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Results for “Signal Recognition Particle”

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PubMed1982

A bacterial secretory protein requires signal recognition particle for translocation across mammalian endoplasmic reticulum.

In vitro transcription of DNA from plasmid pBR322 was coupled to cell-free translation in a wheat germ system. The major translation product was pre-beta-lactamase. Upon addition of dog pancreas microsomes, the precursor was processed to authentic beta-lactamase as shown by partial NH2-terminal sequence analysis. Processing was linked to translocation into the microsomal vesicles. Salt-extracted microsomes did not process pre-beta lactamase but could be reactivated by purified signal recognition particle, which is the functional component of the salt wash (Walter, P., and Blobel, G. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 7112-7116). Signal recognition particle alone caused a drastic translation arrest that could be released by salt-depleted membranes. These data are consistent with those obtained for eukaryotic proteins and suggest that co-translational translocation of both bacterial and eukaryotic secretory proteins across the endoplasmic reticulum require identical components.

Animals↗

PubMed2002

Signal recognition particle (SRP)-mediated targeting and Sec-dependent translocation of an extracellular Escherichia coli protein.

Hemoglobin protease (Hbp) is a hemoglobin-degrading protein that is secreted by a human pathogenic Escherichia coli strain via the autotransporter mechanism. Little is known about the earliest steps in autotransporter secretion, i.e. the targeting to and translocation across the inner membrane. Here, we present evidence that Hbp interacts with the signal recognition particle (SRP) and the Sec-translocon early during biogenesis. Furthermore, Hbp requires a functional SRP targeting pathway and Sec-translocon for optimal translocation across the inner membrane. SecB is not required for targeting of Hbp but can compensate to some extent for the lack of SRP. Hbp is synthesized with an unusually long signal peptide that is remarkably conserved among a subset of autotransporters. We propose that these autotransporters preferentially use the co-translational SRP/Sec route to avoid adverse effects of the exposure of their mature domains in the cytoplasm.

Adenosine Triphosphatases↗

PubMed1982

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

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.

Endoplasmic Reticulum↗

PubMed1993

Cloning and characterization of a Bacillus subtilis gene encoding a homolog of the 54-kilodalton subunit of mammalian signal recognition particle and Escherichia coli Ffh.

By using a DNA fragment of Escherichia coli ffh as a probe, the Bacillus subtilis ffh gene was cloned. The complete nucleotide sequence of the cloned DNA revealed that it contained three open reading frames (ORFs). Their order in the region, given by the gene product, was suggested to be ORF1-Ffh-S16, according to their similarity to the gene products of E. coli, although ORF1 exhibited no significant identity with any other known proteins. The orf1 and ffh genes are organized into an operon. Genetic mapping of the ffh locus showed that the B. subtilis ffh gene is located near the pyr locus on the chromosome. The gene product of B. subtilis ffh shared 53.9 and 32.6% amino acid identity with E. coli Ffh and the canine 54-kDa subunit of signal recognition particle, respectively. Although there was low amino acid identity with the 54-kDa subunit of mammalian signal recognition particle, three GTP-binding motifs in the NH2-terminal half and amphipathic helical cores in the COOH-terminus were conserved. The depletion of ffh in B. subtilis led to growth arrest and drastic morphological changes. Furthermore, the translocation of beta-lactamase and alpha-amylase under the depleted condition was also defective.

Amino Acid Sequence↗

PubMed1994

The stress response to loss of signal recognition particle function in Saccharomyces cerevisiae.

