scholarly journals Protein translocation across the endoplasmic reticulum. I. Detection in the microsomal membrane of a receptor for the signal recognition particle.

1982 ◽  
Vol 95 (2) ◽  
pp. 463-469 ◽  
Author(s):  
R Gilmore ◽  
G Blobel ◽  
P Walter
Keyword(s):  
Membrane Fraction ◽  
Protein Translocation ◽  
Signal Recognition Particle ◽  
Secretory Protein ◽  
Microsomal Membrane ◽  
Supernatant Fraction ◽  
Signal Recognition ◽  
Cell Biol ◽  
Protease Digestion ◽  
Microsomal Membranes

Salt-extracted microsomal membranes (K-RM) contain an activity that is capable of releasing the signal recognition particle (SRP)-mediated elongation arrest of the synthesis of secretory polypeptides (Walter, P., and G. Blobel, 1981, J. Cell Biol., 91:557-561). This arrest-releasing activity was shown to be a function of an integral microsomal membrane protein, termed the SRP receptor (Gilmore, R., P. Walter, and G. Blobel, 1982, J. Cell Biol., 95:470-477). We attempted to solubilize the arrest-releasing activity of the SRP receptor by mild protease digestion of K-RM using either trypsin or elastase. We found, however, that neither a trypsin, nor an elastase "solubilized" supernatant fraction exhibited the arrest-releasing activity. Only when either the trypsin- or elastase-derived supernatant fraction was combined with the trypsinized membrane fraction, which by itself was also inactive, was the arrest-releasing activity restored. Release of the elongation arrest was followed by the translocation of the secretory protein across the microsomal membrane and the removal of the signal peptide. Thus, although we have been unable to proteolytically sever the arrest-releasing activity from K-RM and thereby to uncouple the release of the elongation arrest from the process of chain translocation, we have been able to proteolytically dissect and reconstitute the arrest-releasing activity. Furthermore, we found that the arrest-releasing activity of the SRP receptor can be inactivated by alkylation of K-RM with N-ethylmaleimide.

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 Signal sequence recognition and targeting of ribosomes to the endoplasmic reticulum by the signal recognition particle do not require GTP.

1994 ◽  
Vol 5 (8) ◽  
pp. 887-897 ◽  
Author(s):  
P J Rapiejko ◽  
R Gilmore
Keyword(s):  
Protein Translocation ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Beta Subunits ◽  
Signal Recognition ◽  
Binding Assays ◽  
Nascent Polypeptide ◽  
Sequence Recognition ◽  
Gtp Binding ◽  
Microsomal Membranes

The identification of GTP-binding sites in the 54-kDa subunit of the signal recognition particle (SRP) and in both the alpha and beta subunits of the SRP receptor has complicated the task of defining the step in the protein translocation reaction that is controlled by the GTP-binding site in the SRP. Ribonucleotide binding assays show that the purified SRP can bind GDP or GTP. However, crosslinking experiments show that SRP54 can recognize the signal sequence of a nascent polypeptide in the absence of GTP. Targeting of SRP-ribosome-nascent polypeptide complexes, formed in the absence of GTP, to microsomal membranes likewise proceeds normally. To separate the GTPase cycles of SRP54 and the alpha subunit of the SRP receptor (SR alpha), we employed an SR alpha mutant that displays a markedly reduced affinity for GTP. We observed that the dissociation of SRP54 from the signal sequence and the insertion of the nascent polypeptide into the translocation site could only occur when GTP binding to SR alpha was permitted. These data suggest that the GTP binding and hydrolysis cycles of both SRP54 and SR alpha are initiated upon formation of the SRP-SRP receptor complex.

scholarly journals Protein translocation across the yeast microsomal membrane is stimulated by a soluble factor.

1986 ◽  
Vol 103 (6) ◽  
pp. 2629-2636 ◽  
Author(s):  
M G Waters ◽  
W J Chirico ◽  
G Blobel
Keyword(s):  
Heat Treatment ◽  
Saccharomyces Cerevisiae ◽  
Protein Translocation ◽  
Sedimentation Coefficient ◽  
Protein S ◽  
Microsomal Membrane ◽  
Supernatant Fraction ◽  
Soluble Factor ◽  
Microsomal Membranes ◽  
Stimulation Of

We have found that a soluble activity present in the postribosomal supernatant fraction of Saccharomyces cerevisiae stimulates posttranslational translocation of yeast prepro-alpha-factor across yeast microsomal membranes. Stimulation of translocation is not due to a nonspecific affect on ATP levels. The activity is likely to be due to protein(s) as it is destroyed by N-ethylmaleimide, protease, or heat treatment but not by incubation with RNase. Its apparent sedimentation coefficient is approximately 9.6 S.

