scholarly journals Translocation of proteins across the endoplasmic reticulum III. Signal recognition protein (SRP) causes signal sequence-dependent and site-specific arrest of chain elongation that is released by microsomal membranes.

1981 ◽  
Vol 91 (2) ◽  
pp. 557-561 ◽  
Author(s):  
P Walter ◽  
G Blobel
Keyword(s):  
Signal Sequence ◽  
Ribosomal Subunit ◽  
Inhibitory Effect ◽  
Secretory Protein ◽  
Chain Elongation ◽  
Large Ribosomal Subunit ◽  
Site Specific ◽  
Amino Terminal ◽  
Nascent Chain ◽  
Microsomal Membranes

The previously observed (Walter, et al. 1981 J. Cell Biol. 91:545-550) inhibitory effect of SRP selectively on the cell-free translation of mRNA for secretory protein (preprolactin) was shown here to be caused by a signal sequence-induced and site-specific arrest in polypeptide chain elongation. The Mr of the SRP-arrested nascent preprolactin chain was estimated to be 8,000 corresponding to approximately 70 amino acid residues. Because the signal sequence of preprolactin comprises 30 residues and because approximately 40 residues of the nascent chain are buried (protected from protease) in the large ribosomal subunit, we conclude that it is the interaction of SRP with the amino-terminal signal peptide of the nascent chain (emerged from the large ribosomal subunit) that modulates translation and thereby causes an arrest in chain elongation. This arrest is released upon SRP-mediated binding of the elongation-arrested ribosomes to the microsomal membrane, resulting in chain completion and translocation into the microsomal vesicle.

scholarly journals Biosynthesis of rice seed alpha-amylase: proteolytic processing and glycosylation of precursor polypeptides by microsomes.

1983 ◽  
Vol 96 (3) ◽  
pp. 802-806 ◽  
Author(s):  
S Miyata ◽  
T Akazawa
Keyword(s):  
Signal Sequence ◽  
Inhibitory Effect ◽  
Secretory Protein ◽  
Proteolytic Processing ◽  
Alpha Amylase ◽  
Rice Seed ◽  
Microsomal Membranes ◽  
Enzyme Molecules ◽  
Triton X ◽  
Polypeptide Chains

Microsomes prepared from the rice seed scutellum were incubated in wheat germ extracts (S-100 fraction) to direct the synthesis of alpha-amylase, a secretory protein subject to proteolytic processing (cleavage of the N-terminal signal sequence) as well as glycosylation during its biosynthesis. The characterization and identification of the immunoprecipitable products synthesized were performed by SDS gel electrophoresis and subsequent fluorography. The molecular weight of the alpha-amylase synthesized by the microsomes was found to be identical with that of the mature secretory form of the enzyme on the basis of electrophoretic mobilities. A significant portion of the enzyme molecules synthesized was shown to be segregated into the microsomal vesicles and protected against digestion by endo-beta-N-acetylglucosaminidase, indicating that both proteolytic processing and glycosylation of the precursor polypeptide chains take place in the microsomes. The modification of the polypeptide chains was further examined by disrupting the microsomal membranes with Triton X-100. Detergent treatment of the microsomes prior to protein synthesis caused an inhibition of both proteolytic processing and glycosylation of the polypeptide chains, leading to the synthesis of the unprocessed nascent (precursor I), processed but nonglycosylated nascent (precursor II) forms, in addition to the mature form of alpha-amylase. Furthermore, the results of time-sequence analysis of the inhibitory effect of Triton X-100 on the modification of the polypeptide chains have led us to conclude that both proteolytic processing and subsequent glycosylation occur in the microsomes during the biosynthesis of alpha-amylase.

scholarly journals Ligand crowding at a nascent signal sequence

2003 ◽  
Vol 163 (1) ◽  
pp. 35-44 ◽  
Author(s):  
Gottfried Eisner ◽  
Hans-Georg Koch ◽  
Konstanze Beck ◽  
Joseph Brunner ◽  
Matthias Müller
Keyword(s):  
Escherichia Coli ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Secretory Protein ◽  
Trigger Factor ◽  
Cross Linking ◽  
Signal Recognition ◽  
Site Specific ◽  
E Coli ◽  
Nascent Chain

