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Results for “Signal Recognition Particle”

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PubMed1985

Escherichia coli 6S RNA is not essential for growth or protein secretion.

The function of the stable 6S RNA of Escherichia coli is not known. Recently, it was proposed that the 6S RNA is a component of a bacterial signal recognition particle required for protein secretion. To test this proposal, we isolated a mutant that lacks the 6S RNA. Studies of the mutant show that the 6S RNA is not essential for growth or for protein secretion. The gene for the 6S RNA (ssr) maps near serA at 63 min on the E. coli genetic map.

Bacterial Proteins↗

PubMed1995

Nascent polypeptide-associated complex protein prevents mistargeting of nascent chains to the endoplasmic reticulum.

We show that, after removal of the nascent polypeptide-associated complex (NAC) from ribosome-associated nascent chains, ribosomes synthesizing proteins lacking signal peptides are efficiently targeted to the endoplasmic reticulum (ER) membrane. After this mistargeting, translocation across the ER membrane occurs, albeit less efficiently than for a nascent secretory polypeptide, perhaps because the signal peptide is needed to catalyze the opening of the translocation pore. The mistargeting was prevented by the addition of purified NAC and was shown not to be mediated by the signal recognition particle and its receptor. Instead, it appears to be a consequence of the intrinsic affinity of ribosomes for membrane binding sites, since it can be blocked by competing ribosomes that lack associated nascent polypeptides. We propose that, when bound to a signalless ribosome-associated nascent polypeptide, NAC sterically blocks the site in the ribosome for membrane binding.

Animals↗

PubMed2006

Role of protein kinase C-mediated protein phosphorylation in mitochondrial translocation of mouse CYP1A1, which contains a non-canonical targeting signal.

A large number of mitochondrial proteins lack canonical mitochondrial-targeting signals. The bimodal transport of cytochromes P450 (CYPs) to endoplasmic reticulum and mitochondria (MT), reported previously by us, likely represents one mode of non-canonical protein targeting to MT. Herein, we have studied the mechanism of mouse MT-CYP1A1 targeting to gain insight into the regulatory features and evolutionary conservation of bimodal targeting mechanism. Mouse MT-CYP1A1 consists of two NH2-terminal-truncated molecular species, +91A1 and +331A1. Mutations Pro-2 --> Leu and Tyr-5 --> Leu, which increase the signal recognition particle (SRP) binding, diminished MT targeting of the protein in intact cells. By contrast, mutations Leu-7 --> Asn and Leu-17 --> Asn, which decreased SRP-binding affinity, enhanced MT targeting, thus suggesting that SRP binding is an important regulatory step that modulates bimodal targeting. Protein kinase C (PKC)-mediated phosphorylation of nascent chains at Thr-35 vastly decreased affinity for SRP binding suggesting an important regulatory step. In support of these results, COS cell transfection experiments show that phosphomimetic mutation Thr-35 --> Asp or induced cellular PKC caused increased CYP1A1 targeting to MT and correspondingly lower levels to the endoplasmic reticulum. Results suggest evolutionary conservation of chimeric signals and bimodal targeting of CYP1A1 in different species. The mouse MT-CYP1A1 is an extrinsic membrane protein, which exhibited high FDX1 plus FDXR-mediated N-demethylation of a number of tricyclic antidepressants, pain killers, anti-psychotics, and narcotics that are poor substrates for microsomal CYP1A1.

Amino Acid Sequence↗

PubMed2000

Regulation of ribosome detachment from the mammalian endoplasmic reticulum membrane.

In current models, protein translocation in the endoplasmic reticulum (ER) occurs in the context of two cycles, the signal recognition particle (SRP) cycle and the ribosome cycle. Both SRP and ribosomes bind to the ER membrane as a consequence of the targeting process of translocation. Whereas SRP release from the ER membrane is regulated by the GTPase activities of SRP and the SRP receptor, ribosome release from the ER membrane is thought to occur in response to the termination of protein synthesis. We report that ER-bound ribosomes remain membrane-bound following the termination of protein synthesis and in the bound state can initiate the translation of secretory and cytoplasmic proteins. Two principal observations are reported. 1) Membrane-bound ribosomes engaged in the synthesis of proteins lacking a signal sequence are released from the ER membrane as ribosome-nascent polypeptide complexes. 2) Membrane-bound ribosomes translating secretory proteins can access the translocon in an SRP receptor-independent manner. We propose that ribosome release from the ER membrane occurs in the context of protein translation, with release occurring by default in the absence of productive nascent polypeptide-membrane interactions.

