally when the segment preceding the signal was only 11-amino acids long.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Sls1p Stimulates Sec63p-Mediated Activation of Kar2p in a Conformation-Dependent Manner in the Yeast Endoplasmic Reticulum

2000 ◽  
Vol 20 (18) ◽  
pp. 6923-6934 ◽  
Author(s):  
Mehdi Kabani ◽  
Jean-Marie Beckerich ◽  
Claude Gaillardin
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Synthetic Lethality ◽  
In Vitro Assays ◽  
Endoplasmic Reticulum Membrane ◽  
Dependent Manner ◽  
Dose Dependent

ABSTRACT We previously characterized the SLS1 gene in the yeastYarrowia lipolytica and showed that it interacts physically with YlKar2p to promote translocation across the endoplasmic-reticulum membrane (A. Boisramé, M. Kabani, J. M. Beckerich, E. Hartmann, and C. Gaillardin, J. Biol. Chem. 273:30903–30908, 1998). A Y. lipolytica Kar2p mutant was isolated that restored interaction with an Sls1p mutant, suggesting that the interaction with Sls1p could be nucleotide and/or conformation dependent. This result was used as a working hypothesis for more accurate investigations in Saccharomyces cerevisiae. We show by two-hybrid an in vitro assays that the S. cerevisiae homologue of Sls1p interacts with ScKar2p. Using dominant lethal mutants of ScKar2p, we were able to show that ScSls1p preferentially interacts with the ADP-bound conformation of the molecular chaperone. Synthetic lethality was observed between ΔScsls1 and translocation-deficientkar2 or sec63-1 mutants, providing in vivo evidence for a role of ScSls1p in protein translocation. Synthetic lethality was also observed with ER-associated degradation and folding-deficient kar2 mutants, strongly suggesting that Sls1p functions are not restricted to the translocation process. We show that Sls1p stimulates in a dose-dependent manner the binding ofScKar2p on the lumenal J domain of Sec63p fused to glutathione S-transferase. Moreover, Sls1p is shown to promote the Sec63p-mediated activation of Kar2p's ATPase activity. Our data strongly suggest that Sls1p could be the first GrpE-like protein described in the endoplasmic reticulum.

Expression of Viral Membrane Proteins from Cloned cDNA by Microinjection into Eucaryotic Cell Nuclei

1982 ◽  
Vol 40 ◽  
pp. 26-29
Author(s):  
H. Garoff ◽  
Cl. Kondor-Koch
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Signal Sequence ◽  
Endoplasmic Reticulum Membrane ◽  
In Vitro Mutagenesis ◽  
Cell Nuclei ◽  
Eucaryotic Cell ◽  
Cloned Cdna ◽  
The Relationship

The relationship between a protein's structure and its function can be explored in detail by in vitro mutagenesis of a cloned DNA molecule encoding the protein and expression of its mutagenized form in a eucaryotic cell. This will be a useful approach to study the assembly of spanning viral or plasma membrane proteins into the endoplasmic reticulum membrane and their transport to the cell surface. Questions that could be answered using this technique include:Is the signal sequence alone sufficient for translocation of a polypeptide chain across the endoplasmic reticulum membrane? Is a cytoplasmic protein (e.g. a viral capsid protein) translocated when linked to a signal sequence?

scholarly journals Rer1p, a Retrieval Receptor for Endoplasmic Reticulum Membrane Proteins, Is Dynamically Localized to the Golgi Apparatus by Coatomer

2001 ◽  
Vol 152 (5) ◽  
pp. 935-944 ◽  
Author(s):  
Ken Sato ◽  
Miyuki Sato ◽  
Akihiko Nakano
Keyword(s):  
Endoplasmic Reticulum ◽  
Green Fluorescent Protein ◽  
Membrane Proteins ◽  
Golgi Apparatus ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Endoplasmic Reticulum Membrane ◽  
Golgi Membrane ◽  
Green Fluorescent

Rer1p, a yeast Golgi membrane protein, is required for the retrieval of a set of endoplasmic reticulum (ER) membrane proteins. We present the first evidence that Rer1p directly interacts with the transmembrane domain (TMD) of Sec12p which contains a retrieval signal. A green fluorescent protein (GFP) fusion of Rer1p rapidly cycles between the Golgi and the ER. Either a lesion of coatomer or deletion of the COOH-terminal tail of Rer1p causes its mislocalization to the vacuole. The COOH-terminal Rer1p tail interacts in vitro with a coatomer complex containing α and γ subunits. These findings not only give the proof that Rer1p is a novel type of retrieval receptor recognizing the TMD in the Golgi but also indicate that coatomer actively regulates the function and localization of Rer1p.

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