Signal recognition particle: a ribonucleoprotein required for cotranslational translocation of proteins, isolation and properties.
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Animals↗
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Results for “Signal Recognition Particle”
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Human genes and pseudogenes for the 7SL RNA component of signal recognition particle.
Of the several hundred 7SL RNA-like sequences that are dispersed in human DNA, no more than four are likely to represent genes for 7SL RNA; the majority are 7SL pseudogenes which appear to result from the reverse flow of genetic information from 7SL RNA back into genomic DNA. We present the sequence of five 7SL pseudogenes displaying an unprecedented diversity of structures. All are truncated copies of 7SL RNA, but the site of truncation can occur at either the 5' end, the 3' end or at both ends of the RNA sequence. We suggest that such diverse 7SL pseudogenes are generated by different but related pathways. In particular, we argue that two of the loci are secondary 7SL pseudogenes which derive from RNA polymerase III transcripts of primary (preexisting) 7SL pseudogenes. We also report the isolation and characterisation of a human genomic clone carrying two linked 7SL RNA coding regions, 7L30.1 and 7L30.2. The 7L30.2 locus differs by several single base changes from the known human 7SL RNA sequences and does not appear to be expressed at a detectable level in HeLa cells. The 7L30.1 locus is an authentic 7SL RNA gene encoding one of the three sequence variants of human 7SL RNA.
Base Sequence↗
Prokaryotic secretion: a signal recognition particle in Escherichia coli?
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Journal Article↗
Identification of a gene in the euryarchaeal Thermococcus species AN1 encoding a protein homologous to the alpha subunit of the eukaryal signal recognition particle (SRP) receptor.
We describe here the sequence and transcriptional analysis of a gene from the euryarchaeal Thermococcus species AN1 (DSM 2770) encoding a protein homologous to the alpha subunit of the eukaryal SRP receptor (SR alpha). The AN1 protein is found to share the highest degree of homology with the only other described archaeal SR alpha homolog characterized from the hyperthermophilic crenarchaeal Sulfolobus solfataricus. Sequence analysis of the translation of the AN1 gene reveals the presence of the following previously described domains: an N-terminal alpha domain rich in acidic and basic residues; an X domain and four GTP binding motifs (G1-G4). A putative guanine nucleotide dissociation stimulator binding element is also present. The AN1 SR alpha protein would now represent the shortest variant of this expanding family with 329 residues and a predicted molecular weight of 36.4 kDa. A Northern analysis indicates that the AN1 SR alpha protein gene transcript is present at low levels suggesting that SR alpha is likely to be only a minor cell constituent. The presence of an SR alpha homolog in another kingdom within the archaeal domain possessing the full suite of conserved motifs is significant in several respects. It not only supports the monophyletic character of the domain Archaea but suggests that these homologs have similar functions in these organisms and emphasises the ancient origins of the protein export machinery.
Amino Acid Sequence↗
Nuclear export of yeast signal recognition particle lacking srp54p by the Xpo1p/Crm1p NES-dependent pathway
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Journal Article↗
Did the last common ancestor have a biological membrane?
All theories about the origin and evolution of membrane bound cells necessarily have to cope with the nature of the last common ancestor of cellular life. One of the most important aspect of this ancestor, whether it had a closed biological membrane or not, has recently been intensely debated. Having a consensus about it would be an important step towards an eventual (though probably still remote) synthesis of the best elements of the current multitude of cell evolution models. Here I analyse the structural and functional conservation of the few universally distributed proteins that were undoubtedly present in the last common ancestor and that carry out membrane-associated functions. These include the SecY subunit of the protein-conducting channel, the signal recognition particle, the signal recognition particle receptor, the signal peptidase, and the proton ATPase. The conserved structural and functional aspects of these proteins indicate that the last common ancestor was associated with a hydrophobic layer with two hydrophilic sides (an inside and an outside) that had a full-fledged and asymmetric protein insertion and translocation machinery and served as a permeability barrier for protons and other small molecules. It is difficult to escape the conclusion that the last common ancestor had a closed biological membrane from which all cellular membranes evolved.
Journal Article↗
Translocation of preproinsulin across the endoplasmic reticulum membrane. The relationship between nascent polypeptide size and extent of signal recognition particle-mediated inhibition of protein synthesis.
