SEARCH · PubMed Health

Results for “Signal Recognition Particle”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 55 records · Page 3Linked to original sources

PubMed1989

Evidence for a two-step mechanism involved in assembly of functional signal recognition particle receptor.

The signal recognition particle (SRP) and SRP receptor act sequentially to target nascent secretory proteins to the membrane of the ER. The SRP receptor consists of two subunits, SR alpha and SR beta, both tightly associated with the ER membrane. To examine the biogenesis of the SRP receptor we have developed a cell-free assay system that reconstitutes SR alpha membrane assembly and permits both anchoring and functional properties to be assayed independently. Our experiments reveal a mechanism involving at least two distinct steps, targeting to the ER and anchoring of the targeted molecule on the cytoplasmic face of the membrane. Both steps can be reconstituted in vitro to restore translocation activity to ER microsomes inactivated by alkylation with N-ethyl-maleimide. The characteristics elucidated for this pathway distinguish it from SRP-dependent targeting of secretory proteins, SRP-independent ER translocation of proteins such as prepromellitin, and direct insertion mechanisms of the type exemplified by cytochrome b5.

Endoplasmic Reticulum↗

PubMed1997

Interaction of rice and human SRP19 polypeptides with signal recognition particle RNA.

The signal recognition particle (SRP) controls the transport of secretory proteins into and across lipid bilayers. SRP-like ribonucleoprotein complexes exist in all organisms, including plants. We characterized the rice SRP RNA and its primary RNA binding protein, SRP19. The secondary structure of the rice SRP RNA was similar to that found in other eukaryotes; however, as in other plant SRP RNAs, a GUUUCA hexamer sequence replaced the highly conserved GNRA-tetranucleotide loop motif at the apex of helix 8. The small domain of the rice SRP RNA was reduced considerably. Structurally, rice SRP19 lacked two small region that can be present in other SRP19 homologues. Conservative structure prediction and site-directed mutagenesis of rice and human SRP19 polypeptides indicated that binding to the SRP RNAs occurred via a loop that is present in the N-domain of both proteins. Rice SRP19 protein was able to form a stable complex with the rice SRP RNA in vitro. Furthermore, heterologous ribonucleoprotein complexes with components of the human SRP were assembled, thus confirming a high degree of structural and functional conservation between plant and mammalian SRP components.

Amino Acid Sequence↗

PubMed1991

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.

Animals↗

PubMed1982

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

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.

Centrifugation, Density Gradient↗

PubMed1992

Protein translocation across the ER requires a functional GTP binding site in the alpha subunit of the signal recognition particle receptor.

The signal recognition particle (SRP)-mediated translocation of proteins across the RER is a GTP dependent process. Analysis of the primary amino acid sequence of one protein subunit of SRP (SRP54), as well as the alpha subunit of the SRP receptor (SR alpha), has indicated that these proteins contain predicted GTP binding sites. Several point mutations confined to the GTP binding consensus elements of SR alpha were constructed by site specific mutagenesis to define a role for the GTP binding site in SR alpha during protein translocation. The SR alpha mutants were analyzed using an in vitro system wherein SR alpha-deficient microsomal membranes were repopulated with SR alpha by in vitro translation of wild-type or mutant mRNA transcripts. SRP receptors containing SR alpha point mutants were analyzed for their ability to function in protein translocation and to form guanylyl-5'-imidodiphosphate (Gpp[NH]p) stabilized complexes with the SRP. Mutations in SR alpha produced SRP receptors that were either impaired or inactive in protein translocation. These SRP receptors were likewise unable to form Gpp(NH)p stabilized complexes with the SRP. One SR alpha point mutant, Thr 588 to Asn 588, required 50- to 100-fold higher concentrations of GTP relative to the wild-type SR alpha to function in protein translocation. This mutant has provided information on the reaction step in protein translocation that involves the GTP binding site in the alpha subunit of the SRP receptor.

Animals↗

PubMedDate not supplied

The signal sequence of nascent preprolactin interacts with the 54K polypeptide of the signal recognition particle.

Hydrophobic signal sequences direct the translocation of nascent secretory proteins and many membrane proteins across the membrane of the endoplasmic reticulum. Initiation of this process involves the signal recognition particle (SRP), which consists of six polypeptide chains and a 7S RNA and interacts with ribosomes carrying nascent secretory polypeptide chains. In the case of aminoterminal, cleavable signal sequences, in the absence of microsomal membranes it exerts a site-specific translational arrest in vitro. The size of the arrested fragment (60-70 amino-acid residues) suggests that elongation stops when the signal sequence has emerged fully from the ribosome. However, a direct interaction between the signal sequence and SRP has not previously been demonstrated and has even been questioned recently. We now show for the first time a direct interaction between the signal sequence of a secretory protein and a component of SRP, the 45K polypeptide (relative molecular mass (Mr) 54,000). This was achieved by means of a new method of affinity labelling which involves the translational incorporation of an amino acid, carrying a photoreactive group, into nascent polypeptides.

