scholarly journals Characterization of secretory protein translocation: ribosome-membrane interaction in endoplasmic reticulum.

1986 ◽  
Vol 103 (1) ◽  
pp. 241-253 ◽  
Author(s):  
M Hortsch ◽  
D Avossa ◽  
D I Meyer
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Signal Recognition Particle ◽  
Secretory Protein ◽  
Signal Peptidase ◽  
Secretory Proteins ◽  
Peptidase Activity ◽  
Ribosome Binding ◽  
Functional Inactivation ◽  
Docking Protein

Secretory proteins are synthesized on ribosomes bound to the membrane of the endoplasmic reticulum (ER). After the selection of polysomes synthesizing secretory proteins and their direction to the membrane of the ER via signal recognition particle (SRP) and docking protein respectively, the polysomes become bound to the ER membrane via an unknown, protein-mediated mechanism. To identify proteins involved in protein translocation, beyond the (SRP-docking protein-mediated) recognition step, controlled proteolysis was used to functionally inactivate rough microsomes that had previously been depleted of docking protein. As the membranes were treated with increasing levels of protease, they lost their ability to be functionally reconstituted with the active cytoplasmic fragment of docking protein (DPf). This functional inactivation did not correlate with a loss of either signal peptidase activity, nor with the ability of the DPf to reassociate with the membrane. It did correlate, however, with a loss of the ability of the microsomes to bind ribosomes. Ribophorins are putative ribosome-binding proteins. Immunoblots developed with monoclonal antibodies against canine ribophorins I and II demonstrated that no correlation exists between the protease-induced inability to bind ribosomes and the integrity of the ribophorins. Ribophorin I was 85% resistant and ribophorin II 100% resistant to the levels of protease needed to totally eliminate ribosome binding. Moreover, no direct association was found between ribophorins and ribosomes; upon detergent solubilization at low salt concentrations, ribophorins could be sedimented in the presence or absence of ribosomes. Finally, the alkylating agent N-ethylmaleimide was shown to be capable of inhibiting translocation (beyond the SRP-docking protein-mediated recognition step), but had no affect on the ability of ribosomes to bind to ER membranes. We conclude that potentially two additional proteinaceous components, as yet unidentified, are involved in protein translocation. One is protease sensitive and possibly involved in ribosome binding, the other is N-ethylmaleimide sensitive and of unknown function.

scholarly journals Elongation arrest is not a prerequisite for secretory protein translocation across the microsomal membrane.

1985 ◽  
Vol 100 (6) ◽  
pp. 1913-1921 ◽  
Author(s):  
V Siegel ◽  
P Walter
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Signal Recognition Particle ◽  
Integral Membrane Protein ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Signal Recognition ◽  
7Sl Rna ◽  
Nascent Chain ◽  
Specific Proteins

Signal recognition particle (SRP) is a ribonucleoprotein consisting of six distinct polypeptides and one molecule of small cytoplasmic 7SL RNA. It was previously shown to promote the co-translational translocation of secretory proteins across the endoplasmic reticulum by (a) arresting the elongation of the presecretory nascent chain at a specific point, and (b) interacting with the SRP receptor, an integral membrane protein of the endoplasmic reticulum which is active in releasing the elongation arrest. Recently a procedure was designed by which the particle could be disassembled into its protein and RNA components. We have further separated the SRP proteins into four homogeneous fractions. When recombined with each other and with 7SL RNA, they formed fully active SRP. Particles missing specific proteins were assembled in the hope that some of these would retain some functional activity. SRP(-9/14), the particle lacking the 9-kD and 14-kD polypeptides, was fully active in promoting translocation, but was completely inactive in elongation arrest. This implied that elongation arrest is not a prerequisite for protein translocation. SRP receptor was required for SRP(-9/14)-mediated translocation to occur, and thus must play some role in the translocation process in addition to releasing the elongation arrest.

scholarly journals Protein translocation mutants defective in the insertion of integral membrane proteins into the endoplasmic reticulum.

1992 ◽  
Vol 3 (2) ◽  
pp. 129-142 ◽  
Author(s):  
C J Stirling ◽  
J Rothblatt ◽  
M Hosobuchi ◽  
R Deshaies ◽  
R Schekman
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Protein Translocation ◽  
Secretory Protein ◽  
Polyclonal Antiserum ◽  
Secretory Proteins ◽  
Temperature Sensitive ◽  
Endoplasmic Reticulum Membrane ◽  
Membrane Spanning ◽  
Coa Reductase