Signal recognition particle (SRP) induces elongation arrest of nascent presecretory proteins as the signal peptide protrudes from the large ribosomal subunit. To examine the relationship between the size of the precursor and extent of SRP mediated inhibition of polypeptide chain elongation, we performed in vitro translation experiments in the presence of SRP using a series of truncated preproinsulin mRNA molecules. These precursors possessed the same NH2 terminus as native preproinsulin followed by progressively shorter COOH termini. SRP inhibited translation of precursors as short as 64 amino acids in length, however, the extent of inhibition diminished for shorter precursors. This correlated with a reduction in the time required for ribosomes to transit through the mRNA encoding the shortened precursors. By exploiting a chimeric protein comprising the first 71 residues of preproinsulin fused to the bacterial cytoplasmic enzyme chloramphenicol acetyltransferase, we demonstrate that the largest size a nascent chain can reach and still be susceptible to SRP-mediated elongation arrest is approximately 17 kDa. Our data support the model that SRP binding to the signal peptide is a reversible process even in the absence of microsomal membranes, and that SRP can arrest polypeptide chain elongation at multiple stages during translation.
Animals↗
Regulation of the GTPase cycle in post-translational signal recognition particle-based protein targeting involves cpSRP43.
The chloroplast signal recognition particle consists of a conserved 54-kDa GTPase and a novel 43-kDa chromodomain protein (cpSRP43) that together bind light-harvesting chlorophyll a/b-binding protein (LHCP) to form a soluble targeting complex that is subsequently directed to the thylakoid membrane. Homology-based modeling of cpSRP43 indicates the presence of two previously identified chromodomains along with a third N-terminal chromodomain. Chromodomain deletion constructs were used to examine the role of each chromodomain in mediating distinct steps in the LHCP localization mechanism. The C-terminal chromodomain is completely dispensable for LHCP targeting/integration in vitro. The central chromodomain is essential for both targeting complex formation and integration because of its role in binding the M domain of cpSRP54. The N-terminal chromodomain (CD1) is unnecessary for targeting complex formation but is required for integration. This correlates with the ability of CD1 along with the ankyrin repeat region of cpSRP43 to regulate the GTPase cycle of the cpSRP-receptor complex.
Amino Acid Sequence↗
Formation of a functional ribosome-membrane junction during translocation requires the participation of a GTP-binding protein.
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.
Adenosine Triphosphate↗
Signal recognition particle-dependent protein targeting, universal to all kingdoms of life.
The signal recognition particle (SRP) and its membrane-bound receptor represent a ubiquitous protein-targeting device utilized by organisms as different as bacteria and humans, archaea and plants. The unifying concept of SRP-dependent protein targeting is that SRP binds to signal sequences of newly synthesized proteins as they emerge from the ribosome. In eukaryotes this interaction arrests or retards translation elongation until SRP targets the ribosome-nascent chain complexes via the SRP receptor to the translocation channel. Such channels are present in the endoplasmic reticulum of eukaryotic cells, the thylakoids of chloroplasts, or the plasma membrane of prokaryotes. The minimal functional unit of SRP consists of a signal sequence-recognizing protein and a small RNA. The as yet most complex version is the mammalian SRP whose RNA, together with six proteinaceous subunits, undergo an intricate assembly process. The preferential substrates of SRP possess especially hydrophobic signal sequences. Interactions between SRP and its receptor, the ribosome, the signal sequence, and the target membrane are regulated by GTP hydrolysis. SRP-dependent protein targeting in bacteria and chloroplasts slightly deviate from the canonical mechanism found in eukaryotes. Pro- and eukaryotic cells harbour regulatory mechanisms to prevent a malfunction of the SRP pathway.
Animals↗
Conformational changes in the bacterial SRP receptor FtsY upon binding of guanine nucleotides and SRP.
In cotranslational preprotein targeting in Escherichia coli, the signal recognition particle (SRP) binds to the signal peptide emerging from the ribosome and, subsequently, interacts with the signal recognition particle receptor, FtsY, at the plasma membrane. Both FtsY and the protein moiety of the signal recognition particle, Ffh, are GTPases, and GTP is required for the formation of the SRP-FtsY complex. We have studied the binding of GTP/GDP to FtsY as well as the SRP-FtsY complex formation by monitoring the fluorescence of tryptophan 343 in the I box of mutant FtsY. Thermodynamic and kinetic parameters of the FtsY complexes with GDP, GTP, and signal recognition particle are reported. Upon SRP-FtsY complex formation in the presence of GTP, the fluorescence of tryptophan 343 increased by 50 % and was blue-shifted by 10 nm. We conclude that GTP-dependent SRP-FtsY complex formation leads to an extensive conformational change in the I box insertion in the effector region of FtsY.
Amino Acid Substitution↗
Use of thioredoxin as a reporter to identify a subset of Escherichia coli signal sequences that promote signal recognition particle-dependent translocation.
