scholarly journals Mechanism of compartmentation of secretory proteins: transport of exocrine pancreatic proteins across the microsomal membrane

1980 ◽  
Vol 87 (3) ◽  
pp. 611-628 ◽  
Author(s):  
G Scheele ◽  
R Jacoby ◽  
T Carne
Keyword(s):  
Protein Synthesis ◽  
Guinea Pig ◽  
Rat Liver ◽  
Micrococcal Nuclease ◽  
Secretory Proteins ◽  
Nascent Polypeptide ◽  
Amino Terminal ◽  
Microsomal Membranes ◽  
Dog Pancreas ◽  
Polypeptide Chains

The mechanism by which secretory proteins are segregated within the cisternal space of microsomal vesicles was studied using dog pancreas mRNA which directs the synthesis of 14 well-characterized nonglycosylated pancreatic exocrine proteins. In the absence of microsomal membranes, each of the proteins was synthesized as larger polypeptide chains (presecretory proteins). 1,000-2,000 daltons larger than their authentic counterparts as judged by polyacrylamide gel electrophoresis in SDS. Conditions optimal for the study of reconstituted rough microsomes in the reticulocyte lysate system were examined in detail using mRNA and microsomal membranes isolated from dog pancreas. Functional reconstitution of rough microsomes was considerably more efficient in the presence of micrococcal nuclease- treated membranes than in the presence of EDTA-treated membranes. Analysis for segregation of nascent secretory proteins by microsomal vesicles, using post-translational incubation in the presence of trypsin and chymotrypsin, 50 μg/ml each, was shown to be inadequate, because of the disruption of vesicles by protease activity. Addition of 1-3 mM tetracaine or 1 mM dibucaine stabilized microsomal membranes incubated in the presence of trypsin and chymotrypsin at either 0 degrees or 22 degrees C. Each of the pancreatic presecretory proteins studied was correctly processed to authentic secretory proteins by nuclease-treated microsomal membranes, as judged by both one-dimensional and two-dimensional gel electophoresis. Post-translational addition of membranes did not result in either segregation or processing of nascent polypeptide chains. Post- translational proteolysis, carried out in the presence of 3 mM tetracaine, indicated that each of the 14 characterized dog pancreas secretory proteins was quantitatively segregated by nuclease-treated microsomal vesicles. Segregation of nascent secretory proteins was irreversible, since radioactive amylase, as well as the other labeled secretory proteins, remained quantitatively sequestered in microsomal vesicles during a 90-min incubation at 22 degrees C after the cessation of protein synthesis. Studies employing synchronized protein synthesis and delayed addition of membranes indicated that all pancreatic presecretory proteins contain amino terminal peptide extensions. These peptide extensions are shown to mediate the cotranslational binding of presecretory proteins to microsomal membranes and the transport of nascent secretory proteins to the vesicular space. The maximum chain lengths which, during synthesis, allow segregation of nascent polypeptide chains varied between 61 (pretrypsinogen 2 + 3) and 88 (preprocarboxypeptidase A1) amino acid residues among dog pancreas presecretory proteins. Reconstitution studies using homologous and heterologous mixtures of mRNA (dog, guinea pig, and rat pancreas; rat liver) and micrococcal nuclease-treated microsomal membranes (dog, guinea pig, and rat liver; dog pancreas), in the presence of placental ribonuclease inhibitor, suggest that the translocation mechanism described is common to the rough endoplasmic reticulum of all mammalian tissues.

scholarly journals Preparation and characterization of dog pancreas microsomal membranes specifically depleted of protein disulphide-isomerase

1989 ◽  
Vol 257 (3) ◽  
pp. 657-663 ◽  
Author(s):  
J L Paver ◽  
H C Hawkins ◽  
R B Freedman
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Enzyme Assay ◽  
Experimental System ◽  
Secretory Proteins ◽  
Immunoblotting Analysis ◽  
Protein Disulphide Isomerase ◽  
Microsomal Membranes ◽  
Dog Pancreas