It has been shown previously that growth and endoplasmic reticulum (ER) translocation defects occur in response to depletion of the 54-kDa subunit of signal recognition particle (SRP54) in Saccharomyces cerevisiae (Hann, B. B., and Walter, P. (1991) Cell 67, 131-144). We report here that cells depleted of SRP54p undergo a general stress response, the onset of which is observed almost two-cell doublings after SRP54 protein levels fall below the limits of detection. The stress response to SRP54p depletion occurs in two distinct phases, unlike the response to other stressors such as heat shock. In the initial phase, the cytoplasmic Hsp70 levels are drastically increased coincident with an abrupt slowing of growth and accumulation of untranslocated species of the ER-resident chaperone BiP. During this first response, levels of the yeast DnaJ homolog Ydj1p are also increased. In the second phase, which is detected 5 h later, levels of the cytoplasmic heat shock proteins Hsp82 and Hsp104 are increased. BiP is also induced during this second phase, while the ER levels of the resident foldase protein disulfide isomerase are significantly reduced. Since only those cytoplasmic stress proteins which have been shown to participate in membrane translocation are induced in the first phase, these findings indicate the presence of a stress response specific to accumulation of secretory protein precursors in the cytoplasm.

Blotting, Western↗

PubMed2004

The core Escherichia coli signal recognition particle receptor contains only the N and G domains of FtsY.

Previous studies have proposed that the N-terminal A domain (approximately 200 amino acid residues) of the Escherichia coli signal recognition particle (SRP) receptor, FtsY, is required for membrane targeting. In contrast to this suggestion, we show that A domain-truncated versions of FtsY, harboring only domains N and G, are functional. Therefore, we propose that N and G domains constitute the core SRP receptor.

Bacterial Proteins↗

PubMed2003

The beta-subunit of the signal recognition particle receptor is a novel GTP-binding protein without intrinsic GTPase activity.

The beta-subunit of the signal recognition particle receptor (SRbeta), a member of the Ras family of small molecular weight GTPases, is involved in the targeting of nascent polypeptide chains to the protein translocation machinery in the endoplasmic reticulum membrane. We purified SRbeta from an expressing strain of Escherichia coli and investigated the properties of the isolated GTPase. We find that, unlike other Ras family GTPases, most SRbeta purifies bound to GTP, and SRbeta-bound GTP is not easily exchanged with solution GTP. SRbeta possesses no detectable GTPase activity. Although a stable interaction between SRbeta and ribosomes is observed, SRbeta is not stimulated to hydrolyze GTP when incubated with ribosomes or ribosome-nascent chains. A GTPase mutant harboring a mutation in a region predicted to be functionally important, based on observations made in related GTPases, binds GTP with faster kinetics and appears to be a less stable protein but otherwise displays similar properties to the wild-type SRbeta GTPase. Our results demonstrate that as an isolated GTPase, SRbeta functions differently from the Arf- and Ras-type GTPases that it is most closely related to by sequence.

Animals↗

PubMed1997

Expression, crystallization and preliminary X-ray diffraction study of FtsY, the docking protein of the signal recognition particle of E. coli.

FtsY is the docking protein or SR alpha homologue in E. coli. It is involved in targeting secretory proteins to the cytoplasmic membrane by interacting with the signal recognition particle, controlled by guanosine 5'-triphosphate. Two different constructs have been used in crystallization studies: the full-length protein and a truncated fragment with a his-tag at the C terminus. Only the second construct resulted in crystals suitable for x-ray diffraction. The crystals belong to the monoclinic space group P2(1) with cell dimensions a = 32.20 A, b = 79.57 A, c = 59.21 A, and beta = 94.45, and contain one molecule per asymmetric unit. At cryogenic temperatures the crystals diffract to a resolution limit of 2.5 A by using a rotating anode, and beyond 1.8 A by using synchrotron radiation.

Bacterial Proteins↗

PubMed2003

The trypanosomatid signal recognition particle consists of two RNA molecules, a 7SL RNA homologue and a novel tRNA-like molecule.

