The signal sequence interacts with the methionine-rich domain of the 54-kD protein of signal recognition particle.

The signal sequence of nascent preprolactin interacts with the 54-kD protein of the signal recognition particle (SRP54). To identify the domain or site on SRP54 that interacts with the signal sequence we used a photocross-linking approach followed by limited proteolysis and immunoprecipitation using anti-peptide antibodies specific for defined regions of SRP54. We found that the previously identified methionine-rich RNA-binding domain of SRP54 (SRP54M domain) also interacts with the signal sequence. The smallest fragment that was found to be crosslinked to the signal sequence comprised the COOH-terminal 6-kD segment of the SRP54M domain. No cross-link to the putative GTP-binding domain of SRP54 (SRP54G domain) was found. Proteolytic cleavage between the SRP54M domain and SRP54G domain did not impair the subsequent interaction between the signal sequence and the SRP54M domain. Our results show that both the RNA binding and signal sequence binding functions of SRP54 are performed by the SRP54M domain.

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The 54-kD protein of signal recognition particle contains a methionine-rich RNA binding domain.

The Journal of Cell Biology ◽

10.1083/jcb.111.5.1793 ◽

1990 ◽

Vol 111 (5) ◽

pp. 1793-1802 ◽

Cited By ~ 97

Author(s):

K Römisch ◽

J Webb ◽

K Lingelbach ◽

H Gausepohl ◽

B Dobberstein

Keyword(s):

Amino Acids ◽

Rna Binding ◽

Signal Sequence ◽

Signal Recognition Particle ◽

Binding Domain ◽

Secretory Proteins ◽

Signal Recognition ◽

Rna Binding Domain ◽

Necessary And Sufficient

Signal recognition particle (SRP) plays the key role in targeting secretory proteins to the membrane of the endoplasmic reticulum (Walter, P., and V. R. Lingappa. 1986. Annu. Rev. Cell Biol. 2:499-516). It consists of SRP7S RNA and six proteins. The 54-kD protein of SRP (SRP54) recognizes the signal sequence of nascent polypeptides. The 19-kD protein of SRP (SRP19) binds to SRP7S RNA directly and is required for the binding of SRP54 to the particle. We used deletion mutants of SRP19 and SRP54 and an in vitro assembly assay in the presence of SRP7S RNA to define the regions in both proteins which are required to form a ribonucleoprotein particle. Deletion of the 21 COOH-terminal amino acids of SRP19 does not interfere with its binding to SRP7S RNA. Further deletions abolish SRP19 binding to SRP7S RNA. The COOH-terminal 207 amino acids of SRP54 (M domain) were found to be necessary and sufficient for binding to the SRP19/7S RNA complex in vitro. Limited protease digestion of purified SRP confirmed our results for SRP54 from the in vitro binding assay. The SRP54M domain could also bind to Escherichia coli 4.5S RNA that is homologous to part of SRP7S RNA. We suggest that the methionine-rich COOH terminus of SRP54 is a RNA binding domain and that SRP19 serves to establish a binding site for SRP54 on the SRP7S RNA.

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The methionine-rich domain of the 54 kd protein subunit of the signal recognition particle contains an RNA binding site and can be crosslinked to a signal sequence.

The EMBO Journal ◽

10.1002/j.1460-2075.1990.tb07902.x ◽

1990 ◽

Vol 9 (13) ◽

pp. 4511-4517 ◽

Cited By ~ 155

Author(s):

D. Zopf ◽

H. D. Bernstein ◽

A. E. Johnson ◽

P. Walter

Keyword(s):

Binding Site ◽

Rna Binding ◽

Signal Sequence ◽

Signal Recognition Particle ◽

Signal Recognition ◽

Protein Subunit ◽

Rich Domain

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

Molecular Biology of the Cell ◽

10.1091/mbc.5.8.887 ◽

1994 ◽

Vol 5 (8) ◽

pp. 887-897 ◽

Cited By ~ 17

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.

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

The Journal of Cell Biology ◽

10.1083/jcb.103.6.2253 ◽

1986 ◽

Vol 103 (6) ◽

pp. 2253-2261 ◽

Cited By ~ 115

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.

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SRP RNA Remodeling by SRP68 Explains Its Role in Protein Translocation

Science ◽

10.1126/science.1249094 ◽

2014 ◽

Vol 344 (6179) ◽

pp. 101-104 ◽

Cited By ~ 26

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.

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

eLife ◽

10.7554/elife.04418 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 28

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.

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Mechanisms of membrane protein biogenesis

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s205327331408838x ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1161-C1161

Author(s):

Irmgard Sinning

Keyword(s):

Membrane Proteins ◽

Signal Sequence ◽

Signal Recognition Particle ◽

Signal Recognition ◽

Protein Biogenesis ◽

Cargo Proteins ◽

Target Membrane ◽

Hydrophobic Nature ◽

Correct Target ◽

Srp Rna

More than 25% of the cellular proteome comprise membrane proteins that have to be inserted into the correct target membrane. Most membrane proteins are delivered to the membrane by the signal recognition particle (SRP) pathway which relies on the recognition of an N-terminal signal sequence. In contrast to this co-translational mechanism, which avoids problems due to the hydrophobic nature of the cargo proteins, tail-anchored (TA) membrane proteins utilize a post-translational mechanism for membrane insertion – the GET pathway (guided entry of tail-anchored membrane proteins). The SRP and GET pathways are both regulated by GTP and ATP binding proteins of the SIMIBI family. However, in the SRP pathway the SRP RNA plays a unique regulatory role. Recent insights into eukaryotic SRP will be discussed.

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