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 Formation of a functional ribosome-membrane junction during translocation requires the participation of a GTP-binding protein.

1986 ◽  
Vol 103 (6) ◽  
pp. 2253-2261 ◽  
Author(s):  
T Connolly ◽  
R Gilmore
Keyword(s):  
Signal Sequence ◽  
Binding Protein ◽  
Signal Recognition Particle ◽  
Secretory Protein ◽  
Signal Recognition ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Protease Digestion ◽  
Nascent Chain

The requirement for ribonucleotides and ribonucleotide hydrolysis was examined at several distinct points during translocation of a secretory protein across the endoplasmic reticulum. We monitored binding of in vitro-assembled polysomes to microsomal membranes after removal of ATP and GTP. Ribonucleotides were not required for the initial low salt-insensitive attachment of the ribosome to the membrane. However, without ribonucleotides the nascent secretory chains were sensitive to protease digestion and were readily extracted from the membrane with either EDTA or 0.5 M KOAc. In contrast, nascent chains resisted extraction with either EDTA or 0.5 M KOAc and were insensitive to protease digestion after addition of GTP or nonhydrolyzable GTP analogues. Translocation of the nascent secretory polypeptide was detected only when ribosome binding was conducted in the presence of GTP. Thus, translocation-competent binding of the ribosome to the membrane requires the participation of a novel GTP-binding protein in addition to the signal recognition particle and the signal recognition particle receptor. The second event we examined was translocation and processing of a truncated secretory polypeptide. Membrane-bound polysomes bearing an 86-residue nascent chain were generated by translation of a truncated preprolactin mRNA. Ribonucleotide-independent translocation of the polypeptide was detected by cleavage of the 30-residue signal sequence after puromycin termination. Nascent chain transport, per se, is apparently dependent upon neither ribonucleotide hydrolysis nor continued elongation of the polypeptide once a functional ribosome-membrane junction has been established.

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.

SRP RNA Remodeling by SRP68 Explains Its Role in Protein Translocation

Science ◽  
2014 ◽  
Vol 344 (6179) ◽  
pp. 101-104 ◽  
Author(s):  
Jan Timo Grotwinkel ◽  
Klemens Wild ◽  
Bernd Segnitz ◽  
Irmgard Sinning
Keyword(s):  
Ribosomal Rna ◽  
Protein Targeting ◽  
Rna Binding ◽  
Protein Translocation ◽  
Signal Recognition Particle ◽  
Structural Basis ◽  
Signal Recognition ◽  
Rna Remodeling ◽  
Srp Rna ◽  
Arginine Rich Motif

The signal recognition particle (SRP) is central to membrane protein targeting; SRP RNA is essential for SRP assembly, elongation arrest, and activation of SRP guanosine triphosphatases. In eukaryotes, SRP function relies on the SRP68-SRP72 heterodimer. We present the crystal structures of the RNA-binding domain of SRP68 (SRP68-RBD) alone and in complex with SRP RNA and SRP19. SRP68-RBD is a tetratricopeptide-like module that binds to a RNA three-way junction, bends the RNA, and inserts an α-helical arginine-rich motif (ARM) into the major groove. The ARM opens the conserved 5f RNA loop, which in ribosome-bound SRP establishes a contact to ribosomal RNA. Our data provide the structural basis for eukaryote-specific, SRP68-driven RNA remodeling required for protein translocation.

scholarly journals Real-time observation of signal recognition particle binding to actively translating ribosomes

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Thomas R Noriega ◽  
Jin Chen ◽  
Peter Walter ◽  
Joseph D Puglisi
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Initial Step ◽  
Signal Recognition ◽  
Fluorescence Measurements ◽  
Nascent Chain ◽  
Time Observation

The signal recognition particle (SRP) directs translating ribosome-nascent chain complexes (RNCs) that display a signal sequence to protein translocation channels in target membranes. All previous work on the initial step of the targeting reaction, when SRP binds to RNCs, used stalled and non-translating RNCs. This meant that an important dimension of the co-translational process remained unstudied. We apply single-molecule fluorescence measurements to observe directly and in real-time E. coli SRP binding to actively translating RNCs. We show at physiologically relevant SRP concentrations that SRP-RNC association and dissociation rates depend on nascent chain length and the exposure of a functional signal sequence outside the ribosome. Our results resolve a long-standing question: how can a limited, sub-stoichiometric pool of cellular SRP effectively distinguish RNCs displaying a signal sequence from those that are not? The answer is strikingly simple: as originally proposed, SRP only stably engages translating RNCs exposing a functional signal sequence.

Sign in / Sign up

Export Citation Format

Share Document