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 415 records · Page 23Linked to original sources

PubMed1989

SecB functions as a cytosolic signal recognition factor for protein export in E. coli.

A purified 64 kd protein, consisting of four identical subunits of the 16 kd SecB, binds to the signal sequence of preproteins prior to their translocation across inverted vesicles (INV) derived from the E. coli plasma membrane. The purified SecB tetramer competes with canine signal recognition particle (SRP) in signal sequence binding and thus behaves as a prokaryotic equivalent of SRP. As shown by cell fractionation and immunoblot analysis with anti-SecB antibodies, SecB is a cytosolic protein. An E. coli supernatant depleted of SecB after passage through an anti-SecB Sepharose column retains full translation activity but is unable to support translocation into added INV. Translocation into INV is fully restored by readdition of purified SecB.

Bacterial Proteins↗

PubMed2002

Mapping the signal sequence-binding site on SRP reveals a significant role for the NG domain.

We present evidence that the signal recognition particle (SRP) recognizes signal sequences via the NG domain on the SRP54 protein subunit. Using a recently developed cross-linking method (Fancy, D. A., and Kodadek, T. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 6020-6024; Correction (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 1317), we find that signal peptides cross-link to the Escherichia coli SRP protein Ffh (the homologue of the mammalian SRP54 subunit) via the NG domain. Within the NG domain, the cross-linking site maps to the ras-like C-terminal subdomain termed the G domain. This result stands in contrast to previous studies, which concluded based on nascent chain cross-linking that the signal sequence bound to the adjacent M domain. As independent evidence of a direct binding interaction between the NG domain and the signal sequence, we find that the NG domain of Ffh binds signal peptides as an isolated entity. Our results suggest that the NG domain forms a substantial part of the binding site for the signal sequence.

Amino Acid Sequence↗

PubMed1997

A highly conserved nucleotide in the Alu domain of SRP RNA mediates translation arrest through high affinity binding to SRP9/14.

Binding of the signal recognition particle (SRP) to signal sequences during translation leads to an inhibition of polypeptide elongation known as translation arrest. The arrest activity is mediated by a discrete domain comprised of the Alu portion of SRP RNA and a 9 and 14 kDa polypeptide heterodimer (SRP9/14). Although very few nucleotides in SRP RNA are conserved throughout evolution, the remarkable conservation of G24, which resides in the region of SRP9/14 interaction, suggests that it is essential for translation arrest. To understand the functional significance of the G24 residue, we made single base substitutions in SRP RNA at this position and analyzed the ability of the mutants to bind SRP9/14 and to reconstitute functional SRPs. Mutation of G24 to C reduced binding to SRP9/14 by at least 50-fold, whereas mutation to A and U reduced binding approximately 2- and 5-fold respectively. The mutant RNAs could nevertheless assemble into SRPs at high subunit concentrations. SRPs reconstituted with mutant RNAs were not significantly defective in translation arrest assays, indicating that the conserved guanosine does not interact directly with the translational machinery. Taken together, these results demonstrate that G24 plays an important role in the translation arrest function of SRP by mediating high affinity binding of SRP9/14.

Base Sequence↗

PubMed2000

Chloroplast Oxa1p homolog albino3 is required for post-translational integration of the light harvesting chlorophyll-binding protein into thylakoid membranes.

Multiple sorting pathways operate in chloroplasts to localize proteins to the thylakoid membrane. The signal recognition particle (SRP) pathway in chloroplasts employs the function of a signal recognition particle (cpSRP) to target light harvesting chlorophyll-binding protein (LHCP) to the thylakoid membrane. In assays that reconstitute stroma-dependent LHCP integration in vitro, the stroma is replaceable by the addition of GTP, cpSRP, and an SRP receptor homolog, cpFtsY. Still lacking is an understanding of events that take place at the thylakoid membrane including the identification of membrane proteins that may function at the level of cpFtsY binding or LHCP integration. The identification of Oxa1p in mitochondria, an inner membrane translocase component homologous to predicted proteins in bacteria and to the albino3 (ALB3) protein in thylakoids, led us to investigate the potential role of ALB3 in LHCP integration. Antibody raised against a 50-amino acid region of ALB3 (ALB3-50aa) identified a single 45-kDa thylakoid protein. Treatment of thylakoids with antibody to ALB3-50aa inhibited LHCP integration, whereas the same antibody treatment performed in the presence of antigen reversed the inhibition. In contrast, transport by the thylakoid Sec or Delta pH pathways was unaffected. These data support a model whereby a distinct translocase containing ALB3 is used to integrate LHCP into thylakoid membranes.

