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Results for “Signal Recognition Particle”

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PubMed2003

Export of beta-lactamase is independent of the signal recognition particle.

In Escherichia coli, three different types of proteins engage the SecY translocon of the inner bacterial membrane for translocation or insertion: 1) polytopic membrane proteins that prior to their insertion into the membrane are targeted to the translocon using the bacterial signal recognition particle (SRP) and its receptor; 2) secretory proteins that are targeted to and translocated across the SecY translocon in a SecA- and SecB-dependent reaction; and 3) membrane proteins with large periplasmic domains, requiring SRP for targeting and SecA for the translocation of the periplasmic moiety. In addition to its role as a targeting device for membrane proteins, a function of the bacterial SRP in the export of SecB-independent secretory proteins has also been postulated. In particular, beta-lactamase, a hydrolytic enzyme responsible for cleavage of the beta-lactam ring containing antibiotics, is considered to be recognized and targeted by SRP. To examine the role of the SRP pathway in beta-lactamase targeting and export, we performed a detailed in vitro analysis. Chemical cross-linking and membrane binding assays did not reveal any significant interaction between SRP and beta-lactamase nascent chains. More importantly, membrane vesicles prepared from mutants lacking a functional SRP pathway did block the integration of SRP-dependent membrane proteins but supported the export of beta-lactamase in the same way as that of the SRP-independent protein OmpA. These data demonstrate that in contrast to previous results, the bacterial SRP is not involved in the export of beta-lactamase and further suggest that secretory proteins of Gram-negative bacteria in general are not substrates of SRP.

Biological Transport↗

PubMed2002

Signal recognition particle RNA localization within the nucleolus differs from the classical sites of ribosome synthesis.

The nucleolus is the site of ribosome biosynthesis, but is now known to have other functions as well. In the present study we have investigated how the distribution of signal recognition particle (SRP) RNA within the nucleolus relates to the known sites of ribosomal RNA synthesis, processing, and nascent ribosome assembly (i.e., the fibrillar centers, the dense fibrillar component (DFC), and the granular component). Very little SRP RNA was detected in fibrillar centers or the DFC of the nucleolus, as defined by the RNA polymerase I-specific upstream binding factor and the protein fibrillarin, respectively. Some SRP RNA was present in the granular component, as marked by the protein B23, indicating a possible interaction with ribosomal subunits at a later stage of maturation. However, a substantial portion of SRP RNA was also detected in regions of the nucleolus where neither B23, UBF, or fibrillarin were concentrated. Dual probe in situ hybridization experiments confirmed that a significant fraction of nucleolar SRP RNA was not spatially coincident with 28S ribosomal RNA. These results demonstrate that SRP RNA concentrates in an intranucleolar location other than the classical stations of ribosome biosynthesis, suggesting that there may be nucleolar regions that are specialized for other functions.

Animals↗

PubMed1996

The nascent polypeptide-associated complex modulates interactions between the signal recognition particle and the ribosome.

BACKGROUND: The first step in the co-translational targeting of secretory proteins to the endoplasmic reticulum membrane involves the recognition of signal sequences by the 54 kDa subunit of the signal recognition particle (SRP) as they emerge from the ribosome. It has recently been proposed that the nascent polypeptide-associated complex (NAC) contributes to the fidelity of targeting by modulating interactions that occur between the ribosome-nascent chain complex, the SRP and the endoplasmic reticulum membrane. Precisely how NAC influences SRP function is presently unclear. RESULTS: We have used immunoblotting experiments to monitor interactions between the SRP and the ribosome-nascent chain complex, in the absence and presence of NAC. In the absence of NAC, SRP binds in a high-salt-resistant manner only to ribosomes that contain a signal sequence, confirming the specificity of SRP for signal sequences. Binding of SRP to signalless ribosome nascent chains is observed at lower salt concentrations; however, the amount of SRP bound to this complex is indistinguishable from that bound to ribosomes lacking nascent chains. Thus, this salt-sensitive binding is likely to be the result of interactions between SRP and the ribosome that occur independently of the nascent chain. A minimal particle consisting of SRP54 and SRP RNA is sufficient to confer salt-resistant binding to ribosomes that contain signal sequences, whereas all of the SRP subunits are required for salt-sensitive binding to ribosomes that lack nascent chains. This salt-sensitive binding by SRP is inhibited by the addition of purified NAC. CONCLUSIONS: Based on our results, we define two distinct modes of interaction between SRP and the ribosome-nascent chain complex: salt-resistant interactions between SRP54 and signal sequences, and salt-sensitive interactions between additional components of SRP and the ribosome. We conclude that NAC does not directly influence signal sequence recognition by SRP but, rather, that it negatively modulates interactions that occur between SRP and the ribosome itself. These results are discussed in terms of a model wherein SRP and NAC regulate each others' activity during protein targeting.