Animals↗

PubMed1991

A nascent membrane protein is located adjacent to ER membrane proteins throughout its integration and translation.

The immediate environment of nascent membrane proteins undergoing integration into the ER membrane was investigated by photocrosslinking. Nascent polypeptides of different lengths, each containing a single IgM transmembrane sequence that functions either as a stop-transfer or a signal-anchor sequence, were synthesized by in vitro translation of truncated mRNAs in the presence of N epsilon-(5-azido-2-nitrobenzoyl)-Lys-tRNA, signal recognition particle, and microsomal membranes. This yielded nascent chains with photoreactive probes at one end of the transmembrane sequence where two lysine residues are located. When irradiated, these nascent chains reacted covalently with several ER proteins. One prominent crosslinking target was a glycoprotein similar in size to a protein termed mp39, shown previously to be situated adjacent to a secretory protein during its translocation across the ER membrane (Krieg, U. C., A. E. Johnson, and P. Walter. 1989. J. Cell Biol. 109:2033-2043; Wiedmann, M., D. Goerlich, E. Hartmann, T. V. Kurzchalia, and T. A. Rapoport. 1989. FEBS (Fed. Eur. Biochem. Soc.) Lett. 257:263-268) and likely to be identical to a protein previously designated the signal sequence receptor (Wiedmann, M., T. V. Kurzchalia, E. Hartmann, and T. A. Rapoport. 1987. Nature (Lond.). 328:830-833). Changing the orientation of the transmembrane domain in the bilayer, or making the transmembrane domain the first topogenic sequence in the nascent chain instead of the second, did not significantly alter the identities of the ER proteins that were the primary crosslinking targets. Furthermore, the nascent chains crosslinked to the mp39-like glycoprotein and other microsomal proteins even after the cytoplasmic tail of the nascent chain had been lengthened by nearly 100 amino acids beyond the stop-transfer sequence. Yet when the nascent chain was allowed to terminate normally, the major photocrosslinks were no longer observed, including in particular that to the mp39-like glycoprotein. These results show that the transmembrane segment of a nascent membrane protein is located adjacent to the mp39-like glycoprotein and other ER proteins during the integration process, and that at least a portion of the nascent chain remains in close proximity to these ER proteins until translation has been completed.

Endoplasmic Reticulum↗

PubMed1988

A chemically synthesized radiolabeled signal peptide: design, preparation, and biological evaluation of an iodinated analog of preproparathyroid hormone.

The chemically synthesized signal peptide (native-sequence signal peptide) of preproparathyroid hormone exhibits signal sequence-like activity by inhibiting the translocation/processing of precursor proteins to their mature forms in an in vitro translation system. In order to prepare a biologically functional radiolabeled form of this peptide, we undertook structure-function studies of the native-sequence signal peptide. Since conventional iodination of peptides is performed under oxidizing conditions, chemical design efforts were focused on the oxidation-labile residues, methionine and cysteine, present in the native sequence. Substitution of the three methionines with norleucine and the single cysteine with alanine yielded a surfur-free analog, [Nle-(-25), Nle-(-21),Nle-(-18),Ala-(-14),D-Tyr-(+1)]pre-proPTH-(-29-+1)amide, which is resistant to oxidation and active in the inhibition of processing assay. An interaction between the signal region and one of the components of the intracellular secretory apparatus, signal recognition particle (SRP), was demonstrated: iodinated sulfur-free analog was cross-linked (using the homo-bifunctional reagent disuccinimidyl suberate) to the 54 kilodalton (kDa) subunit of SRP. The 68 kDa and 72 kDa subunits of SRP were also labeled, but to a lesser extent, by the iodinated peptide.