1. The selective release of protein disulphide-isomerase from dog pancreas and rat liver microsomal membranes was studied to throw light on the mechanisms of retention of this enzyme within the endoplasmic reticulum, and in order to prepare microsomal membranes specifically depleted of the enzyme. 2. Protein disulphide-isomerase was quantitatively released from dog pancreas microsomal membranes by washing at pH 9 and above, as demonstrated both by enzyme assay and by immunoblotting analysis. 3. Integral membrane proteins implicated in the process of translocation and segregation of secretory proteins were retained in pH 9-washed dog pancreas microsomal membranes. 4. After pH 9 washing, dog pancreas microsomal membranes were fully active in the translocation, segregation and processing of nascent secretory proteins; these membranes therefore provide a useful experimental system for testing the action of protein disulphide-isomerase on nascent secretory proteins. 5. Protein disulphide-isomerase was not released from rat liver microsomal membranes by pH 9 washing, and was much less readily released from these membranes by sonication, washing etc. than from dog pancreas microsomal membranes. 6. The mechanism of retention of protein disulphide-isomerase, and of other resident proteins of the lumen of the endoplasmic reticulum, is discussed in the light of these findings.

scholarly journals Initiation and processing in vitro of the primary translation products of guinea-pig caseins

1979 ◽  
Vol 184 (2) ◽  
pp. 261-267 ◽  
Author(s):  
R K Craig ◽  
P A J Perera ◽  
A Mellor ◽  
A E Smith
Keyword(s):  
Guinea Pig ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Secretory Proteins ◽  
Post Translational Modifications ◽  
Microsomal Membranes

1. Guinea-pig caseins synthesized in a mRNA-directed wheat-germ cell-free protein-synthesizing system represent the primary translation products, even though they appear to be of lower molecular weight when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in parallel with caseins isolated from guinea-pig milk. 2. Identification of the N-terminal dipeptide of the primary translational product of caseins A, B and C and alpha-lactalbumin showed that all shared a common sequence, which was identified as either Met-Arg or Met-Lys. 3. Procedures utilizing methionyl-tRNAfMet or methionyl-tRNAMet in the presence or absence of microsomal membranes during translation provide a rapid method of distinguishing between N-terminal processing of peptides synthesized in vitro and other post-translational modifications (glycosylation, phosphorylation), which also result in a change in mobility of peptides when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. The results demonstrate that guinea-pig caseins, in common with most other secretory proteins, are synthesized with transient N-terminal ‘signal’-peptide extensions, which are cleaved during synthesis in the presence of microsomal membranes.

scholarly journals Biosynthesis of rat liver pI-6.1 esterase, a carboxylesterase of the cisternal space of the endoplasmic reticulum

1987 ◽  
Vol 248 (2) ◽  
pp. 545-550 ◽  
Author(s):  
M Robbi ◽  
H Beaufay
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Rabbit Antiserum ◽  
Secretory Proteins ◽  
Structural Element ◽  
Oligosaccharide Moiety ◽  
Reticulocyte Lysate ◽  
Dog Pancreas ◽  
Processed Products

Biosynthesis of the rat liver microsomal esterase with pI 6.1 was investigated in cell-free systems and in cultured hepatocytes, by using a rabbit antiserum. Protein synthesis directed by total rat liver RNA in wheatgerm extract or reticulocyte lysate generated a single immunoprecipitable product, also found with the RNA extracted from bound, but not from free, polysomes. When dog pancreas microsomal fractions were included, reticulocyte lysates gave two processed products, a prominent one slightly larger, and another slightly smaller, than the precursor, both resistant to exogenous proteinases and, hence, segregated within vesicles. The processing was co-translational; it consisted of the removal of a peptide fragment and, for the large component, the addition of a single oligosaccharide chain. Indeed, this component bound to concanavalin A-Sepharose and gave the small one (approximately 2000 Mr loss) by cleavage with endo-beta-N-acetylglucosaminidase H (endo-H). A single labelled peptide was precipitated from hepatocytes incubated with [35S]methionine. Its apparent Mr was decreased by approximately 2000 after treatment with endo-H; it was then identical with that of an unglycosylated form produced in hepatocytes poisoned with tunicamycin. Even in that case, immunoreactive peptides were not detected in the culture medium. Whether synthesized in reticulocyte lysate or in hepatocytes, the glycosylated forms migrated in SDS/polyacrylamide-gel electrophoresis as the purified enzyme labelled with [3H]di-isopropyl fluorophosphate. Thus, although pI-6.1 esterase is not secreted, its biosynthesis is, as yet, indistinguishable from that of secretory proteins. Its oligosaccharide moiety is apparently not the structural element that retains it in the endoplasmic reticulum.