Peptides↗

PubMed1996

Substitution of fifty four homologue (Ffh) in Escherichia coli with the mammalian 54-kDa protein of signal-recognition particle.

The fifty four homologue (Ffh) of Escherichia coli promotes the translocation of a subset of periplasmic, membrane and secreted proteins across the cytoplasmic membrane. The ffh gene product is essential for cell viability and efficient protein export. Here we show that the mammalian homologue signal-recognition particle (SRP) 54 kDa is not able to suppress the translocation defect in an Ffh conditional mutant Wam 113 [Phillips, G.J. & Silhavy, T.J. (1992) Nature 359, 744-746]. The expression of SRP 54kDa, which is increased when Ffh is suppressed in the Wam 113 strain, causes a pleiotropic defect characterised by cell elongation, and increased accumulation of precursor proteins. The accumulation of precursors of outer membrane protein A (Omp A) and maltose-binding protein (MBP), See-B dependent pre-proteins, was less than the Ffh-dependent proteins ribose-binding protein (RBP) and beta-lactamase. Sec B expression was suppressed by Ffh expression. The recombinant SRP 54 kDa, which forms a ribonucleoprotein complex in E coli, was shown to bind to precursor proteins, but is unable to interact with the filamentous temperature-sensitive Y (Fts Y) membrane receptor of the translocation machinery.

Amino Acid Sequence↗

PubMed2006

Compartmentalization directs assembly of the signal recognition particle.

Many ribonucleoprotein complexes assemble stepwise in distinct cellular compartments, a process that usually involves bidirectional transport of both RNA and proteins between the nucleus and cytoplasm. The biological rationale for such complex transport steps in RNP assembly is obscure. One important example is the eukaryotic signal recognition particle (SRP), a cytoplasmic RNP consisting of one RNA and six proteins. Prior in vivo studies support an "SRP54-late" assembly model in which all SRP proteins, except SRP54, are imported from the cytoplasm to the nucleus to bind SRP RNA. This partially assembled complex is then exported to the cytoplasm where SRP54 binds and forms the SRP holocomplex. Here we show that native SRP assembly requires segregated and ordered binding by its protein components. A native ternary complex forms in vitro when SRP19 binds the SRP RNA prior to binding by SRP54, which approximates the eukaryotic cellular pathway. In contrast, the presence of SRP54 disrupts native assembly of SRP19, such that two RNA-binding loops in SRP19 misfold. These results imply that SRP54 must be sequestered during early SRP assembly steps, as apparently occurs in vivo, for proper assembly of the SRP to occur. Our findings emphasize that spatial compartmentalization provides an additional level of regulation that prevents competition among components and can function to promote native assembly of the eukaryotic SRP.

Active Transport, Cell Nucleus↗

PubMed1995

The SRP9/14 subunit of the signal recognition particle (SRP) is present in more than 20-fold excess over SRP in primate cells and exists primarily free but also in complex with small cytoplasmic Alu RNAs.