Amino Acid Sequence↗

PubMedDate not supplied

Ribonucleoparticle-independent import of proteins into mammalian microsomes involves a membrane protein which is sensitive to chemical alkylation.

Import of the small precursor protein preprocecropin A (ppcec A) into dog pancreas microsomes under post-translational conditions does not involve the ribonucleoparticles, signal recognition particle and ribosome, and their receptors on the microsomal surface, docking protein and ribosome receptor. In this study, we attempted to obtain direct evidence for the involvement of proteinaceous membrane components in this alternative import mechanism by utilizing various sulfhydryl-modifying reagents (N-ethylmaleimide (NEM), N-iodoacetylaminoethyl-5-naphthylamine-1- sulphonic acid (AEDANS) and iodoacetamide (IAA]. As a result of observing the inhibitory effect of 2 reagents (NEM and AEDANS) on the microsomes with respect to ppcec A-import, we concluded that there is at least one membrane protein with a cytoplasmically exposed sulfhydryl involved in ppcep A-import. This membrane protein(s) is (are) distinct from membrane proteins which are known to be involved in protein import, such as the signal peptidase and the so-called signal sequence receptor.

Alkylating Agents↗

PubMed2004

LuxS-mediated signaling in Streptococcus mutans is involved in regulation of acid and oxidative stress tolerance and biofilm formation.

LuxS-mediated quorum sensing has recently been shown to regulate important physiologic functions and virulence in a variety of bacteria. In this study, the role of luxS of Streptococcus mutans in the regulation of traits crucial to pathogenesis was investigated. Reporter gene fusions showed that inactivation of luxS resulted in a down-regulation of fructanase, a demonstrated virulence determinant, by more than 50%. The LuxS-deficient strain (TW26) showed increased sensitivity to acid killing but could still undergo acid adaptation. Northern hybridization revealed that the expression of RecA, SmnA (AP endonuclease), and Nth (endonuclease) were down-regulated in TW26, especially in early-exponential-phase cells. Other down-regulated genes included ffh (a signal recognition particle subunit) and brpA (biofilm regulatory protein A). Interestingly, the luxS mutant showed an increase in survival rate in the presence of hydrogen peroxide (58.8 mM). The luxS mutant formed less biofilm on hydroxylapatite disks, especially when grown in biofilm medium with sucrose, and the mutant biofilms appeared loose and hive-like, whereas the biofilms of the wild type were smooth and confluent. The mutant phenotypes were complemented by exposure to supernatants from wild-type cultures. Two loci, smu486 and smu487, were identified and predicted to encode a histidine kinase and a response regulator. The phenotypes of the smu486 smu487 mutant were, in almost all cases, similar to those of the luxS mutant, although our results suggest that this is not due to AI-2 signal transduction via Smu486 and Smu487. This study demonstrates that luxS-dependent signaling plays critical roles in modulating key virulence properties of S. mutans.

Bacterial Proteins↗

PubMed1995

Expression and in vitro targeting of a sunflower oleosin.

Two distinct cDNAs encoding oleosins (oil body proteins) have been identified by degenerate PCR as transcripts present in the developing seeds of sunflower (Helianthus annuus L. cv. Dwarf Sunbred). One (pSOM) of these is closely related to a reported sunflower oleosin, whilst the other (pSO5) has not been previously described. Different expression patterns were observed for the two cDNAs, pSO5 being expressed earlier than pSOM in seed maturation and oil deposition. The results support the contention that oleosin proteins are synthesised either during or closely after the formation of the oil body. Translation in vitro of synthetic oleosin transcripts was enhanced by the addition of microsomes but suppressed by the addition of purified signal recognition particle (SRP) complex. Deletion of 62 amino acid residues at the C-terminus of the oleosin did not alter the in vitro targeting of the protein to the microsomal membrane. Taken together these data support the idea that oleosins are targeted to the ER membrane as part of oil body biogenesis.

Amino Acid Sequence↗

PubMed1990

Structure and biosynthesis of the signal-sequence receptor.