Acetylation of nascent polypeptide chains on rat liver polyribosomes in vivo and in vitro

Biochemistry ◽  
1975 ◽  
Vol 14 (7) ◽  
pp. 1404-1412 ◽  
Author(s):  
Angel Pestana ◽  
Henry C. Pitot
Keyword(s):  
Rat Liver ◽  
Nascent Polypeptide ◽  
Polypeptide Chains

Study of the precursors of ovine lactoproteins: primary structures of the ‘signals’ and enzymic processing of prelactoproteins by mammary microsomal membranes

1979 ◽  
Vol 46 (2) ◽  
pp. 175-180 ◽  
Author(s):  
Pierre Gaye
Keyword(s):  
Germ Cell ◽  
Wheat Germ ◽  
Secretory Proteins ◽  
Signal Peptides ◽  
Amino Acid Residues ◽  
Free System ◽  
Cell Free System ◽  
Amino Terminal ◽  
Microsomal Membranes ◽  
Β Lactoglobulin

SUMMARYThe radiolabelled primary translation products of ovine mammary mRNAs synthesized in a wheat germ cell-free system were isolated by immuno-precipitation and analysed by automated Edman degradation. The 3 ‘Ca-sensitive’ caseins (α, α andβ ), k-casein, β-lactoglobulin and α-lactalbumin were found to be synthesized as precursors with amino terminal extensions of 15, 21, 18 and 19 amino acid residues respectively. The extra pieces of these various lactoproteins are similar to ‘signal’ peptides of other secretory proteins in their length and hydro-phobicity. The occurrence of an alanyl residue at the C-termini of the extra pieces of the 6 ovine prelactoproteins suggests that the mammary proteinase responsible for the cleavage of the signal peptides may have an elastase-like specificity.When mammary mRNAs were translated in a wheat germ cell-free system in the presence of mammary microsomal membranes, pre-β-casein was converted into authentic β-casein as demonstrated by amino terminal sequence analyses. Additionally, pre-β-casein was post-translationally converted into authentic β-casein by a specific proteinase(s) extracted from rough microsomes with Na deoxycholate.

scholarly journals Elongation inhibitors do not prevent the release of puromycylated nascent polypeptide chains from ribosomes

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Benjamin D Hobson ◽  
Linghao Kong ◽  
Erik W Hartwick ◽  
Ruben L Gonzalez ◽  
Peter A Sims
Keyword(s):  
Protein Synthesis ◽  
Messenger Rna ◽  
Mammalian Cells ◽  
Acyl Transfer ◽  
Proximity Ligation Assay ◽  
Nascent Polypeptide ◽  
Rna Translation ◽  
Biochemical Assays ◽  
Messenger Rna Translation ◽  
Polypeptide Chains

Puromycin is an amino-acyl transfer RNA analog widely employed in studies of protein synthesis. Since puromycin is covalently incorporated into nascent polypeptide chains, anti-puromycin immunofluorescence enables visualization of nascent protein synthesis. A common assumption in studies of local messenger RNA translation is that the anti-puromycin staining of puromycylated nascent polypeptides in fixed cells accurately reports on their original site of translation, particularly when ribosomes are stalled with elongation inhibitors prior to puromycin treatment. However, when we attempted to implement a proximity ligation assay to detect ribosome-puromycin complexes, we found no evidence to support this assumption. We further demonstrated, using biochemical assays and live cell imaging of nascent polypeptides in mammalian cells, that puromycylated nascent polypeptides rapidly dissociate from ribosomes even in the presence of elongation inhibitors. Our results suggest that attempts to define precise subcellular translation sites using anti-puromycin immunostaining may be confounded by release of puromycylated nascent polypeptide chains prior to fixation.

scholarly journals Elongation Inhibitors do not Prevent the Release of Puromycylated Nascent Polypeptide Chains from Ribosomes

2020 ◽  
Author(s):  
Benjamin D. Hobson ◽  
Linghao Kong ◽  
Erik W. Hartwick ◽  
Ruben L. Gonzalez ◽  
Peter A. Sims
Keyword(s):  
Protein Synthesis ◽  
Messenger Rna ◽  
Acyl Transfer ◽  
Proximity Ligation Assay ◽  
Nascent Polypeptide ◽  
Rna Translation ◽  
Proximity Ligation ◽  
Biochemical Assays ◽  
Messenger Rna Translation ◽  
Polypeptide Chains