Arabidopsis↗

PubMed2003

SecYEG proteoliposomes catalyze the Deltaphi-dependent membrane insertion of FtsQ.

In Escherichia coli, the insertion of most inner membrane proteins is mediated by the Sec translocase. Ribosome-bound nascent chains of Sec-dependent inner membrane proteins are targeted to the SecYEG complex via the signal recognition particle pathway. We now demonstrate that the signal recognition particle-dependent co-translational membrane targeting and membrane insertion of FtsQ can be reconstituted with proteoliposomes containing purified SecYEG. SecA and a transmembrane electrical potential are essential for the translocation of the large periplasmic domain of FtsQ, whereas co-reconstituted YidC has an inhibitory effect. These data demonstrate that membrane protein insertion can be reconstituted with a minimal set of purified Sec components.

Adenosine Triphosphatases↗

PubMed1999

Effect of transforming RNA on the synthesis of a protein with a secretory signal sequence in vitro.

U5 small nuclear RNA itself can act as a clastogenic and transforming agent when transfected into cells. In the previous work, the 3' half of the U5 small nuclear RNA first stem structure (designated RNA3S) was capable of driving normal cells into tumorigenic cells when expressed with a poly(A) tail (RNA3S+). This transformation critically depended upon the polypurine sequence GGAGAGGAA in RNA3S+. In this work, we first examined the pre-beta-lactamase and luciferase (model secretory and nonsecretory proteins) translation with the in vitro synthesized RNA3S in rabbit reticulocyte lysate. The capped RNA3S with a poly(A) tail suppressed the translation. In addition, the polypurine sequence played a crucial role in affecting the secretory protein synthesis, indicating a primary action of RNA3S+. Further studies revealed that the oligodeoxynucleotides, corresponding to the polypurine and its antisense sequences, directly contacted 28 S rRNA in ribosome and 7SL RNA in signal recognition particle, respectively, and differentially affected the nascent chain elongation of secretory protein synthesis. These results suggest that RNA3S+ blocks a physiological regulatory function played by signal recognition particle and the ribosome in the secretory protein synthesis and support the idea that the transformation might result from a repressed cellular activity.

Animals↗

PubMed2002

Bimodal targeting of microsomal CYP2E1 to mitochondria through activation of an N-terminal chimeric signal by cAMP-mediated phosphorylation.

Cytochrome P450 2E1 (CYP2E1) plays an important role in alcohol-induced toxicity and oxidative stress. Recently, we showed that this predominantly microsomal protein is also localized in rat hepatic mitochondria. In this report, we show that the N-terminal 30 amino acids of CYP2E1 contain a chimeric signal for bimodal targeting of the apoprotein to endoplasmic reticulum (ER) and mitochondria. We demonstrate that the cryptic mitochondrial targeting signal at sequence 21-31 of the protein is activated by cAMP-dependent phosphorylation at Ser-129. S129A mutation resulted in lower affinity for binding to cytoplasmic Hsp70, mitochondrial translocases (TOM40 and TIM44) and reduced mitochondrial import. S129A mutation, however, did not affect the extent of binding to the signal recognition particle and association with ER membrane translocator protein Sec61. Addition of saturating levels of signal recognition particle caused only a partial inhibition of CYP2E1 translation under in vitro conditions, and saturating levels of ER resulted only in partial membrane integration. cAMP enhanced the mitochondrial CYP2E1 (referred to as P450MT5) level but did not affect its level in the ER. Our results provide new insights on the mechanism of cAMP-mediated activation of a cryptic mitochondrial targeting signal and regulation of P450MT5 targeting to mitochondria.

Amino Acid Sequence↗

PubMed1992

Evolutionary conserved nucleotides within the E.coli 4.5S RNA are required for association with P48 in vitro and for optimal function in vivo.

E.coli 4.5S RNA is homologous to domain IV of eukaryotic SPR7S RNA, the RNA component of the signal recognition particle. The 4.5S RNA is associated in vivo with a 48kD protein (P48), which is homologous to a protein component of the signal recognition particle, SRP54. In addition to secondary structural features, a number of nucleotides are conserved between the 4.5S RNA and domain IV of all other characterised SRP-like RNAs from eubacteria, arachaebacteria and eukaryotes. This domain consists of an extended stem-loop structure; conserved nucleotides lie within the terminal loop and within single-stranded regions bulged from the stem immediately preceding the loop. This conserved region is a candidate for the SRP54/P48 binding site. To determine the functional importance of this region within the 4.5S RNA, mutations were introduced into the 4.5S RNA coding sequence. Mutated alleles were tested for their function in vivo and for the ability of the corresponding RNAs to bind P48 in vitro. Single point mutations in conserved nucleotides within the terminal tetranucleotide loop do not affect P48 binding in vitro and produce only slight growth defects. This suggests that the sequence of the loop may be important for the structure of the molecule rather than for specific interactions with P48. On the other hand, nucleotides within the single-stranded regions bulged from the stem were found to be important both for the binding of P48 to the RNA and for optimal function of the RNA in vivo.