Trypanosomatids are ancient eukaryotic parasites affecting humans and livestock. Here we report that the trypanosomatid signal recognition particle (SRP), unlike all other known SRPs in nature, contains, in addition to the 7SL RNA homologue, a short RNA molecule, termed sRNA-85. Using conventional chromatography, we discovered a small RNA molecule of 85 nucleotides co-migrating with the Leptomonas collosoma 7SL RNA. This RNA molecule was isolated, sequenced, and used to clone the corresponding gene. sRNA-85 was identified as a tRNA-like molecule that deviates from the canonical tRNA structure. The co-existence of these RNAs in a single complex was confirmed by affinity selection using an antisense oligonucleotide to sRNA-85. The two RNA molecules exist in a particle of approximately 14 S that binds transiently to ribosomes. Mutations were introduced in sRNA-85 that disrupted its putative potential to interact with 7SL RNA by base pairing; such mutants were unable to bind to 7SL RNA and to ribosomes and were aberrantly distributed within the cell. We postulate that sRNA-85 may functionally replace the truncated Alu domain of 7SL RNA. The discovery of sRNA-85 raises the intriguing possibility that sRNA-85 functional homologues may exist in other lower eukaryotes and eubacteria that lack the Alu domain.

Animals↗

PubMed2006

Membrane composition changes and physiological adaptation by Streptococcus mutans signal recognition particle pathway mutants.

Previously, we presented evidence that the oral cariogenic species Streptococcus mutans remains viable but physiologically impaired and sensitive to environmental stress when genes encoding the minimal conserved bacterial signal recognition particle (SRP) elements are inactivated. Two-dimensional gel electrophoresis of isolated membrane fractions from strain UA159 and three mutants (Deltaffh, DeltascRNA, and DeltaftsY) grown at pH 7.0 or pH 5.0 allowed us to obtain insight into the adaptation process and the identities of potential SRP substrates. Mutant membrane preparations contained increased amounts of the chaperones DnaK and GroES and ClpP protease but decreased amounts of transcription- and translation-related proteins, the beta subunit of ATPase, HPr, and several metabolic and glycolytic enzymes. Therefore, the acid sensitivity of SRP mutants might be caused in part by diminished ATPase activity, as well as the absence of an efficient mechanism for supplying ATP quickly at the site of proton elimination. Decreased amounts of LuxS were also observed in all mutant membranes. To further define physiological changes that occur upon disruption of the SRP pathway, we studied global gene expression in S. mutans UA159 (parent strain) and AH333 (Deltaffh mutant) using microarray analysis. Transcriptome analysis revealed up-regulation of 81 genes, including genes encoding chaperones, proteases, cell envelope biosynthetic enzymes, and DNA repair and replication enzymes, and down-regulation of 35 genes, including genes concerned with competence, ribosomal proteins, and enzymes involved in amino acid and protein biosynthesis. Quantitative real-time reverse transcription-PCR analysis of eight selected genes confirmed the microarray data. Consistent with a demonstrated defect in competence and the suggested impairment of LuxS-dependent quorum sensing, biofilm formation was significantly decreased in each SRP mutant.

Adaptation, Physiological↗

PubMed2005

The signal recognition particle and its interactions during protein targeting.

The synthesis of secretory or integral membrane proteins can be directly coupled to their translocation across or insertion into membranes. In co-translational targeting, the translation machine, the ribosome, is transferred to the respective membrane by the signal recognition particle (SRP) and its receptor (SR) as soon as a signal sequence emerges. Protein synthesis can continue at the membrane, with the nascent peptide chain directly inserting into the ribosome-bound protein-conducting channel, the Sec61 complex. During the past two years, several structures have been solved by crystallography and cryo-electron microscopy that represent distinct functional states of the SRP cycle. On this basis, the first structure-based models can be suggested that explain important aspects of protein targeting, such as the SRP-ribosome and SRP-SR interactions.

Binding Sites↗

PubMed1998

Leader peptide efficiency correlates with signal recognition particle dependence in Saccharomyces cerevisiae.