ABSTRACTPuromycin is an amino-acyl transfer RNA analog widely employed in studies of protein synthesis. Since puromycin is covalently incorporated into nascent polypeptide chains, anti-puromycin immunofluorescence enables visualization of nascent protein synthesis. A common assumption in studies of local messenger RNA translation is that the anti-puromycin staining of puromycylated nascent polypeptides in fixed cells accurately reports on their original site of translation, particularly when ribosomes are stalled with elongation inhibitors prior to puromycin treatment. However, when we attempted to implement a proximity ligation assay to detect ribosome-puromycin complexes, we found no evidence to support this assumption. We further demonstrated, using biochemical assays and live cell imaging of nascent polypeptides, that puromycylated nascent polypeptides rapidly dissociate from ribosomes even in the presence of elongation inhibitors. Our results suggest that attempts to define precise subcellular translation sites using anti-puromycin immunostaining may be confounded by release of puromycylated nascent polypeptide chains prior to fixation.

Formation of New Proteins Seen from the Beginning

2010 ◽  
Vol 18 (3) ◽  
pp. 6-8
Author(s):  
Stephen W. Carmichael
Keyword(s):  
Signal Peptide ◽  
Polypeptide Chain ◽  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Direct Visualization ◽  
Conducting Channel ◽  
Nascent Polypeptide ◽  
Amino Terminal ◽  
Cryo Electron Microscopy ◽  
Polypeptide Chains

All cells have the ability to synthesize and secrete proteins. Although many details of this process are well-known, Martin Kampmann and Günter Blobel recently highlighted two “landmark papers” that used cryo-electron microscopy (cryoEM) to obtain information at subnanometer resolution, which provided direct visualization of nascent polypeptide chains in the tunnel with ribosomes . It is known that the signal peptide (the first few amino acids on the amino terminal that do not become part of the final polypeptide) emerges from a ribosome and engages the signal recognition particle (SRP) in the cytoplasm, and this complex is directed to the SRP receptor on the endoplasmic reticulum (ER). The SRP is released, the signal peptide enters the protein-conducting channel (PCC), and the nascent polypeptide chain (that will become the protein) enters the lumen of the ER.

scholarly journals Immunochemical characterization and biosynthesis of pI-6.4 esterase, a carboxylesterase of rat liver microsomal extracts

1988 ◽  
Vol 254 (1) ◽  
pp. 51-57 ◽  
Author(s):  
M Robbi ◽  
H Beaufay
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Culture Media ◽  
Intact Cell ◽  
Molecular Size ◽  
Secretory Proteins ◽  
Cell Culture Media ◽  
Reticulocyte Lysates ◽  
Dog Pancreas ◽  
Processing Steps

Rat liver pI-6.4 esterase was purified from microsomes (microsomal extracts) and used to generate antibodies in the rabbit. Two active enzyme forms, similarly sensitive to endo-H (endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96), but differing slightly in polypeptide chain length, were present in the preparation. In microsomes, immunoblots revealed a single form, with Mr congruent to 62,000, identical with the large component of the purified enzyme, indicating that the second component is an artefact. Rabbit reticulocyte lysates and wheat germ extracts programmed with RNA extracted from total or bound polysomes synthesized a single immunoreactive 61 kDa polypeptide, which was not formed with RNA extracted from free polysomes. The immunoreactive product synthesized in the presence of dog pancreas microsomes was slightly larger (62 kDa); like the authentic enzyme, it bound to concanavalin A and was decreased in molecular size to 60 kDa by the action of endo-H. Thus the enzyme is synthesized with a short cleavable sequence and bears at least one high-mannose oligosaccharide chain. Metabolic labelling in hepatocytes cultured with [35S]methionine also generated a single immunoreactive polypeptide of 62 kDa, which was decreased to 60 kDa in size by treatment with endo-H or addition of tunicamycin to the culture medium. This confirms the molecular homogeneity and the glycosylation of the enzyme in the intact cell. Culture media contained no pI-6.4-esterase-related protein, whether tunicamycin was present or not. The processing steps in the synthesis of pI-6.4 esterase are thus, as for other esterases of the endoplasmic reticulum [Robbi & Beaufay (1986) Eur. J. Biochem. 158, 187-194; (1987) Biochem. J. 248, 545-550] indistinguishable from those occurring early in the synthesis of secretory proteins. Glycosylation is apparently not the sorting signal responsible for their retention in the endoplasmic reticulum.

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