The signal-sequence receptor (SSR) has previously been shown to be a component of the environment which nascent polypeptides meet on passage through the endoplasmic reticulum (ER) membrane. We report here on the primary structure of the SSR as deduced from cDNA clones and from direct protein sequencing. The glycoprotein is synthesized with a cleavable amino-terminal signal sequence and contains only one classical membrane-spanning segment. Its insertion into the ER membrane during biosynthesis depends on the function of the signal-recognition particle. SSR shows a remarkable charge distribution with the amino terminus being highly negatively charged, and the cytoplasmic carboxyl terminus positively charged. The SSR can be phosphorylated in its cytoplasmic tail both in intact cells and in a cell-free system, suggesting a regulation of its function. The localization of the protein in the ER membrane was confirmed by immunofluorescence microscopy.

Amino Acid Sequence↗

PubMed2002

Components required for membrane assembly of newly synthesized K+ channel KcsA.

An Escherichia coli in vitro transcription-translation system was used to study the components involved in the biogenesis of the homotetrameric potassium channel KcsA. We show that a functional signal recognition particle pathway is essential for tetramer formation, probably to direct correct monomer insertion in the membrane. In the absence of YidC or at reduced SecYEG levels, KcsA assembly occurs with lower efficiency. Strikingly, the highest efficiency of tetramerization was observed when transcription-translation was carried out in the presence of pure lipid vesicles, demonstrating that a phospholipid bilayer is the minimal membrane requirement to form the KcsA tetramer. It is concluded that SecYEG and YidC are not required for the formation of tetrameric KcsA in vitro.

Bacterial Proteins↗

PubMed1992

Sea urchin small RNA ribonucleoprotein particles: identification, synthesis, and subcellular localization during early embryonic development.

Small RNAs in sea urchins were examined in order to characterize developmental changes in their level, subcellular localization, synthesis, and association with proteins and other RNAs. Small RNAs such as the U snRNAs, 5S and 5.8S rRNAs, and 7S RNAs were identified by their mobility on highly cross-linked acrylamide gels. In addition, 7SL and U1 RNAs were identified by northern blot hybridization to cloned human and sea urchin probes, respectively. The level, subcellular localization, and association with proteins or RNA do not change for most small RNAs from fertilization to blastula, even though this is the time when the stored maternal pool of many small RNAs is being supplemented and replaced by embryonically synthesized RNAs. New embryonic synthesis of small RNAs was first detected at the 8-12 hr blastula stage. Although the predicted subsets of the total small RNA pool can be found in the appropriate subcellular compartments, newly synthesized small RNAs have a predominantly cytoplasmic localization: All of the newly synthesized small RNAs were found to be constituents of small RNPs. The RNPs containing newly synthesized small RNAs had sedimentation rates indistinguishable from their maternal counterparts. Thus, on the basis of sedimentation rate, no gross differences could be detected between maternal and embryonic small RNP pools. These small RNPs include a cytoplasmic RNP containing newly synthesized U1 snRNA and the sea urchin signal recognition particle (SRP) containing the 7SL, RNA. We have also identified a small RNP bearing the 5S rRNA which is present in both eggs and embryos. The presence of multiple, abundant, small RNAs and RNPs that are maintained at constant levels in particular subcellular fractions throughout development suggests that small RNAs may be involved in many more cellular activities than have so far been described.

Animals↗

PubMed2000

Expression profiling of pancreatic beta cells: glucose regulation of secretory and metabolic pathway genes.

Pancreatic beta cells respond to changes in blood glucose by secreting insulin and increasing insulin synthesis. To identify genes used in these responses, we have carried out expression profiling of beta cells exposed to high (25 mM) or low (5.5 mM) glucose by using oligonucleotide microarrays. Functional clustering of genes that averaged a 2.2-fold or greater change revealed large groups of secretory pathway components, enzymes of intermediary metabolism, cell-signaling components, and transcription factors. Many secretory pathway genes were up-regulated in high glucose, including seven members of the endoplasmic reticulum (ER) translocon. In agreement with array analysis, protein levels of translocon components were increased by high glucose. Most dramatically, the alpha subunit of the signal recognition particle receptor was increased over 20-fold. These data indicate that the translocon and ribosome docking are major regulatory targets of glucose in the beta cell. Analysis of genes encoding enzymes of intermediary metabolism indicated that low glucose brought about greater utilization of amino acids as an energy source. This conclusion was supported by observations of increased urea production under low-glucose conditions. The above results demonstrate genome-wide integration of beta-cell functions at the level of transcript abundance and validate the efficacy of expression profiling in identifying genes involved in the beta-cell glucose response.