We have systematically analyzed the molecular environment of the signal sequence of a growing secretory protein from Escherichia coli using a stage- and site-specific cross-linking approach. Immediately after emerging from the ribosome, the signal sequence of pOmpA is accessible to Ffh, the protein component of the bacterial signal recognition particle, and to SecA, but it remains attached to the surface of the ribosome via protein L23. These contacts are lost upon further growth of the nascent chain, which brings the signal sequence into sole proximity to the chaperone Trigger factor (TF). In its absence, nascent pOmpA shows extended contacts with L23, and even long chains interact in these conditions proficiently with Ffh. Our results suggest that upon emergence from the ribosome, the signal sequence of an E. coli secretory protein gradually becomes sequestered by TF. Although TF thereby might control the accessibility of pOmpA's signal sequence to Ffh and SecA, it does not influence interaction of pOmpA with SecB.

scholarly journals Antiribophorin antibodies inhibit the targeting to the ER membrane of ribosomes containing nascent secretory polypeptides.

1990 ◽  
Vol 111 (4) ◽  
pp. 1335-1342 ◽  
Author(s):  
Y H Yu ◽  
D D Sabatini ◽  
G Kreibich
Keyword(s):  
Protein Synthesis ◽  
Polyclonal Antibodies ◽  
Liver Microsomes ◽  
Inhibitory Effect ◽  
Secretory Protein ◽  
Rat Liver Microsomes ◽  
Membrane Glycoproteins ◽  
Site Specific ◽  
Dog Pancreas ◽  
Er Membrane

Polyclonal antibodies directed against ribophorins I and II, two membrane glycoproteins characteristic of the rough endoplasmic reticulum, inhibit the cotranslational translocation of a secretory protein growth hormone into the lumen of dog pancreas or rat liver microsomes. As expected, site-specific antibodies to epitopes located within the cytoplasmic domain of ribophorin I, but not antibodies to epitopes in the luminal domain of this protein, were effective in inhibiting translocation. Since monovalent Fab fragments were as inhibitory as intact IgG molecules, ribophorins must be closely associated with the translocation site and, therefore, are likely to function at some stage in the translocation process. In all cases, the antibodies that inhibited translocation also caused a significant reduction in total protein synthesis and treatments that neutralized their capacity to inhibit translocation also prevented their inhibitory effect on protein synthesis. This would be expected if the antibodies blocked the membrane-mediated relief of the SRP-induced arrest of polypeptide elongation. The antibodies were effective only when added before translocation was allowed to begin. In this case, they prevented the targeting of active ribosomes containing mRNA and nascent chains to the ER membrane. Thus, ribophorins must either directly participate in targeting or be so close to the targeting site that the antibodies sterically blocked this early phase of the translocation process.

scholarly journals A nascent membrane protein is located adjacent to ER membrane proteins throughout its integration and translation.

1991 ◽  
Vol 112 (5) ◽  
pp. 809-821 ◽  
Author(s):  
R N Thrift ◽  
D W Andrews ◽  
P Walter ◽  
A E Johnson
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Secretory Protein ◽  
In Vitro Translation ◽  
Nascent Chain ◽  
Transmembrane Sequence ◽  
Er Membrane

The immediate environment of nascent membrane proteins undergoing integration into the ER membrane was investigated by photocrosslinking. Nascent polypeptides of different lengths, each containing a single IgM transmembrane sequence that functions either as a stop-transfer or a signal-anchor sequence, were synthesized by in vitro translation of truncated mRNAs in the presence of N epsilon-(5-azido-2-nitrobenzoyl)-Lys-tRNA, signal recognition particle, and microsomal membranes. This yielded nascent chains with photoreactive probes at one end of the transmembrane sequence where two lysine residues are located. When irradiated, these nascent chains reacted covalently with several ER proteins. One prominent crosslinking target was a glycoprotein similar in size to a protein termed mp39, shown previously to be situated adjacent to a secretory protein during its translocation across the ER membrane (Krieg, U. C., A. E. Johnson, and P. Walter. 1989. J. Cell Biol. 109:2033-2043; Wiedmann, M., D. Goerlich, E. Hartmann, T. V. Kurzchalia, and T. A. Rapoport. 1989. FEBS (Fed. Eur. Biochem. Soc.) Lett. 257:263-268) and likely to be identical to a protein previously designated the signal sequence receptor (Wiedmann, M., T. V. Kurzchalia, E. Hartmann, and T. A. Rapoport. 1987. Nature (Lond.). 328:830-833). Changing the orientation of the transmembrane domain in the bilayer, or making the transmembrane domain the first topogenic sequence in the nascent chain instead of the second, did not significantly alter the identities of the ER proteins that were the primary crosslinking targets. Furthermore, the nascent chains crosslinked to the mp39-like glycoprotein and other microsomal proteins even after the cytoplasmic tail of the nascent chain had been lengthened by nearly 100 amino acids beyond the stop-transfer sequence. Yet when the nascent chain was allowed to terminate normally, the major photocrosslinks were no longer observed, including in particular that to the mp39-like glycoprotein. These results show that the transmembrane segment of a nascent membrane protein is located adjacent to the mp39-like glycoprotein and other ER proteins during the integration process, and that at least a portion of the nascent chain remains in close proximity to these ER proteins until translation has been completed.