Secretion of bovine pancreatic trypsin inhibitor (BPTI) in Saccharomyces cerevisiae was examined with four different leader peptides: the invertase signal peptide, the mfalpha1 signal peptide, a synthetic signal peptide, and a synthetic pre pro leader. BPTI secretion from a low-copy CEN plasmid varies from 1.8 to 10.4 microgram/mL among these constructs. Secretion titers correlate with dependence on signal recognition particle (SRP), with greatest secretion from the most SRP-dependent construct. Examination of co- vs post-translational translocation pathways and overall translocation efficiency by ubiquitin translocation assay (UTA) does not provide insight into the variation in BPTI secretion efficiency, perhaps due to alteration in translocation kinetics from the additional polypeptide fusion required by the assay. BPTI translocation efficiency (as measured by UTA) is found to drop markedly upon depletion of Srp54p, prior to any observable growth defect. Subsequent to stress response induction and the onset of slow growth (15-h doubling time), BPTI translocation efficiency recovers to the level observed prior to SRP depletion.

Amino Acid Sequence↗

PubMed2002

Functional analysis of the signal recognition particle in Escherichia coli by characterization of a temperature-sensitive ffh mutant.

The Ffh protein of Escherichia coli is a 48-kDa polypeptide that is homologous to the SRP54 subunit of the eukaryotic signal recognition particle (SRP). Efforts to understand the function of Ffh in bacteria have depended largely on the use of E. coli strains that allow depletion of the wild-type gene product. As an alternative approach to studying Ffh, a temperature-sensitive ffh mutant was isolated. The ffh-10(Ts) mutation results in two amino acid changes in conserved regions of the Ffh protein, and characterization of the mutant revealed that the cells rapidly lose viability at the nonpermissive temperature of 42 degrees C as well as show reduced growth at the permissive temperature of 30 degrees C. While the ffh mutant is defective in insertion of inner membrane proteins, the export of proteins with cleavable signal sequences is not impaired. The mutant also shows elevated expression of heat shock proteins and accumulates insoluble proteins, especially at 42 degrees C. It was further observed that the temperature sensitivity of the ffh mutant was suppressed by overproduction of 4.5S RNA, the RNA component of the bacterial SRP, by stabilizing the thermolabile protein. Collectively, these results are consistent with a model in which Ffh is required only for localization of proteins integral to the cytoplasmic membrane and suggest new genetic approaches to the study of how the structure of the SRP contributes to its function.

Alleles↗

PubMed2006

L25 functions as a conserved ribosomal docking site shared by nascent chain-associated complex and signal-recognition particle.

The nascent chain-associated complex (NAC) is a dimeric protein complex of archaea and eukarya that interacts with ribosomes and translating polypeptide chains. We show that, in yeast, NAC and the signal-recognition particle (SRP) share the universally conserved ribosomal protein L25 as a docking site, which is in close proximity to the ribosomal exit tunnel. The amino-terminal segment of beta-NAC was found to be required for L25 binding. Purified NAC can prevent protein aggregation in vitro and thus shows certain properties of a molecular chaperone. Interestingly, the alpha-subunit of NAC interacts with the 54 kDa subunit of SRP. Consistent with a regulatory role of NAC in protein translocation into the endoplasmic reticulum (ER), we find that deletion of NAC results in an induction of the ER stress-response pathway. These results identify L25 as a conserved interaction platform for specific cytosolic factors that guide nascent polypeptides to their proper cellular destination.

Archaeal Proteins↗

PubMed1997

The NG domain of the prokaryotic signal recognition particle receptor, FtsY, is fully functional when fused to an unrelated integral membrane polypeptide.

Recent studies have revealed that Escherichia coli possesses an essential targeting system for integral membrane proteins, similar to the mammalian signal recognition particle (SRP) machinery. One essential protein in this system is FtsY, a homologue of the alpha-subunit of the mammalian SRP-receptor (SR-alpha). However, E. coli does not possess a close homologue of the integral membrane protein SR-beta, which anchors SR-alpha to the membrane. Moreover, although FtsY can be found as a peripheral membrane protein, the majority is found soluble in the cytoplasm. In this study, we obtained genetic and biochemical evidence that FtsY must be targeted to the membrane for proper function. We demonstrate that the essential membrane targeting activity of FtsY is mediated by a 198-residue-long acidic N-terminal domain. This domain can be functionally replaced by unrelated integral membrane polypeptides, thus avoiding the need for specific FtsY membrane targeting factors. Therefore, the N terminus of FtsY constitutes an independent domain, which is required only for the targeting of the C-terminal NG domain of FtsY to the membrane.