Animals↗

PubMed1995

Targeting and topology in the membrane of plant 3-hydroxy-3-methylglutaryl coenzyme A reductase.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyzes the synthesis of mevalonate. This is the first committed step of isoprenoid biosynthesis. A common feature of all known plant HMGR isoforms is the presence of two highly conserved hydrophobic sequences in the N-terminal quarter of the protein. Using an in vitro system, we showed that the two hydrophobic sequences of Arabidopsis HMGR1S function as internal signal sequences. Specific recognition of these sequences by the signal recognition particle mediates the targeting of the protein to microsomes derived from the endoplasmic reticulum. Arabidopsis HMGR is inserted into the microsomal membrane, and the two hydrophobic sequences become membrane-spanning segments. The N-terminal end and the C-terminal catalytic domain of Arabidopsis HMGR are positioned on the cytosolic side of the membrane, whereas only a short hydrophilic sequence is exposed to the lumen. Our results suggest that the plant HMGR isoforms known to date are primarily targeted to the endoplasmic reticulum and have the same topology in the membrane. This reinforces the hypothesis that mevalonate is synthesized only in the cytosol. The possibility that plant HMGRs might be located in different regions of the endomembrane system is discussed.

Amino Acid Sequence↗

PubMed2006

Myositis specific autoantibodies.

Recent studies have continued to examine the clinical associations of the group of autoantibodies that occurs predominantly in patients who have myositis (antibodies to aminoacyl-tRNA synthetases, to signal recognition particle [SRP], and to the nuclear helicase Mi-2). The anti-synthetase syndrome has been further studied, and the value of tacrolimus in treatment of the associated interstitial lung disease has been noted. The low frequency of myositis specific autoantibodies in non-myositis neuromuscular disorders has been more clearly demonstrated. The clinical associations of anti-Mi-2 and anti-SRP were further studied, and patients with antibodies without myositis were reported. Evidence suggested that ultraviolet light exposure may influence the expression of dermatomyositis and anti-Mi-2. A new classification for myositis using overlap clinical features and autoantibodies was proposed. A new autoantibody, anti-caDM-140, was described, associated with clinically amyopathic dermatomyositis and interstitial lung disease. The possibility was raised that increased antigen expression in regenerating muscle may help to perpetuate the disease. These antibodies continue to be the subject of active investigation.

Amino Acyl-tRNA Synthetases↗

PubMed2005

Grass evolution inferred from chromosomal rearrangements and geometrical and statistical features in RNA structure.