The heterodimeric protein SRP9/14 bound to the Alu sequences of SRP RNA is essential for the translational control function of the signal recognition particle (SRP). The Alu RNAs of primate cells are believed to be derived from SRP RNA and have been shown to bind to an SRP14-related protein in vitro. We have used antibodies to characterize SRP9/14 and examine its association with small RNAs in vivo. Although SRP9 proteins are the same size in both rodent and primate cells, SRP14 subunits are generally larger in primate cells. An additional alanine-rich domain at the C-terminus accounts for the larger size of one human isoform. Although the other four SRP proteins are largely assembled into SRP in both rodent and primate cells, we found that the heterodimer SRP9/14 is present in 20-fold excess over SRP in primate cells. An increased synthesis rate of both proteins may contribute to their accumulation. The majority of the excess SRP9/14 is cytoplasmic and does not appear to be bound to any small RNAs; however, a significant fraction of a small cytoplasmic Alu RNA is complexed with SRP9/14 in a 8.5 S particle. Our findings that there is a large excess of SRP9/14 in primate cells and that Alu RNAs are bound to SRP9/14 in vivo suggest that this heterodimeric protein may play additional roles in the translational control of gene expression and/or Alu transcript metabolism.

Amino Acid Sequence↗

PubMed1989

Homology of 54K protein of signal-recognition particle, docking protein and two E. coli proteins with putative GTP-binding domains.

Most proteins exported from mammalian cells contain a signal sequence which mediates targeting to and insertion into the membrane of the endoplasmic reticulum (ER). Involved in this process are the signal-recognition particle (SRP) and docking protein (DP), the receptor for SRP in the ER membrane. SRP interacts with the signal sequence on nascent polypeptide chains and retards their further elongation, which resumes only after interaction of the arrested ribosomal complex with the docking protein. SRP is a ribonucleoprotein particle comprising a 7S RNA and six polypeptides with relative molecular masses (Mr) of 9,000 (9K) 14K, 19K, 54K, 68K and 72K (ref. 1). The 9K and 14K proteins are essential for elongation arrest and the 68K-72K heterodimer is required for docking to the ER membrane. The 54K protein binds to the signal sequence when it emerges from the ribosome. Docking protein consists of two polypeptides, a 72K alpha-subunit (DP alpha) and a 30K beta-subunit (DP beta). No components structurally homologous to SRP and docking protein have yet been found in yeast or Escherichia coli. To understand the molecular nature of the interaction between the signal sequence and its receptor(s) we have characterized a complementary DNA coding for the 54K protein of SRP. Significant sequence homology was found to part of DP alpha and two E. coli proteins of unknown function. The homologous region includes a putative GTP-binding domain.

Amino Acid Sequence↗

PubMed2004

Unraveling the interface of signal recognition particle and its receptor by using chemical cross-linking and tandem mass spectrometry.

Among the methods used to unravel protein interaction surfaces, chemical cross-linking followed by identification of the cross-linked peptides by mass spectrometry has proven especially useful in dynamic and complex systems. During the signal recognition particle (SRP)-dependent targeting of proteins to the bacterial plasma membrane, the specific interaction between Ffh (the protein component of SRP) and FtsY (the SRP receptor) is known to be essential for the efficiency and fidelity of this process. In this work, we studied the Escherichia coli and Thermus aquaticus Ffh.FtsY complexes by using chemical cross-linking and tandem mass spectrometry to identify nine intermolecular cross-linked peptides. This information was used in conjunction with a previously undescribed model-building approach that combines geometric restraint optimization with macromolecular docking. The resulting model of the Ffh.FtsY complex is in good agreement with the crystal structure solved shortly thereafter. Intriguingly, four of the cross-linked pairs involve the M domain of Ffh, which is absent from the crystal structure, providing previously undocumented experimental evidence that the M domain is positioned in close proximity to the Ffh.FtsY interface in the complex.

Bacterial Proteins↗

PubMed1997

The signal recognition particle receptor alpha subunit of the hyperthermophilic archaeon Acidianus ambivalens exhibits an intrinsic GTP-hydrolyzing activity.