Yeast mutants defective in the translocation of soluble secretory proteins into the lumen of the endoplasmic reticulum (sec61, sec62, sec63) are not impaired in the assembly and glycosylation of the type II membrane protein dipeptidylaminopeptidase B (DPAPB) or of a chimeric membrane protein consisting of the multiple membrane-spanning domain of yeast hydroxymethylglutaryl CoA reductase (HMG1) fused to yeast histidinol dehydrogenase (HIS4C). This chimera is assembled in wild-type or mutant cells such that the His4c protein is oriented to the ER lumen and thus is not available for conversion of cytosolic histidinol to histidine. Cells harboring the chimera have been used to select new translocation defective sec mutants. Temperature-sensitive lethal mutations defining two complementation groups have been isolated: a new allele of sec61 and a single isolate of a new gene sec65. The new isolates are defective in the assembly of DPAPB, as well as the secretory protein alpha-factor precursor. Thus, the chimeric membrane protein allows the selection of more restrictive sec mutations rather than defining genes that are required only for membrane protein assembly. The SEC61 gene was cloned, sequenced, and used to raise polyclonal antiserum that detected the Sec61 protein. The gene encodes a 53-kDa protein with five to eight potential membrane-spanning domains, and Sec61p antiserum detects an integral protein localized to the endoplasmic reticulum membrane. Sec61p appears to play a crucial role in the insertion of secretory and membrane polypeptides into the endoplasmic reticulum.

scholarly journals The signal sequence receptor, unlike the signal recognition particle receptor, is not essential for protein translocation

1992 ◽  
Vol 117 (1) ◽  
pp. 15-25 ◽  
Author(s):  
G Migliaccio ◽  
CV Nicchitta ◽  
G Blobel
Keyword(s):  
Membrane Protein ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Chain Complex ◽  
Secretory Protein ◽  
Signal Recognition ◽  
Ribosome Binding ◽  
Signal Recognition Particle Receptor ◽  
Nascent Chain

Detergent extracts of canine pancreas rough microsomal membranes were depleted of either the signal recognition particle receptor (SR), which mediates the signal recognition particle (SRP)-dependent targeting of the ribosome/nascent chain complex to the membrane, or the signal sequence receptor (SSR), which has been proposed to function as a membrane bound receptor for the newly targeted nascent chain and/or as a component of a multi-protein translocation complex responsible for transfer of the nascent chain across the membrane. Depletion of the two components was performed by chromatography of detergent extracts on immunoaffinity supports. Detergent extracts lacking either SR or SSR were reconstituted and assayed for activity with respect to SR dependent elongation arrest release, nascent chain targeting, ribosome binding, secretory precursor translocation, and membrane protein integration. Depletion of SR resulted in the loss of elongation arrest release activity, nascent chain targeting, secretory protein translocation, and membrane protein integration, although ribosome binding was unaffected. Full activity was restored by addition of immunoaffinity purified SR before reconstitution of the detergent extract. Surprisingly, depletion of SSR was without effect on any of the assayed activities, indicating that SSR is either not required for translocation or is one of a family of functionally redundant components.

The role of molecular chaperones in protein transport into the endoplasmic reticulum

1993 ◽  
Vol 339 (1289) ◽  
pp. 335-341 ◽  
Keyword(s):  
Endoplasmic Reticulum ◽  
Molecular Chaperones ◽  
Protein Transport ◽  
Protein Translocation ◽  
Signal Recognition Particle ◽  
Microsomal Membrane ◽  
Nucleoside Triphosphate ◽  
Secretory Proteins ◽  
Hsc 70 ◽  
Hydrolysis Of

In eukaryotic cells export of the vast majority of newly synthesized secretory proteins is initiated at the level of the membrane of the endoplasmic reticulum (microsomal membrane). The precursors of secretory proteins are not transported across the microsomal m em brane in their native state. Typically, signal peptides in the precursor proteins are involved in preserving the transport-competent state. Furthermore, there are two alternatively acting mechanisms involved in preserving transport competence in the cytosol. The first mechanism involves two ribonucleoparticles (ribosome and signal recognition particle) and their receptors on the microsomal surface and requires the hydrolysis of GTP. The second mechanism does not involve ribonucleoparticles and their receptors but depends on the hydrolysis of A TP and on cA-acting molecular chaperones, such as heat shock cognate protein 70 (hsc 70). In both mechanisms a translocase in the microsomal membrane mediates protein translocation. This translocase includes a signal peptide receptor on the cu-side of the microsomal membrane and a component that also depends on the hydrolysis of ATP. At least in certain cases, an additional nucleoside triphosphate-requiring step is involved which is related to the trans -acting molecular chaperone BiP.

scholarly journals Identification of signal sequence binding proteins integrated into the rough endoplasmic reticulum membrane

1987 ◽  
Vol 242 (3) ◽  
pp. 767-777 ◽  
Author(s):  
A Robinson ◽  
M A Kaderbhai ◽  
B M Austen
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Signal Peptide ◽  
Signal Sequence ◽  
Secretory Protein ◽  
Phospholipid Bilayer ◽  
Signal Peptidase ◽  
Secretory Proteins ◽  
Endoplasmic Reticulum Membrane