scholarly journals Effect of modeccin on the steps of peptide-chain elongation

1978 ◽  
Vol 176 (2) ◽  
pp. 371-379 ◽  
Author(s):  
L Montanaro ◽  
S Sperti ◽  
M Zamboni ◽  
M Denaro ◽  
G Testoni ◽  
...  
Keyword(s):  
Ribosomal Subunit ◽  
Elongation Factor ◽  
Peptide Bond ◽  
Inhibitory Effect ◽  
Artemia Salina ◽  
Chain Elongation ◽  
Peptide Bond Formation ◽  
Experimental Conditions ◽  
Related Plant ◽  
The Individual

Modeccin inhibits polypeptide-chain elongation catalysed by Artemia salina (brine shrimp) ribosomes by inactivating the 60 S ribosomal subunit. Among the individual steps of elongation, peptide-bond formation, catalysed by 60 S peptidyltransferase, is unaffected by the toxin, whereas the binding of EF 2 (elongation factor 2) to ribosomes is strongly inhibited. Modeccin does not affect the poly(U)-dependent non-enzymic binding of either deacylated tRNAPhe or phenylalanyl-tRNA to ribosomes. The inhibitory effect of modeccin on the EF 1 (elongation factor 1)-dependent binding of phenylalanyl-tRNA is discussed, since it is decreased by tRNAPhe, which stimulates the binding reaction. The analysis of the distribution of ribosome-bound radioactivity during protein synthesis shows that modeccin consistently inhibits the radioactivity bound as long-chain peptides, but depending on the experimental conditions, can leave unchanged or even greatly stimulates the radioactivity bound as phenylalanyl-tRNA and/or short-chain peptides. It is concluded that, during the complete elongation cycle, modeccin does not affect the binding of the first aminoacyl-tRNA to ribosomes, but inhibits some step in the subsequent repetitive activity of either EF 1 or EF 2. The results obtained indicate that the mechanism of action of modeccin is very similar to that of ricin and related plant toxins such as abrin and crotin.

The Role of Intracellular Membranes in Protein Processing

1981 ◽  
Vol 39 ◽  
pp. 514-515
Author(s):  
Dennis Shields ◽  
Thomas G. Warren ◽  
Sara E. Roth ◽  
Reza F. Green
Keyword(s):  
Endoplasmic Reticulum ◽  
Signal Peptide ◽  
Messenger Rna ◽  
Polypeptide Chain ◽  
Secretory Protein ◽  
Free Protein ◽  
Amino Terminal ◽  
Free Translation ◽  
Microsomal Membranes

Most polypeptides destined for secretion are synthesized on polyribosomes bound to the membrane of the endoplasmic reticulum (E.R.), in contrast, cytosolic proteins are made on free ribosomes. When the messenger RNA (mRNA) for a secretory protein is translated in a cell-free protein synthesizing system, the product is usually larger than the mature protein by about 3,000 daltons. Numerous studies have demonstrated that the higher molecular weight of the cell-free translation product can be attributed to an amino terminal extension of about 20-30 amino acids termed the “signal peptide”. This signal peptide is thought to mediate binding of ribosomes bearing the nascent polypeptide chain to the membrane of the endoplasmic reticulum. Upon interaction with the E.R., the polypeptide chain is translocated across the membrane usually resulting in proteolytic removal of the signal peptide and segregation of the “processed” polypeptide into the ER. cisternae. This series of reactions can be followed in vitro by supplementing the cell-free protein synthesizing system with heterologous microsomal membranes which have been stripped of their endogenous ribosomes.