We have previously reported that the DsbA signal sequence promotes efficient, cotranslational translocation of the cytoplasmic protein thioredoxin-1 via the bacterial signal recognition particle (SRP) pathway. However, two commonly used signal sequences, those of PhoA and MalE, which promote export by a posttranslational mechanism, do not export thioredoxin. We proposed that this difference in efficiency of export was due to the rapid folding of thioredoxin in the cytoplasm; cotranslational export by the DsbA signal sequence avoids the problem of cytoplasmic folding (C. F. Schierle, M. Berkmen, D. Huber, C. Kumamoto, D. Boyd, and J. Beckwith, J. Bacteriol. 185:5706-5713, 2003). Here, we use thioredoxin as a reporter to distinguish SRP-dependent from non-SRP-dependent cleavable signal sequences. We screened signal sequences exhibiting a range of hydrophobicity values based on a method that estimates hydrophobicity. Successive iterations of screening and refining the method defined a threshold hydrophobicity required for SRP recognition. While all of the SRP-dependent signal sequences identified were above this threshold, there were also a few signal sequences above the threshold that did not utilize the SRP pathway. These results suggest that a simple measure of the hydrophobicity of a signal sequence is an important but not a sufficient indicator for SRP recognition. In addition, by fusing a number of both classes of signal sequences to DsbA, we found that DsbA utilizes an SRP-dependent signal sequence to achieve efficient export to the periplasm. Our results suggest that those proteins found to be exported by SRP-dependent signal sequences may require this mode of export because of their tendency to fold rapidly in the cytoplasm.
Blotting, Western↗
Synthetic signal peptide and analogs display different activities in mammalian and plant in vitro secretion systems.
The biological properties of four chemically synthesized signal peptides were compared in mammalian (rabbit reticulocyte) and plant (wheat germ) cell-free protein secretion systems. The precursor-specific region of bovine pre-proparathyroid hormone (preproPTH), [D-Tyr-(+1)]preproPTH-(-29-+1)amide, and a sulfur-free analog, [Nle-(-25), Nle-(-21), Nle-(-18), Ala-(-14), D-Tyr-(+1)]preproPTH-(-29-+1)amide, inhibit the processing of an unrelated precursor protein (pre-prolactin) to its mature secreted form (prolactin) in the mammalian system. In the plant system supplemented with signal recognition particle, the signal peptides arrest translation of both secretory (preprolactin) and cytoplasmic (globin) proteins. One analog, [Nle-(-25), Nle-(-21), Asp-(-18), Ala-(-14), D-Tyr-(+1)]preproPTH-(-29-+1)amide, inhibits preprotein processing in the mammalian system but fails to induce translation arrest in the plant system. A truncated peptide, [N alpha-AcLeu-(-17), Ala-(-14), D-Tyr-(+1)]preproPTH-(-17-+1)amide, lacking the N-terminal (positively charged) region and a portion of the hydrophobic core region, is inactive in both systems. These studies demonstrate that the chemically synthesized signal region of a precursor protein interacts directly with signal recognition particle and functionally mimics the proposed properties of a native signal sequence linked to a nascent protein as it emerges from the ribosome during biosynthesis, and an analog of the signal peptide reveals fundamental differences between the components involved in the protein secretion apparatus in mammals and plants.
Amino Acid Sequence↗
Integration of membrane proteins into the endoplasmic reticulum requires GTP.
We have examined the requirement for ribonucleotides and ribonucleotide triphosphate hydrolysis during early events in the membrane integration of two membrane proteins: the G protein of vesicular stomatitis virus and the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus. Both proteins contain a single transmembrane-spanning segment but are integrated in the membrane with opposite orientations. The G protein has an amino-terminal signal sequence and a stop-transfer sequence located near the carboxy terminus. The HN glycoprotein has a single sequence near the amino terminus that functions as both a signal-sequence and a transmembrane-spanning segment. Membrane insertion was explored using a cell-free system directed by transcribed mRNAs encoding amino-terminal segments of the two proteins. Ribosome-bound nascent polypeptides were assembled, ribonucleotides were removed by gel filtration chromatography, and the ribosomes were incubated with microsomal membranes under conditions of defined ribonucleotide content. Nascent chain insertion into the membrane required the presence of both the signal recognition particle and a functional signal recognition particle receptor. In the absence of ribonucleotides, insertion of nascent membrane proteins was not detected. GTP or nonhydrolyzable GTP analogues promoted efficient insertion, while ATP was comparatively ineffective. Surprisingly, the majority of the HN nascent chain remained ribosome associated after puromycin treatment. Ribosome-associated HN nascent chains remained competent for membrane insertion, while free HN chains were not competent. We conclude that a GTP binding protein performs an essential function during ribosome-dependent insertion of membrane proteins into the endoplasmic reticulum that is unrelated to protein synthesis.
Cell Membrane↗
Signal recognition particle-dependent insertion of coronavirus E1, an intracellular membrane glycoprotein.