Affinity Labels↗

PubMed1997

A truncation in the 14 kDa protein of the signal recognition particle leads to tertiary structure changes in the RNA and abolishes the elongation arrest activity of the particle.

The signal recognition particle (SRP) provides the molecular link between synthesis of polypeptides and their concomitant translocation into the endoplasmic reticulum. During targeting, SRP arrests or delays elongation of the nascent chain, thereby presumably ensuring a high translocation efficiency. Components of the Alu domain, SRP9/14 and the Alu sequences of SRP RNA, have been suggested to play a role in the elongation arrest function of SRP. We generated a truncated SRP14 protein, SRP14-20C, which forms, together with SRP9, a stable complex with SRP RNA. However, particles reconstituted with SRP9/14-20C, RC(9/14-20C), completely lack elongation arrest activity. RC(9/14-20C) particles have intact signal recognition, targeting and ribosome binding activities. SRP9/14-20C therefore only impairs interactions with the ribosome that are required to effect elongation arrest. This result provides evidence that direct interactions between the Alu domain components and the ribosome are required for this function. Furthermore, SRP9/14-20C binding to SRP RNA results in tertiary structure changes in the RNA. Our results strongly indicate that these changes account for the negative effect of SRP14 truncation on elongation arrest, thus revealing a critical role of the RNA in this function.

Amino Acid Sequence↗

PubMed1987

Wild type and mutant signal peptides of Escherichia coli outer membrane lipoprotein interact with equal efficiency with mammalian signal recognition particle.

The signal peptide of the outer membrane lipoprotein (OMLP) of Escherichia coli was shown to be capable of promoting protein translocation across mammalian microsomal membranes in vitro. We assayed translocation of a fusion protein containing the OMLP signal peptide and nine amino acids of OMLP fused in frame to beta-lactamase. The efficiency with which the mammalian translocation machinery recognizes and accepts the OMLP signal peptide as substrate is indistinguishable from that of mammalian secretory proteins. Upon translocation mammalian signal peptidase processes the pre-OMLP-beta-lactamase protein at different sites than are utilized in vivo by E. coli OMLP signal peptidase (signal peptidase II) but that can be predicted as mammalian signal peptidase cleavage sites. Mutants in the OMLP signal peptide were tested for their ability to promote translocation of the fusion protein in this assay system. It has been shown previously that mutants in the positively charged amino acids at the amino terminus of the signal peptide severely delay the translocation of OMLP in vivo in E. coli. However, these mutants had no detectable effect either on signal recognition by mammalian signal recognition particle or on the efficiency of translocation itself.

Amino Acid Sequence↗

PubMed2006

Alternate recruitment of signal recognition particle and trigger factor to the signal sequence of a growing nascent polypeptide.

Different from cytoplasmic membrane proteins, presecretory proteins of bacteria usually do not require the signal recognition particle for targeting to the Sec translocon. Nevertheless signal sequences of presecretory proteins have been found in close proximity to signal recognition particle immediately after they have emerged from the ribosome. We show here that at the ribosome, the molecular environment of a signal sequence depends on the nature of downstream sequence elements that can cause an alternate recruitment of signal recognition particle and the ribosome-associated chaperone Trigger factor to a growing nascent chain. While signal recognition particle and Trigger factor might remain bound to the same ribosome, both ligands are clearly able to displace each other from a nascent chain. The data also imply that a signal sequence owes its molecular environment to the fact that it remains closely apposed to the ribosomal exit site during growth of a nascent secretory protein.

Adenosine Triphosphatases↗

PubMed1985

Structure of the signal recognition particle by electron microscopy.

The signal recognition particle (SRP) is a ribonucleoprotein consisting of six distinct polypeptide components and one molecule of small cytoplasmic RNA (7SL RNA). The particle was previously shown to function in protein translocation across and protein integration into the endoplasmic reticulum membrane. Homogeneous signal recognition particle preparations were visualized by electron microscopy (i) after negative staining, (ii) by dark-field imaging of unstained specimens, and (iii) by platinum-shadowing. The results of each of these different techniques indicate that the signal recognition particle is a rod-shaped particle 5-6 nm wide and 23-24 nm long.

Animals↗

PubMed1993

Assembly of the 68- and 72-kD proteins of signal recognition particle with 7S RNA.