Alleles↗

PubMed1988

Integration of a small integral membrane protein, M2, of influenza virus into the endoplasmic reticulum: analysis of the internal signal-anchor domain of a protein with an ectoplasmic NH2 terminus.

The M2 protein of influenza A virus is a small integral membrane protein of 97 residues that is expressed on the surface of virus-infected cells. M2 has an unusual structure as it lacks a cleavable signal sequence yet contains an ectoplasmic amino-terminal domain of 23 residues, a 19 residue hydrophobic transmembrane spanning segment, and a cytoplasmic carboxyl-terminal domain of 55 residues. Oligonucleotide-mediated deletion mutagenesis was used to construct a series of M2 mutants lacking portions of the hydrophobic segment. Membrane integration of the M2 protein was examined by in vitro translation of synthetic mRNA transcripts prepared using bacteriophage T7 RNA polymerase. After membrane integration, M2 was resistant to alkaline extraction and was converted to an Mr approximately equal to 7,000 membrane-protected fragment after digestion with trypsin. In vitro integration of M2 requires the cotranslational presence of the signal recognition particle. Deletion of as few as two residues from the hydrophobic segment of M2 markedly decreases the efficiency of membrane integration, whereas deletion of six residues completely eliminates integration. M2 proteins containing deletions that eliminate stable membrane anchoring are apparently not recognized by signal recognition particles, as these polypeptides remain sensitive to protease digestion, indicating that in addition they do not have a functional signal sequence. These data thus indicate that the signal sequence that initiates membrane integration of M2 resides within the transmembrane spanning segment of the polypeptide.

Animals↗

PubMed2005

N-myristoylation determines dual targeting of mammalian NADH-cytochrome b5 reductase to ER and mitochondrial outer membranes by a mechanism of kinetic partitioning.

Mammalian NADH-cytochrome b5 reductase (b5R) is an N-myristoylated protein that is dually targeted to ER and mitochondrial outer membranes. The N-linked myristate is not required for anchorage to membranes because a stretch of hydrophobic amino acids close to the NH2 terminus guarantees a tight interaction of the protein with the phospholipid bilayer. Instead, the fatty acid is required for targeting of b5R to mitochondria because a nonmyristoylated mutant is exclusively localized to the ER. Here, we have investigated the mechanism by which N-linked myristate affects b5R targeting. We find that myristoylation interferes with interaction of the nascent chain with signal recognition particle, so that a portion of the nascent chains escapes from cotranslational integration into the ER and can be post-translationally targeted to the mitochondrial outer membrane. Thus, competition between two cotranslational events, binding of signal recognition particle and modification by N-myristoylation, determines the site of translation and the localization of b5R.

Animals↗

PubMed1989

Uncoupling of co-translational translocation from signal peptidase processing in a mutant rat preapolipoprotein-A-IV with a deletion that includes the COOH-terminal region of its signal peptide.

In order to characterize the function of the COOH-terminal (c) region of eukaryotic signal peptides, a 14-amino acid long segment was deleted from a secreted rat liver and intestinal protein, preapolipoprotein-A-IV. This deletion spanned the c-region plus all potential signal peptidase cleavage sites. The functional consequences of this mutation were assessed using an in vitro transcription/translation/microsomal membrane processing system. Removal of these residues had no effect on interaction of the nascent preprotein with signal recognition particle as measured by a translational arrest assay. Although no signal peptidase cleavage of the mutant was detected, the efficiency of its co-translational translocation was similar to the wild type protein. A postinitiation translocation assay was utilized to compare the translocation capabilities of nascent wild type and mutant proteins as their chain lengths were progressively increased. No difference was detected between the two species suggesting that their initial conformations are functionally equivalent as measured by the translocation machinery. We conclude from these studies of preapolipoprotein-A-IV that (i) the efficiency of translocation is not dependent on signal peptidase cleavage and (ii) structural features present in the c-region of the signal peptide are not necessary for interaction with signal recognition particle or for subsequent targeting to, and translocation across, microsomal membranes.