Two adjacent genes of the acidophilic and hyperthermophilic crenarchaeon Acidianus ambivalens were cloned and sequenced. The 1.6 kb genomic nucleotide sequence under investigation consists of the 1.12 kb SRa gene encoding the putative signal recognition particle receptor alpha subunit (SR alpha, 42.2 kDa) and the 186 basepair secE gene coding for the putative secretory component secE subunit (6800 Da). The SR alpha protein is structured by three distinct regions: the N-terminal hydrophilic H-region, the following X-region and the C-terminal GTP-binding domain. A polyclonal anti-E. coli lacZ/A. ambivalens SR alpha antiserum detects a 51 kDa cell protein (p51) on immunoblots. Proteolysis of the recombinant SR alpha protein by Proteinase K produces a 31.6 kDa protease-resistant protein fragment comprising X-region and G-domain. The protein binds tightly to the GTP-agarose affinity matrix in a temperature-dependent manner. It hydrolyzes GTP readily at higher temperatures only in the presence of Mg2+. Point mutations (T326N) and (D329A) in the G-4 element of A. ambivalens SR alpha G-domain diminish the GTPase activity significantly. In contrast, the deletion mutant protein SR alpha (delta1-92) lacking the hydrophilic H-region displays a higher GTP-hydrolyzing activity when compared to the unmodified recombinant protein. Addition of GDP greatly inhibits GTP hydrolysis in mutant and unmodified A. ambivalens SR alpha.

Amino Acid Sequence↗

PubMed1997

Crystal structure of the NG domain from the signal-recognition particle receptor FtsY.

Newly synthesized proteins destined either for secretion or incorporation into membranes are targeted to the membrane translocation machinery by a ubiquitous system consisting of a signal-recognition particle (SRP) and its receptor. Both the SRP receptor and the protein within the SRP that binds the signal sequence contain GTPases. These two proteins, together with the RNA component of the SRP, form a complex and thereby regulate each other's GTPase activity. Here we report the structure of the GTPase-containing portion of FtsY, the functional homologue of the SRP receptor of Escherichia coli, at 2.2 A resolution without bound nucleotide. This so-called NG domain displays similarities to the Ras-related GTPases, as well as features unique to the SRP-type GTPases, such as a separate amino-terminal domain, an insertion within the p21ras (Ras) effector domain, and a wide-open GTP-binding region. The structure explains the low affinity of FtsY for GTP, and suggests rearrangements that may occur on nucleotide binding. It also identifies regions potentially involved in the transmission of signals between domains and in interactions with regulatory proteins.

Amino Acid Sequence↗

PubMed1987

A 5 S rRNA-like secondary structure in the 7 SL RNA may define a ribosomal binding site of the signal recognition particle.

A new secondary structure model for parts of the 7 SL RNA is proposed which indicates for a stretch of at least 40 bases a strong structural homology to the ribosomal protein L5 binding site of eukaryotic 5 S rRNA. It is suggested that the 5 S rRNA-like structural part of 7 SL RNA mediates binding of the signal recognition particle near to the peptidyl transferase center of the ribosome.

Animals↗

PubMed2006

Sequence-specific interactions of nascent Escherichia coli polypeptides with trigger factor and signal recognition particle.

As nascent polypeptides exit the ribosomal tunnel they immediately associate with chaperones, folding catalysts, and targeting factors. These interactions are decisive for the future conformation and destination of the protein that is being synthesized. Using Escherichia coli as a model organism, we have systematically analyzed how the earliest contacts of nascent polypeptides with cytosolic factors depend on the nature and future destination of the emerging sequence using a photo cross-linking approach. Together, the data suggest that the chaperone trigger factor is adjacent to emerging sequences by default, consistent with both its placement near the nascent chain exit site and its cellular abundance. The signal recognition particle (SRP) effectively competes the contact with TF when a signal anchor (SA) sequence of a nascent inner membrane protein appears outside the ribosome. The SRP remains in contact with the SA and downstream sequences during further synthesis of approximately 30 amino acids. The contact with trigger factor is then restored unless another transmembrane segment reinitiates SRP binding. Importantly and in contrast to published data, the SRP appears perfectly capable of distinguishing SA sequences from signal sequences in secretory proteins at this early stage in biogenesis.

Amino Acid Sequence↗

PubMed1997

A GTP-binding protein of Mycoplasma hominis: a small sized homolog to the signal recognition particle receptor FtsY.