The grasses (Poaceae) represent a monophyletic lineage that arose about 70 million years ago. The lineage contains about 10,000 species that differ widely in morphology and physiology. Species show striking differences in genome size, a feature important in the context of conservation of gene content and order (synteny and colinearity) and in the extension of genomic information directly from one grass species to another using comparative approaches. Grass diversification has been a contentious issue, as the exact branching order of the various subfamilies has been difficult to establish with standard methods. This motivated an evolutionary study of deep phylogenetic relationships based on the structure of coding and non-coding RNA molecules and on chromosomal rearrangements. Phylogenetic relationships in the grass family were inferred directly from the structure of RNA using cladistic principles and considerations in statistical mechanics. Coded attributes describing topological and thermodynamic information embedded in RNA molecules were treated as linearly ordered multi-state characters and were polarized by fixing the direction of character transformation toward molecular order. Intrinsically rooted phylogenies derived from the structure of signal recognition particle (SRP) RNA, the mRNA encoded by the early nodulation gene enod40, the small subunit of ribosomal RNA (rRNA), and the internal transcribed spacer ITS1 of rRNA established an order for the diversification of major grass lineages, suggesting a sister relationship of the Pooideae and the PACCAD clade. This same conclusion was reached when large-scale chromosomal rearrangements derived from the comparative genetic mapping of cereal genomes were studied. Chromosomal complements aligned in the most parsimonious manner allowed identification and coding of characters depicting chromosomal translocations, insertions, and linkage block arrangements and the reconstruction of phylogenetic trees based on large-scale chromosomal structure. Congruent reconstruction of deep branching relationships using geometrical and statistical features of RNA structure and orthology and large scale chromosomal recombination events support assumptions of polarization in character argumentation, and fail to falsify the claim that extant grass chromosomes can be considered combinations of linkage blocks of an ancestor of the rice genome. Congruence also suggests that the universal tendency toward order in RNA and the search for the most parsimonious organization of be genome architecture appear to be mutually supported drivers of molecular evolution. The study clarifies the relationship of major clades in the grasses, shows that phylogenetic history can be reconstructed effectively from the combinatorial exchange of chromosomal linkage blocks, and reveals considerable phylogenetic signal embedded in the structure of signal polypeptide-coding mRNA molecules, describing an instance where mRNA structure is the subject of strong evolutionary constraint.

Base Pairing↗

PubMed1985

Elongation arrest is not a prerequisite for secretory protein translocation across the microsomal membrane.

Signal recognition particle (SRP) is a ribonucleoprotein consisting of six distinct polypeptides and one molecule of small cytoplasmic 7SL RNA. It was previously shown to promote the co-translational translocation of secretory proteins across the endoplasmic reticulum by (a) arresting the elongation of the presecretory nascent chain at a specific point, and (b) interacting with the SRP receptor, an integral membrane protein of the endoplasmic reticulum which is active in releasing the elongation arrest. Recently a procedure was designed by which the particle could be disassembled into its protein and RNA components. We have further separated the SRP proteins into four homogeneous fractions. When recombined with each other and with 7SL RNA, they formed fully active SRP. Particles missing specific proteins were assembled in the hope that some of these would retain some functional activity. SRP(-9/14), the particle lacking the 9-kD and 14-kD polypeptides, was fully active in promoting translocation, but was completely inactive in elongation arrest. This implied that elongation arrest is not a prerequisite for protein translocation. SRP receptor was required for SRP(-9/14)-mediated translocation to occur, and thus must play some role in the translocation process in addition to releasing the elongation arrest.

Animals↗

PubMed1992

The intracellular targeting and membrane topology of 3-hydroxy-3-methylglutaryl-CoA reductase.

We present evidence that the amino-terminal 39 residue region of 3-hydroxy-3-methylglutaryl- (HMG) CoA reductase, which includes the putative first transmembrane span, is a signal sequence for targeting HMG-CoA reductase to the endoplasmic reticulum. This evidence is based upon fractionation, endoglycosidase-H sensitivity and protease protection assays on an in vitro transcription/translocation system programmed with a mutant cDNA of HMG-CoA reductase that is deleted for sequences coding for all of the putative transmembrane spans except the first. We show that the protein product of this mutant cDNA is associated with microsomes, glycosylated, or protected from proteolysis only in the presence of Signal Recognition Particle. Also, we present evidence for a topological model of HMG-CoA reductase that consists of eight transmembrane spans. This evidence is based upon a concanavalin A binding assay for in vivo glycosylation of an engineered glycosylation site in each of a series of mutants of the fusion protein, HMGal (Skalnik, D. G., Narita, H., Kent, C., and Simoni, R. D. (1988) J. Biol. Chem. 263, 6836-6841). This series of mutants was designed such that for each linker segment between transmembrane spans, a mutant was constructed with an engineered glycosylation site introduced into that linker segment. We show that only the mutants with glycosylation sites in the linker segments between transmembrane spans 1 and 2, 3 and 4, and 5 and 6 are glycosylated. These results support an eight transmembrane span model for the topology of HMG-CoA reductase and are inconsistent with a seven-transmembrane span model.

Amino Acid Sequence↗