Amino Acid Sequence↗

PubMed1998

The plurifunctional nucleolus.

The nucleolus of eukaryotic cells was first described in the early 19th century and was discovered in the 1960s to be the seat of ribosome synthesis. Although rRNA transcription, rRNA processing and ribosome assembly have been clearly established as major functions of the nucleolus, recent studies suggest that the nucleolus participates in many other aspects of gene expression as well. Thus, the nucleolus has been implicated in the processing or nuclear export of certain mRNAs. In addition, new results indicate that biosyntheses of signal recognition particle RNA and telomerase RNA involve a nucleolar stage and that the nucleolus is also involved in processing of U6 RNA, one of the spliceosomal small nuclear RNAs. Interestingly, these three nucleolus-associated small nuclear RNAs (signal recognition particle RNA, telomerase RNA and U6 RNA) are components of catalytic ribonucleoprotein machines. Finally, recent work has also suggested that some transfer RNA precursors are processed in the nucleolus. The nucleolus may have evolutionarily descended from a proto-eukaryotic minimal genome that was spatially linked to vicinal RNA processing and ribonucleoprotein assembly events involved in gene read-out. The nucleolus of today's eukaryotes, now surrounded by the chromatin of over 2 billion years of genome expansion, may still perform these ancient functions, in addition to ribosome biosynthesis. The plurifunctional nucleolus concept has a strong footing in contemporary data and adds a new perspective to our current picture of the spatial-functional design of the cell nucleus.

Cell Nucleolus↗

PubMed1992

Signal-sequence recognition by an Escherichia coli ribonucleoprotein complex.

Hydrophobic signal-sequences direct the transfer of secretory proteins across the inner membrane of prokaryotes and the endoplasmic reticulum membranes of eukaryotes. In mammalian cells, signal-sequences are recognized by the 54K protein (M(r) 54,000) of the signal recognition particle (SRP) which is believed to hold the nascent chain in a translocation-competent conformation until it contacts the endoplasmic reticulum membrane. The SRP consists of a 7S RNA and six different polypeptides. The 7S RNA and the 54K signal-sequence-binding protein (SRP54) of mammalian SRP exhibit strong sequence similarity to the 4.5S RNA and P48 protein (Ffh) of Escherichia coli which form a ribonucleoprotein particle. Depletion of 4.5S RNA or overproduction of P48 causes the accumulation of the beta-lactamase precursor, although not of other secretory proteins. Whether 4.5S RNA and P48 are part of an SRP-like complex with a role in protein export is controversial. Here we show that the P48/4.5S RNA ribonucleoprotein complex interacts specifically with the signal sequence of a nascent secretory protein and therefore is a signal recognition particle.

Bacterial Proteins↗

PubMedDate not supplied

Signal sequence mutations disrupt feedback between secretion of an exported protein and its synthesis in E. coli.

Recent studies in a eukaryotic system indicate that a block in secretion can lead to a block in the translation of secretory proteins. This feedback on protein synthesis is thought to be a result of an interaction of the signal recognition particle with the signal sequences of nascent proteins. Genetic studies in the prokaryote Escherichia coli suggest that a complex secretion machinery and a similar feedback mechanism exist. In addition, mutations affecting two genes, secA and secC, thought to encode components of the bacterial secretion machinery, selectively interfere with the synthesis of exported proteins. This selective interference with translation may be a result of recognition by the secretion machinery of signal sequences. If so, alteration of the signal sequence of a particular protein by mutation should eliminate the block in synthesis for that protein. We show here that signal sequence mutants for an exported protein, maltose binding protein, prevent the block in synthesis of this protein in a secA mutant.

ATP-Binding Cassette Transporters↗

PubMedDate not supplied

A structure for the signal sequence binding protein SRP54: 3D reconstruction from STEM images of single molecules.

The 54-kDa subunit SRP54 of the signal recognition particle in eukaryotic cells is responsible for the recognition of nascent proteins destined for secretion or membrane integration. The three-dimensional structure of this protein was determined using computational techniques applied to images of the molecule obtained via high-resolution, low-dose, scanning transmission electron microscopy at low temperature. The reconstructions at spatial resolutions between 12 and 15 A feature two unequal domains joined by a slender linker. The two-domain structure is in agreement with genetic and biochemical data indicating organization of SRP54 into a larger N-terminal GTP-binding region and a smaller C-terminal peptide-binding region. The structure has similarities to other protein domains with related functions and similar amino acid sequences. The larger domain of the 3D reconstruction is consistent in shape and size with the GTP-binding domains of EF-Tu and p21-RAS, while the smaller domain is compatible in structure with part of the peptide-binding protein calmodulin. The overall shape of SRP54 and the deduced location of critical functional regions of the molecule provide a structural framework for its known biochemical properties in the targeting cycle of the signal recognition particle.