A protein homologous to the Escherichia coli FtsY which in turn has characteristics in common with the alpha-subunit of the eukaryotic signal recognition particle receptor (SRalpha) in the membrane of the endoplasmic reticulum, was identified in Mycoplasma hominis and its encoding DNA sequenced. The aa similarity to E. coli FtsY and B. subtilis FtsY was 38% and 51%, respectively. The protein was synthesized in E. coli, purified and shown to bind GTP. Subcellular localization studies revealed that M. hominis FtsY was associated with the cytoplasmic side of the plasma membrane. The molecular mass of M. hominis FtsY was 39.1, which was significantly smaller than FtsY from the gram- E. coli. Analysis of the primary structure showed that M. hominis FtsY had no counterpart to the N-terminal part in E. coli FtsY or mammalian SRalpha, which for the last-mentioned are known to comprise the membrane-anchoring fragment. Comparison of sequenced SRalpha homologue indicates that M. hominis together with Bacillus subtilis comprise a distinct cluster of similar small SRP receptors.

Amino Acid Sequence↗

PubMed2006

A derivative of lipid A is involved in signal recognition particle/SecYEG-dependent and -independent membrane integrations.

A cell-free system was developed that allows the correct integration of single and multispanning membrane proteins of Escherichia coli into proteoliposomes. We found that physiological levels of diacylglycerol were required to prevent spontaneous integration into liposomes even of the polytopic mannitol permease. Using diacylglycerol-containing proteoliposomes, we identified a novel integration-stimulating factor. Integration of mannitol permease was dependent on both the SecYEG translocon and this factor and was mediated by signal recognition particle and signal recognition particle receptor. Integration of M13 procoat, which is independent of both signal recognition particle/signal recognition particle receptor and SecYEG, was also promoted by this factor. Furthermore, the factor stimulated the post-translational translocation of presecretory proteins, suggesting that it also mediates integration of a signal sequence. This factor was found to be a lipid A-derived membrane component possessing a peptide moiety.

Bacterial Outer Membrane Proteins↗

PubMed1996

A putative signal recognition particle receptor alpha subunit (SR alpha) homologue is expressed in the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius.

A 1.64 kb genomic DNA sequence from the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius is composed of two adjacent genes. The first functionally unassigned open reading frame (orf-1) comprises 450 base pairs. The second 1.1 kb large open reading frame encodes the putative signal recognition particle receptor alpha subunit (SR alpha). Both genes are expressed under the heterotrophic growth conditions of the organism. The main transcript of orf-1 appears as a monocistronic RNA in Northern hybridization. With regard to SR alpha the transcription pattern was investigated by reverse transcription polymerase chain reaction and primer extension analysis. A polyclonal antiserum directed against E. coli lacZ'/Sulfolobus SR alpha fusion protein detects a 40.5 kDa protein (p41) in agreement with the 41.4 kDa as deduced from the nucleotide sequence.

Base Sequence↗

PubMedDate not supplied

Removal of the Alu structural domain from signal recognition particle leaves its protein translocation activity intact.

Alu-like elements comprise the most abundant family of interspersed repetitive sequences in primates and rodents, and contain many features of processed genes, suggesting that they were initially derived by reverse transcription of processed RNA transcripts. Transcripts containing Alu family members are represented in heterologous nuclear RNAs, cytoplasmic messenger RNAs and small RNAs, although nothing is known about their function. Evolutionary studies strongly suggest that the parent RNA for the Alu-like elements is the highly conserved 7SL RNA, which is an essential component of signal recognition particle (SRP), a small cytoplasmic ribonucleoprotein whose function is the targeting of nascent secretory and membrane proteins to the rough endoplasmic reticulum (for a review see ref. 6). 7SL RNA is composed of both unique and Alu-like sequences. SRP is rod-shaped and, in addition to its RNA, contains four proteins (two monomers composed of a polypeptide of relative molecular mass (Mr) 19,000 (19K) and one of 54K, and two heterodimers, one composed of a 9K and a 14K polypeptide, and the other composed of a 68K and a 72K polypeptide, respectively). The RNA moiety is required for SRP activity, as well as for structural integrity of the particle. To investigate whether the Alu-like segments of 7SL RNA have a specific role in SRP activity, we have now purified and analysed a SRP subparticle that is created upon extensive digestion with micrococcal nuclease and entirely lacks the Alu-like sequences. We find that it contains the 72/68K, 54K and 19K proteins tightly bound, but lacks the 9/14K protein. In vitro activity assays demonstrated that the subparticle could still promote secretory protein translocation across the microsomal membrane, but could no longer trigger an arrest of pre-secretory protein synthesis. Re-addition of the 9/14K protein did not restore the elongation arrest. We conclude that the region of SRP comprised of the Alu-like RNA and the 9/14K protein exists in a distinct structural domain which is not required for the protein translocation promoted by SRP but apparently confers elongation-arresting activity on the particle.