Animals↗

PubMed2000

Nuclear export of yeast signal recognition particle lacking Srp54p by the Xpo1p/Crm1p NES-dependent pathway.

BACKGROUND: The movement of macromolecules through the nuclear pores requires energy and transport receptors that bind both cargo and nuclear pores. Different molecules/complexes often require different transport receptors. The signal recognition particle (SRP) is a conserved cytosolic ribonucleoprotein that targets proteins to the endoplasmic reticulum. Previous studies have shown that the export of SRP RNA from the nucleus requires trans-acting factors and that SRP may be at least partly assembled in the nucleus, but little else is known about how it is assembled and exported into the cytoplasm. RESULTS: Of the six proteins that constitute the yeast SRP, we found that all except Srp54p were imported into the nucleus. Four of these had nucleolar pools. The same four proteins are required for stability of the yeast SRP RNA scR1, suggesting that they assemble with the RNA in the nucleus to form a central core SRP. This core SRP was a competent export substrate. Of the remaining components, Sec65p entered the nucleus and was assembled onto the core particle there, whereas Srp54p was solely cytoplasmic. The export of SRP from the nucleus required the transport receptor Xpo1p/Crm1p and Yrb2p, both components of the pathway that exports leucine-rich nuclear export signal (NES)-containing proteins from the nucleus. CONCLUSIONS: The SRP is assembled in the nucleus into a complex lacking only Srp54p. It is then exported through the NES pathway into the cytoplasm where Srp54p binds to it. This transport route for a ribonucleoprotein complex is so far unique in yeast.

Carrier Proteins↗

PubMed1999

The ribosome regulates the GTPase of the beta-subunit of the signal recognition particle receptor.

Protein targeting to the membrane of the ER is regulated by three GTPases, the 54-kD subunit of the signal recognition particle (SRP) and the alpha- and beta-subunit of the SRP receptor (SR). Here, we report on the GTPase cycle of the beta-subunits of the SR (SRbeta). We found that SRbeta binds GTP with high affinity and interacts with ribosomes in the GTP-bound state. Subsequently, the ribosome increases the GTPase activity of SRbeta and thus functions as a GTPase activating protein for SRbeta. Furthermore, the interaction between SRbeta and the ribosome leads to a reduction in the affinity of SRbeta for guanine nucleotides. We propose that SRbeta regulates the interaction of SR with the ribosome and thereby allows SRalpha to scan membrane-bound ribosomes for the presence of SRP. Interaction between SRP and SRalpha then leads to release of the signal sequence from SRP and insertion into the translocon. GTP hydrolysis then results in dissociation of SR from the ribosome, and SRP from the SR.

Animals↗

PubMed2003

Structural basis for the function of the beta subunit of the eukaryotic signal recognition particle receptor.

Protein translocation across and insertion into membranes is a process essential to all life forms. In higher eukaryotes, this process is initiated by targeting the translating ribosome to the endoplasmic reticulum via the signal recognition particle (SRP) and its membrane-associated heterodimeric receptor (SR). This targeting step is regulated by three G proteins, SRP54, SR alpha, and SR beta, which act in concert. Little is known about the regulatory role of SR beta. Here, we present the 1.7 A crystal structure of the SR beta-GTP subunit in complex with the interaction domain of SR alpha. Strikingly, the binding interface overlaps largely with the switch 1 region of SR beta. This finding, together with additional biochemical data, shows that the eukaryotic SR is a conditional and not an obligate heterodimer. The results suggest that the GTP/GDP switch cycle of SR beta functions as a regulatory switch for the receptor dimerization. We discuss the implications for the translocation pathway.

Amino Acid Sequence↗