Signal recognition particle (SRP), the cytoplasmic ribonucleoprotein particle that mediates the targeting of proteins to the ER, consists of a 7S RNA and six different proteins. The 68- (SRP68) and 72- (SRP72) kD proteins of SRP are bound to the 7S RNA of SRP as a heterodimeric complex (SRP68/72). Here we describe the primary structure of SRP72 and the assembly of SRP68, SRP72 and 7S RNA into a ribonucleoprotein particle. The amino acid sequence deduced from the cDNA of SRP72 reveals a basic protein of 671 amino acids which shares no sequence similarity with any protein in the sequence data libraries. Assembly of SRP72 into a ribonucleoprotein particle required the presence of 7S RNA and SRP68. In contrast, SRP68 alone specifically bound to 7S RNA. SRP68 contacts the 7S RNA via its NH2-terminal half while COOH-terminal portions of SRP68 and SRP72 are in contact with each other in SRP. SRP68 thus serves as a link between 7S RNA and SRP72. As a large NH2-terminal domain of SRP72 is exposed on SRP it may be a site of contact to other molecules involved in the SRP cycle between the ribosome and the ER membrane.

Amino Acid Sequence↗

PubMed2002

Systematic site-directed mutagenesis of human protein SRP54: interactions with signal recognition particle RNA and modes of signal peptide recognition.

The amino acid residues of human protein SRP54 which are required for binding to SRP RNA were identified by generating 40 nonoverlapping tri-alanine alterations within its methionine-rich M-domain (SRP54M). The mutant polypeptides were expressed in Escherichia coli, and their ability to bind to human and Methanococcus jannaschii SRP RNA were determined in vitro. Residues at positions 379-387, 394-396, 400-405, and 409-411 of human SRP54 were within the predicted RNA binding site, and their alteration abolished the binding activities of the mutant polypeptides as expected. Changes at positions 418-423 had intermediate effects. Polypeptides containing mutations of 328-TLR-330 were inactive although these residues were far away from the presumed RNA binding site in the crystal structure of the free protein. Using the structures of the E. coli Ffh/4.5S core and of the human SRP54m dimer as templates, a molecular model of the complex between human SRP RNA helix 8 and a single SRP54M molecule was constructed in which Leucine 329 was positioned in closer proximity to the RNA binding domain. This representation was supported by studies of the SRP54m monomer/dimer ratio using gel filtration. The results were consistent with a change in the shape of the signal peptide binding groove upon binding of SRP54 to SRP RNA. We propose that the SRP RNA and a small region centered at a bulky nonpolar amino acid residue at position 329 of protein SRP54 play a critical role in the SRP-dependent binding and release of signal peptides.

Amino Acid Sequence↗

PubMed2001

Signal peptides bind and aggregate RNA. An alternative explanation for GTPase inhibition in the signal recognition particle.

N-terminal signal sequences can direct nascent protein chains to the inner membrane of prokaryotes and the endoplasmic reticulum of eukaryotes by interacting with the signal recognition particle. In this study, we show that isolated peptides corresponding to several bacterial signal sequences inhibit the GTPase activity of the Escherichia coli signal recognition particle, as previously reported (Miller, J. D., Bernstein, H. D., and Walter, P. (1994) Nature 367, 657-659), but not by the direct mechanism proposed. Instead, isolated signal peptides bind nonspecifically to the RNA component and aggregate the entire signal recognition particle, leading to a loss of its intrinsic GTPase activity. Surprisingly, only "functional" peptide sequences aggregate RNA; the peptides in general use as "nonfunctional" negative controls (e.g. those with deletions or charged substitutions within the hydrophobic core), are sufficiently different in physical character that they do not aggregate RNA and thus have no effect on the GTPase activity of the signal recognition particle. We propose that the reported effect of functional signal peptides on the GTPase activity of the signal recognition particle is an artifact of the high peptide concentrations and low salt conditions used in these in vitro studies and that signal sequences at the N terminus of nascent chains in vivo do not exhibit this activity.

GTP Phosphohydrolases↗

PubMed2000

Assembly of archaeal signal recognition particle from recombinant components.