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.

scholarly journals Regulation of the Ribosome–Membrane Junction at Early Stages of Presecretory Protein Translocation in the Mammalian Endoplasmic Reticulum

1997 ◽  
Vol 139 (7) ◽  
pp. 1697-1708 ◽  
Author(s):  
Christopher V. Nicchitta ◽  
Tianli Zheng
Keyword(s):  
Endoplasmic Reticulum ◽  
Fusion Protein ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Secretory Protein ◽  
Membrane Topology ◽  
Hydrophobic Core ◽  
Conducting Channel ◽  
Nascent Chain ◽  
Potential Mode

A series of fusion protein constructs were designed to investigate the contribution of secretory nascent chains to regulation of the ribosome–membrane junction in the mammalian endoplasmic reticulum. As a component of these studies, the membrane topology of the signal sequence was determined at stages of protein translocation immediately after targeting and before signal sequence cleavage. Truncated translation products were used to delimit the analysis to defined stages of translocation. In a study of secretory protein precursors, formation of a protease-resistant ribosome–membrane junction, currently thought to define the pathway of the translocating nascent chain, was observed to be precursor- and stage-dependent. Analysis of the binding of early intermediates indicated that the nascent chain was bound to the membrane independent of the ribosome, and that the binding was predominately electrostatic. The membrane topology of the signal sequence was determined as a function of the stage of translocation, and was found to be identical for all assayed intermediates. Unexpectedly, the hydrophobic core of the signal sequence was observed to be accessible to the cytosolic face of the membrane at stages of translocation immediately after targeting as well as stages before signal sequence cleavage. Removal of the ribosome from bound intermediates did not disrupt subsequent translocation, suggesting that the active state of the protein-conducting channel is maintained in the absence of the bound ribosome. A model describing a potential mode of regulation of the ribosome–membrane junction by the nascent chain is presented.

scholarly journals 23S rRNA 2058A→G Alteration Mediates Ketolide Resistance in Combination with Deletion in L22

2006 ◽  
Vol 50 (11) ◽  
pp. 3816-3823 ◽  
Author(s):  
Rita Berisio ◽  
Natascia Corti ◽  
Peter Pfister ◽  
Ada Yonath ◽  
Erik C. Böttger
Keyword(s):  
Mycobacterium Smegmatis ◽  
Deinococcus Radiodurans ◽  
Weak Interactions ◽  
Ribosomal Subunit ◽  
Drug Binding ◽  
23S Rrna ◽  
Large Ribosomal Subunit ◽  
Drug Binding Site ◽  
Nascent Chain ◽  
Carbamate Group

ABSTRACT Resistance to macrolides and ketolides occurs mainly via alterations in RNA moieties of the drug-binding site. Using an A2058G mutant of Mycobacterium smegmatis, additional telithromycin resistance was acquired via deletion of 15 residues from protein L22. Molecular modeling, based on the crystal structure of the large ribosomal subunit from Deinococcus radiodurans complexed with telithromycin, shows that the telithromycin carbamate group is located in the proximity of the tip of the L22 hairpin-loop, allowing for weak interactions between them. These weak interactions may become more important once the loss of A2058 interactions destabilizes drug binding, presumably resulting in a shift of the drug toward the other side of the tunnel, namely, to the vicinity of L22. Hence, the deletion of 15 residues from L22 may further destabilize telithromycin binding and confer telithromycin resistance. Such deletions may also lead to notable differences in the tunnel outline, as well as to an increase of its diameter to a size, allowing the progression of the nascent chain.

Site-Specific Solid-State NMR Studies of “Trigger Factor” in Complex with the Large Ribosomal Subunit 50S

2015 ◽  
Vol 54 (14) ◽  
pp. 4367-4369 ◽  
Author(s):  
Emeline Barbet-Massin ◽  
Chih-Ting Huang ◽  
Venita Daebel ◽  
Shang-Te Danny Hsu ◽  
Bernd Reif
Keyword(s):  
Solid State ◽  
Solid State Nmr ◽  
Ribosomal Subunit ◽  
Trigger Factor ◽  
Large Ribosomal Subunit ◽  
Nmr Studies ◽  
Site Specific
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