An azidophenacyl derivative of a chemically synthesized consensus signal peptide has been prepared. The peptide, when photoactivated in the presence of rough or high-salt-stripped microsomes from pancreas, leads to inhibition of their activity in cotranslational processing of secretory pre-proteins translated from their mRNA in vitro. The peptide binds specifically with high affinity to components in the microsomal membranes from pancreas and liver, and photoreaction of a radioactive form of the azidophenacyl derivative leads to covalent linkage to yield two closely related radiolabelled proteins of Mr about 45,000. These proteins are integrated into the membrane, with large 30,000-Mr domains embedded into the phospholipid bilayer to which the signal peptide binds. A smaller, endopeptidase-sensitive, domain is exposed on the cytoplasmic surface of the microsomal vesicles. The specificity and selectivity of the binding of azidophenacyl-derivatized consensus signal peptide was demonstrated by concentration-dependent inhibition of photolabelling by the ‘cold’ synthetic consensus signal peptide and by a natural internal signal sequence cleaved and isolated from ovalbumin. The properties of the labelled 45,000-Mr protein-signal peptide complexes, i.e. mass, pI, ease of dissociation from the membrane by detergent or salts and immunological properties, distinguish them from other proteins, e.g. subunits of signal recognition particle, docking protein and signal peptidase, already known to be involved in targetting and processing of nascent secretory proteins at the rough endoplasmic reticulum membrane. Although the 45,000-Mr signal peptide binding protein displays properties similar to those of the signal peptidase, a component of the endoplasmic reticulum, the azido-derivatized consensus signal peptide does not interact with it. It is proposed that the endoplasmic reticulum proteins with which the azidophenacyl-derivatized consensus signal peptide interacts to yield the 45,000-Mr adducts may act as receptors for signals in nascent secretory pre-proteins in transduction of changes in the endoplasmic reticulum which bring about translocation of secretory protein across the membrane.

scholarly journals 180-kD ribosome receptor is essential for both ribosome binding and protein translocation.

1993 ◽  
Vol 120 (4) ◽  
pp. 853-863 ◽  
Author(s):  
A J Savitz ◽  
D I Meyer
Keyword(s):  
Monoclonal Antibodies ◽  
Direct Evidence ◽  
Protein Translocation ◽  
Secretory Protein ◽  
Ribosome Binding ◽  
Fab Fragments ◽  
Rough Er

We have previously isolated a 180-kD ribosome receptor (p180) from mammalian rough ER that, when incorporated into liposomes, bound ribosomes with an affinity similar to intact membranes. To directly assess the contribution of p180 to ribosome binding as well as protein translocation, monoclonal antibodies were used to selectively deplete p180 from the detergent extracts of rough ER membranes used in the preparation of translocation-competent proteoliposomes. Proteoliposomes prepared from p180-depleted extracts showed a reduction in ribosome binding to the level of trypsin-inactivated controls as well as a loss in their ability to cotranslationally translocate two different secretory protein precursors. When purified p180 was added back to depleted extracts before proteoliposome formation, both ribosome binding and translocation activity were restored. In addition, the monoclonal antibodies, as well as their Fab' fragments, were able to inhibit ribosome binding and protein translocation when bound to intact rough microsomes. These data provide direct evidence that the 180-kD ribosome receptor is essential for ribosome binding and for the translocation of nascent proteins across the membrane of the rough ER.

scholarly journals Translational Control of Secretory Proteins in Health and Disease

2020 ◽  
Vol 21 (7) ◽  
pp. 2538 ◽  
Author(s):  
Andrey L. Karamyshev ◽  
Elena B. Tikhonova ◽  
Zemfira N. Karamysheva
Keyword(s):  
Translational Control ◽  
Molecular Mechanisms ◽  
Signal Recognition Particle ◽  
Secretory Protein ◽  
Human Diseases ◽  
Secretory Proteins ◽  
Signal Peptides ◽  
Protein Biogenesis ◽  
Health And Disease

Secretory proteins are synthesized in a form of precursors with additional sequences at their N-terminal ends called signal peptides. The signal peptides are recognized co-translationally by signal recognition particle (SRP). This interaction leads to targeting to the endoplasmic reticulum (ER) membrane and translocation of the nascent chains into the ER lumen. It was demonstrated recently that in addition to a targeting function, SRP has a novel role in protection of secretory protein mRNAs from degradation. It was also found that the quality of secretory proteins is controlled by the recently discovered Regulation of Aberrant Protein Production (RAPP) pathway. RAPP monitors interactions of polypeptide nascent chains during their synthesis on the ribosomes and specifically degrades their mRNAs if these interactions are abolished due to mutations in the nascent chains or defects in the targeting factor. It was demonstrated that pathological RAPP activation is one of the molecular mechanisms of human diseases associated with defects in the secretory proteins. In this review, we discuss recent progress in understanding of translational control of secretory protein biogenesis on the ribosome and pathological consequences of its dysregulation in human diseases.

scholarly journals Protein translocation across the endoplasmic reticulum. II. Isolation and characterization of the signal recognition particle receptor.