Signal recognition particle (SRP) takes part in protein targeting and secretion in all organisms. Searches for components of archaeal SRP in primary databases and completed genomes indicated that archaea possess only homologs of SRP RNA, and proteins SRP19 and SRP54. A recombinant SRP was assembled from cloned, expressed and purified components of the hyperthermophilic archaeon Archaeoglobus fulgidus. Recombinant Af-SRP54 associated with the signal peptide of bovine pre-prolactin translated in vitro. As in mammalian SRP, Af-SRP54 binding to Af-SRP RNA required protein Af-SRP19, although notable amounts bound in absence of Af-SRP19. Archaeoglobus fulgidus SRP proteins also bound to full-length SRP RNA of the archaeon Methanococcus jannaschii, to eukaryotic human SRP RNA, and to truncated versions which corresponded to the large domain of SRP. Dependence on SRP19 was most pronounced with components from the same species. Reconstitutions with heterologous components revealed a significant potential of human SRP proteins to bind to archaeal SRP RNAs. Surprisingly, M.jannaschii SRP RNA bound to human SRP54M quantitatively in the absence of SRP19. This is the first report of reconstitution of an archaeal SRP from recombinantly expressed purified components. The results highlight structural and functional conservation of SRP assembly between archaea and eucarya.

Animals↗

PubMed1996

The Signal Recognition Particle Database (SRPDB).

The Signal Recognition Particle Database (SRPDB) provides aligned SRP RNA and SRP protein sequences, annotated and phylogenetically ordered. The current release included 93 RNAs and 29 proteins representing SRP9, SRP14, SRP19, SRP21, SRP54, SRP68 and SRP72. The SRPDB can be downloaded and is accessible via the World Wide Web.

Computer Communication Networks↗

PubMed2003

Role for both DNA and RNA in GTP hydrolysis by the Neisseria gonorrhoeae signal recognition particle receptor.

The prokaryotic signal recognition particle (SRP) targeting system is a complex of two proteins, FtsY and Ffh, and a 4.5S RNA that targets a subset of proteins to the cytoplasmic membrane cotranslationally. We previously showed that Neisseria gonorrhoeae PilA is the gonococcal FtsY homolog. In this work, we isolated the other two components of the gonococcal SRP, Ffh and 4.5S RNA, and characterized the interactions among the three SRP components by using gel retardation and nitrocellulose filter-binding assays and enzymatic analyses of the two proteins. In the current model of prokaryotic SRP function, based on studies of the Escherichia coli and mammalian systems, Ffh binds to 4.5S RNA and the Ffh-4.5S RNA complex binds to the signal sequence of nascent peptides and then docks with FtsY at the membrane. GTP is hydrolyzed by both proteins synergistically, and the nascent peptide is transferred to the translocon. We present evidence that the in vitro properties of the gonococcal SRP differ from those of previously described systems. GTP hydrolysis by PilA, but not that by Ffh, was stimulated by 4.5S RNA, suggesting a direct interaction between PilA and 4.5S RNA that has not been reported in other systems. This interaction was confirmed by gel retardation analyses in which PilA and Ffh, both alone and together, bound to 4.5S RNA. An additional novel finding was that P(pilE) DNA, previously shown by us to bind PilA in vitro, also stimulates PilA GTP hydrolysis. On the basis of these data, we hypothesize that DNA may play a role in targeting proteins via the SRP.

Amino Acid Sequence↗

PubMed1999

The Signal Recognition Particle Database (SRPDB).

The signal recognition particle database (SRPDB) is located at the University of Texas Health Science Center at Tyler and includes tabulations of SRP RNA, SRP protein and SRP receptor sequences. The sequences are annotated with links to the primary databases. They are ordered alphabetically or phylogenetically and are available in aligned form. As of September, 1998, there were 108 SRP RNA sequences, 83 SRP protein sequences and 28 sequences of the SRP receptor alpha subunit and its homologues. In addition, the SRPDB provides search motifs consisting of conserved amino acid and nucleotide residues, and a limited number of SRP RNA secondary structure diagrams and 3-D models. The data are available freely at the URL http://psyche.uthct.edu/dbs/SRPDB/SRPDB.++ +html

Academic Medical Centers↗

PubMed1999

Functional divergence of the plastid and cytosolic forms of the 54-kDa subunit of signal recognition particle.

Chloroplast and cytoplasmic signal recognition particles (cpSRP and cySRP) each contain a similar subunit, SRP54. The chloroplast homologue binds to cpSRP43, which is absent from cytosolic SRP, and cySRP54 binds to SRP-RNA, which appears to be absent from cpSRP. In the presence of cpSRP43, cpSRP54 posttranslationally forms a soluble targeting intermediate, transit complex, with the major light harvesting protein of the thylakoid membrane. In contrast, cySRP54 functions cotranslationally. In this study we investigated whether cytosolic and chloroplast forms of SRP54 were interchangeable in three types of functional assays: complementation of an Escherichia coli SRP54 mutant, formation of the transit complex, and heterologous binding between the SRP54 subunits, cpSRP43, and SRP-RNA. In no cases were the 54-kDa subunits able to substitute for each other suggesting that the two proteins are fundamentally different.

Amino Acid Sequence↗