Amino Acid Sequence↗

PubMed2005

Peculiar properties of DsbA in its export across the Escherichia coli cytoplasmic membrane.

Export of DsbA, a protein disulfide bond-introducing enzyme, across the Escherichia coli cytoplasmic membrane was studied with special reference to the effects of various mutations affecting translocation factors. It was noted that both the internalized precursor retaining the signal peptide and the periplasmic mature product fold rapidly into a protease-resistant structure and they exhibited anomalies in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in that the former migrated faster than the latter. The precursor, once accumulated, was not exported posttranslationally. DsbA export depended on the SecY translocon, the SecA ATPase, and Ffh (signal recognition particle), but not on SecB. SecY mutations, such as secY39 and secY205, that severely impair translocation of a number of secretory substrates by interfering with SecA actions only insignificantly impaired the DsbA export. In contrast, secY125, affecting a periplasmic domain and impairing a late step of translocation, exerted strong export inhibition of both classes of proteins. These results suggest that DsbA uses not only the signal recognition particle targeting pathway but also a special route of translocation through the translocon, which is hence suggested to actively discriminate pre-proteins.

Amino Acid Sequence↗

PubMed1987

A functional interaction between the signal peptide and the translation apparatus is detected by the use of a single point mutation which blocks translocation across mammalian endoplasmic reticulum.

A functional interaction between the signal sequence and the translation apparatus which may serve as a first step in chain targeting to the membrane is described. To this end, we exploited the powerful technique of molecular cloning in a procaryotic system and the well characterized translocation system of mammalian endoplasmic reticulum. The signal peptide of subunit B of the heat labile enterotoxin of Escherichia coli (EltB) was fused to several proteins. Single base substitutions were introduced in the signal peptide and their effect on protein synthesis and translocation was studied. We sought a single amino acid substitution which may define certain steps in the coordinated regulation of chain synthesis and targeting to the membrane. The substitution of proline for leucine at residue -8 in the signal peptide abolished all known functions of the signal peptide. In contrast to wild type signal peptide, the mutant signal peptide did not lead to arrest of nascent chain synthesis by signal recognition particle or translocation of the precursor protein across the membrane of the endoplasmic reticulum. Furthermore, the mutant signal peptide was not cleaved by purified E. coli signal peptidase. Interestingly, the mutation resulted in about a 2-fold increase in the rate of synthesis of the precursor protein, suggesting a role for the signal peptide in regulating the synthesis of the nascent secretory chain as a means of ensuring early and efficient targeting of this chain to the membrane. This role might involve interaction of the signal peptide with components of the translation apparatus and/or endogenous signal recognition particle. These results were obtained with three different fusion proteins carrying the signal peptide of EltB thus leading to the conclusion that the effect of the mutation on the structure and function of the signal peptide is independent of the succeeding sequence to which the signal peptide is attached.

Amino Acid Sequence↗

PubMed1988

Positive charges at the NH2 terminus convert the membrane-anchor signal peptide of cytochrome P-450 to a secretory signal peptide.

The NH2-terminal sequences of cytochromes P-450 resemble signal peptides, but these sequences are not cleaved during the insertion of these integral membrane proteins into the microsomes. To examine whether these putative signal peptides are functionally equivalent to signal peptides of secretory proteins, cDNA coding for a fusion protein was produced, in which the signal peptide for preproparathyroid hormone was replaced with the putative signal peptide of cytochrome P450IIC2. The translational product of RNA synthesized in vitro from the cDNA was neither processed nor translocated by chicken oviduct microsomal membranes in a reticulocyte cell-free system but was resistant to extraction from the membranes by alkaline solutions. In addition, the translation of the hybrid RNA was arrested by signal recognition particle. Unlike most signal peptides, the cytochrome P450IIC2 NH2-terminal sequence does not contain basic amino acids preceding the hydrophobic core. Introduction by oligonucleotide-directed mutagenesis of lysine and arginine at the NH2 terminus resulted in a fusion protein that was partially processed by the microsomal membranes, with translocation across the membrane of both the processed and unprocessed proteins. The positive charges convert the cytochrome P450IIC2 NH2 terminus from a combination membrane insertion-halt transfer signal to a more classical secretory membrane-insertion signal, possibly by altering the orientation of the signal peptide in the membrane.

Amino Acid Sequence↗