1982 ◽  
Vol 95 (2) ◽  
pp. 470-477 ◽  
Author(s):  
R Gilmore ◽  
P Walter ◽  
G Blobel
Keyword(s):  
Gradient Centrifugation ◽  
Signal Recognition Particle ◽  
Preceding Paper ◽  
Signal Peptidase ◽  
Receptor Interaction ◽  
Peptidase Activity ◽  
Signal Recognition ◽  
Isolation And Characterization ◽  
Cell Biol ◽  
Microsomal Membranes

The signal recognition particle (SRP)-mediated elongation arrest of the synthesis of nascent secretory proteins can be released by salt-extracted rough microsomal membranes (Walter, P., and G. Blobel, 1981, J. Cell Biol, 91:557-561). Both the arrest-releasing activity and the signal peptidase activity were solubilized from rough microsomal membranes using the nonionic detergent Nikkol in conjunction with 250 mM KOAc. Chromatography of this extract on SRP-Sepharose separated the arrest-releasing activity from the signal peptidase activity. Further purification of the arrest-releasing activity using sucrose gradient centrifugation allowed the identification of a 72,000-dalton polypeptide as the protein responsible for the activity. Based upon its affinity for SRP, we refer to the 72,000-dalton protein as the SRP receptor. A 60,000-dalton protein fragment (Meyer, D. I., and B. Dobberstein, 1980, J. Cell Biol., 87:503-508) that had been shown previously to reconstitute the translocation activity of protease-digested membranes, was shown here by peptide mapping and immunological criteria to be derived from the SRP receptor. Findings that are in part similar, and in part different from these reported here and in our preceding paper were made independently (Meyer, D. I., E. Krause, and B. Dobberstein, 1982, Nature (Lond.). 297:647-650) and the term "docking protein" was proposed for the SRP receptor. A lower membrane content of both SRP and the SRP receptor than that of membrane bound ribosomes suggests that the SRP-SRP receptor interaction may exist transiently during the formation of a ribosome-membrane junction and during translocation.

scholarly journals Targeting of the hepatitis B virus precore protein to the endoplasmic reticulum membrane: after signal peptide cleavage translocation can be aborted and the product released into the cytoplasm.

1988 ◽  
Vol 106 (4) ◽  
pp. 1093-1104 ◽  
Author(s):  
P D Garcia ◽  
J H Ou ◽  
W J Rutter ◽  
P Walter
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Core Protein ◽  
Signal Sequence ◽  
Signal Peptidase ◽  
Amino Terminus ◽  
Endoplasmic Reticulum Membrane ◽  
Nucleic Acid Binding ◽  
Precore Region ◽  
B Virus

The major hepatitis B virus (HBV) core protein is a viral structural protein involved in nucleic acid binding. Its coding sequence contains an extension of 29 codons (the "precore" region) at the amino terminus of the protein which is present in a fraction of the viral transcripts. This region is evolutionarily conserved among mammalian and avian HBVs, suggesting it has functional importance, although at least for duck HBV it has been shown to be nonessential for replication of infectious virions. Using in vitro assays for protein translocation across the endoplasmic reticulum membrane, we found that the precore region of the HBV genome encodes a signal sequence. This signal sequence was recognized by signal recognition particle, which targeted the nascent precore protein to the endoplasmic reticulum membrane with efficiencies comparable to those of other mammalian secretory proteins. A 19-amino acid signal peptide was removed by signal peptidase on the lumenal side of the microsomal membrane, generating a protein similar to the HBV major core protein, but containing 10 additional amino acids from the precore region at its amino terminus. Surprisingly, we found that 70-80% of this signal peptidase-cleaved product was localized on the cytoplasmic side of the microsomal vesicles and was not associated with the membranes. We conclude that translocation was aborted by an unknown mechanism, then the protein disengaged from the translocation machinery and was released back into the cytoplasm. Thus, a cytoplasmically disposed protein was created whose amino terminus resulted from signal peptidase cleavage. The remaining 20-30% appeared to be completely translocated into the lumen of the microsomes. A deletion mutant lacking the carboxy-terminal nucleic acid binding domain of the precore protein was similarly partitioned between the lumen of the microsomes and the cytoplasmic compartment, indicating that this highly charged domain is not responsible for the aborted translocation. We discuss the implications of our findings for the protein translocation process and suggest a possible role in the virus life cycle.

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