The membrane insertion of the E1 protein of a coronavirus, mouse hepatitis virus A59, was studied in a wheat germ cell-free translation system. E1 is a transmembrane protein spanning the lipid bilayer several times. It is synthesized without a cleavable signal sequence, localized intracellularly, and not transported to the cell surface. It thus represents a model intracellular protein. We found that the synthesis of E1 is specifically and stably blocked by the addition of signal recognition particle to the wheat germ system. Subsequent addition of salt-extracted pancreatic microsomes resulted in the full release of this arrest as well as the completion and the correct membrane integration of E1. Such signal recognition particle-induced arrests failed to produce shorter peptides of a defined length. Addition of signal recognition particle to a synchronized translation at any time during the synthesis of about the first two thirds of E1 (150 amino acids) blocked further translation, suggesting that the most C-terminal of the three internal hydrophobic domains of E1 could function as its signal sequence.
Animals↗
SecA is not required for signal recognition particle-mediated targeting and initial membrane insertion of a nascent inner membrane protein.
In Escherichia coli, signal recognition particle (SRP)-dependent targeting of inner membrane proteins has been described. In vitro cross-linking studies have demonstrated that short nascent chains exposing a highly hydrophobic targeting signal interact with the SRP. This SRP, assisted by its receptor, FtsY, mediates the transfer to a common translocation site in the inner membrane that contains SecA, SecG, and SecY. Here we describe a further in vitro reconstitution of SRP-mediated membrane insertion in which purified ribosome-nascent chain-SRP complexes are targeted to the purified SecYEG complex contained in proteoliposomes in a process that requires the SRP-receptor FtsY and GTP. We found that in this system SecA and ATP are dispensable for both the transfer of the nascent inner membrane protein FtsQ to SecY and its stable membrane insertion. Release of the SRP from nascent FtsQ also occurred in the absence of SecYEG complex indicating a functional interaction of FtsY with lipids. These data suggest that SRP/FtsY and SecB/SecA constitute distinct targeting routes.
Adenosine Triphosphatases↗
Identification of ZipA, a signal recognition particle-dependent protein from Neisseria gonorrhoeae.
A genetic screen designed to identify proteins that utilize the signal recognition particle (SRP) for targeting in Escherichia coli was used to screen a Neisseria gonorrhoeae plasmid library. Six plasmids were identified in this screen, and each is predicted to encode one or more putative cytoplasmic membrane (CM) proteins. One of these, pSLO7, has three open reading frames (ORFs), two of which have no similarity to known proteins in GenBank other than sequences from the closely related N. meningitidis. Further analyses showed that one of these, SLO7ORF3, encodes a protein that is dependent on the SRP for localization. This gene also appears to be essential in N. gonorrhoeae since it was not possible to generate null mutations in the gene. Although appearing unique to Neisseria at the DNA sequence level, SLO7ORF3 was found to share some features with the cell division gene zipA of E. coli. These features included similar chromosomal locations (with respect to linked genes) as well as similarities in the predicted protein domain structures. Here, we show that SLO7ORF3 can complement an E. coli conditional zipA mutant and therefore encodes a functional ZipA homolog in N. gonorrhoeae. This observation is significant in that it is the first ZipA homolog identified in a non-rod-shaped organism. Also interesting is that this is the fourth cell division protein (the others are FtsE, FtsX, and FtsQ) shown to utilize the SRP for localization, which may in part explain why the genes encoding the three SRP components are essential in bacteria.
Amino Acid Sequence↗
Structural and functional characterisation of the signal recognition particle-specific 54 kDa protein (SRP54) of tomato.
Two representative genes for the 54 kDa protein subunit of the signal recognition particle (SRP54) of tomato were cloned. It was shown that both genes are expressed in the tomato cv. Rentita. SRP54 is encoded by nine exons distributed over 10 kb of genomic sequence. The amino acid sequences deduced for the two SRP54 genes are 92% identical and the calculated protein size is 55 kDa. Like the homologous proteins isolated from other eukaryotes, the tomato SRP54 is evidently divided into two domains. As deduced from sequence motif identity, the N-terminally located G-domain can be assumed to have GTPase activity. The C-terminal part of the protein is methionine rich (14% methionine) and represents the M-domain. In in vitro binding experiments, SRP54 of tomato was able to attach to the 7S RNA of tomato, its natural binding partner in the SRP. This interaction can only take place in a trimeric complex consisting of 7S RNA, SRP54 and SRP19. The latter protein subunit of the SRP complex is assumed to induce a conformational change in the 7S RNA. The human SRP19 was able to mediate the binding of the tomato SRP54 to the 7S RNA, irrespective of whether this latter originated from tomato or man.
Amino Acid Sequence↗