Humans↗

PubMed1999

A signal recognition particle receptor gene from the early-diverging eukaryote, Giardia lamblia.

The molecular mechanisms for targeting and translocation of secreted proteins are highly conserved from bacteria to mammalian cells, although the machinery is more complex in higher eukaryotes. To investigate protein transport in the early-diverging eukaryote, Giardia lamblia, we cloned the gene encoding the alpha subunit (SRalpha) of the signal recognition particle (SRP) receptor. SRalpha is a small GTPase that functions in SRP-ribosome targeting to the ER. Sequence and phylogenetic analyses showed that SRalpha from G. lamblia is most homologous to SRalpha proteins from higher eukaryotes, although it lacks some conserved motifs. Specifically, giardial SRalpha has an N-terminal extension that enables SRalpha of higher eukaryotes to interact with a beta subunit that anchors it in the ER membrane. While the C-terminal regions are similar, giardial SRalpha lacks a prominent 13 amino acid regulatory loop that is characteristic of higher eukaryotic versions. Thus, giardial SRalpha resembles that of higher eukaryotes, but likely diverged before the advent of the regulatory loop. The 1.8 kb SRalpha transcript has extremely short untranslated regions (UTRs): a 1-2 nt 5'- and a 9 nt 3' UTR with the polyadenylation signal overlapping with the stop codon. RT-PCR, Northern and Western analyses showed that SRalpha is present at relatively constant levels during vegetative growth and encystation, even though there are extensive changes in endomembrane structures and secretory activity during encystation. Imnuno-EM showed that SRalpha localizes to ER-like structures, strengthening the observation of a typical ER in G. lamlia. Unexpectedly, SRalpha was also found in the lysosome-like peripheral vacuoles, suggesting unusual protein traffic in this early eukaryote. Our results indicate that the eukaryotic type of cotranslational transport appeared early in the evolution of the eukaryotic cell.

Amino Acid Sequence↗

PubMed1999

A chromodomain protein encoded by the arabidopsis CAO gene is a plant-specific component of the chloroplast signal recognition particle pathway that is involved in LHCP targeting.

A recessive mutation in Arabidopsis, named chaos (for chlorophyll a/b binding protein harvesting-organelle specific; designated gene symbol CAO), was isolated by using transposon tagging. Characterization of the phenotype of the chaos mutant revealed a specific reduction of pigment binding antenna proteins in the thylakoid membrane. These nuclear-encoded proteins utilize a chloroplast signal recognition particle (cpSRP) system to reach the thylakoid membrane. Both prokaryotes and eukaryotes possess a cytoplasmic SRP containing a 54-kD protein (SRP54) and an RNA. In chloroplasts, the homolog of SRP54 was found to bind a 43-kD protein (cpSRP43) rather than to an RNA. We cloned the CAO gene, which encodes a protein identified as Arabidopsis cpSRP43. The product of the CAO gene does not resemble any protein in the databases, although it contains motifs that are known to mediate protein-protein interactions. These motifs include ankyrin repeats and chromodomains. Therefore, CAO encodes an SRP component that is unique to plants. Surprisingly, the phenotype of the cpSRP43 mutant (i.e., chaos) differs from that of the Arabidopsis cpSRP54 mutant, suggesting that the functions of the two proteins do not strictly overlap. This difference also suggests that the function of cpSRP43 is most likely restricted to protein targeting into the thylakoid membrane, whereas cpSRP54 may be involved in an additional process(es), such as chloroplast biogenesis, perhaps through chloroplast-ribosomal association with chloroplast ribosomes.

Amino Acid Sequence↗