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Results for “Signal Recognition Particle”

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PubMed2003

Secretion of LamB-LacZ by the signal recognition particle pathway of Escherichia coli.

LamB-LacZ fusion proteins have classically been used in studies of the general secretion pathway of Escherichia coli. Here we describe how increasing signal sequence hydrophobicity routes LamB-LacZ Hyb42-1 to the signal recognition particle (SRP) pathway. Secretion of this hydrophobic fusion variant (H*LamB-LacZ) was reduced in the absence of fully functional Ffh and Ffs, and the translocator jamming caused by Hyb42-1 was prevented by efficient delivery of the fusion to the periplasm. Finally, we found that in the absence of the ribosome-associated chaperone, trigger factor (Tig), LamB-LacZ localized to the periplasm in a SecA-dependent, SRP-independent fashion. Collectively, our results provide compelling in vivo evidence that there is an SRP-dependent cotranslational targeting mechanism in E. coli and argue against a role for trigger factor in pathway discrimination.

Bacterial Outer Membrane Proteins↗

PubMed1983

Subcellular distribution of signal recognition particle and 7SL-RNA determined with polypeptide-specific antibodies and complementary DNA probe.

Signal recognition particle (SRP) is a ribonucleoprotein consisting of six distinct polypeptides and one molecule of small cytoplasmic 7SL-RNA. The particle was previously shown to function in protein translocation across and protein integration into the endoplasmic reticulum membrane. Polypeptide specific antibodies were raised in rabbits against the 72,000-, 68,000-, and 54,000-mol-wt polypeptide of SRP. All three antibodies are shown to neutralize SRP activity in vitro. A solid phase radioimmune assay is described and used to follow SRP in various cell fractions. The partitioning of SRP is shown to be dependent on the ionic conditions of the fractionation. Under conditions approximating physiological ionic strength, SRP is found to be about equally distributed between a membrane associated (38%) and a free (15%) or ribosome associated (47%) state. Furthermore, it is shown that greater than 75% of the total cellular 7SL-RNA is associated with SRP polypeptide in these fractions. Thus it is likely that the major--if not the only--cellular function of 7SL-RNA is as a part of SRP.

Animals↗

PubMed1992

Small cytoplasmic RNA of Bacillus subtilis: functional relationship with human signal recognition particle 7S RNA and Escherichia coli 4.5S RNA.

Small cytoplasmic RNA (scRNA; 271 nucleotides) is an abundant and stable RNA of the gram-positive bacterium Bacillus subtilis. To investigate the function of scRNA in B. subtilis cells, we developed a strain that is dependent on isopropyl-beta-D-thiogalactopyranoside for scRNA synthesis by fusing the chromosomal scr locus with the spac-1 promoter by homologous recombination. Depletion of the inducer leads to a loss of scRNA synthesis, defects in protein synthesis and production of alpha-amylase and beta-lactamase, and eventual cell death. The loss of the scRNA gene in B. subtilis can be complemented by the introduction of human signal recognition particle 7S RNA, which is considered to be involved in protein transport, or Escherichia coli 4.5S RNA. These results provide further evidence for a functional relationship between B. subtilis scRNA, human signal recognition particle 7S RNA, and E. coli 4.5S RNA.

Bacterial Proteins↗

PubMed2006

Following the signal sequence from ribosomal tunnel exit to signal recognition particle.

Membrane and secretory proteins can be co-translationally inserted into or translocated across the membrane. This process is dependent on signal sequence recognition on the ribosome by the signal recognition particle (SRP), which results in targeting of the ribosome-nascent-chain complex to the protein-conducting channel at the membrane. Here we present an ensemble of structures at subnanometre resolution, revealing the signal sequence both at the ribosomal tunnel exit and in the bacterial and eukaryotic ribosome-SRP complexes. Molecular details of signal sequence interaction in both prokaryotic and eukaryotic complexes were obtained by fitting high-resolution molecular models. The signal sequence is presented at the ribosomal tunnel exit in an exposed position ready for accommodation in the hydrophobic groove of the rearranged SRP54 M domain. Upon ribosome binding, the SRP54 NG domain also undergoes a conformational rearrangement, priming it for the subsequent docking reaction with the NG domain of the SRP receptor. These findings provide the structural basis for improving our understanding of the early steps of co-translational protein sorting.

Animals↗

PubMed1984

Role of signal recognition particle in the membrane assembly of Sindbis viral glycoproteins.

We have investigated the role of signal recognition particle (SRP) in the biosynthesis of Sindbis glycoproteins by translating the viral 26S mRNA in a wheat-germ cell-free system. SRP was shown to have no effect on the synthesis or proteolytic processing of the cytoplasmic C protein. In contrast, the membrane integration and the proteolytic processing of the viral glycoproteins PE2 and E1 were demonstrated to be SRP-dependent. In the absence of microsomal membranes, SRP caused an arrest of the synthesis of the viral glycoproteins. This arrest could be released by the addition of salt-extracted microsomal membranes. Synchronization experiments indicated that the uncleaved signal sequence of PE2 was recognized by SRP after at most 130 amino acids of PE2 had been polymerized. No apparent interaction of SRP with a putative signal sequence of E1 and/or a 6-kDa peptide could be detected.

Animals↗

PubMed2006

Does a deficiency of the signal recognition particle (SRP)-pathway affect the biosynthesis of its components in Saccharomyces cerevisiae and Escherichia coli?

We studied the behavior of the signal recognition particle (SRP) components in Saccharomyces cerevisiae upon deficiencies of the protein transport caused by the absence of the SRP membrane receptor alpha-subunit. A decrease in the concentration of the SRP membrane receptor alpha-subunit in the cell significantly decreased the level of an SRP component, protein SRP72, as well as the levels of mRNAs of SRP protein components and the SRP receptor beta-subunit. But the amount of 7SL RNA remained unchanged. In contrast, in Escherichia coli cells the gradual decrease in the level of the protein FtsY (a homolog of the SRP membrane receptor alpha-subunit) was not associated with changes in the Ffh protein level.

Escherichia coli↗

PubMed2005

FtsY, the bacterial signal-recognition particle receptor, interacts functionally and physically with the SecYEG translocon.

Co-translational membrane targeting of proteins by the bacterial signal-recognition particle (SRP) requires the specific interaction of the SRP-ribosome nascent chain complex with FtsY, the bacterial SRP receptor (SR). FtsY is homologous to the SRalpha-subunit of the eukaryotic SR, which is tethered to the endoplasmic-reticulum membrane by its interaction with the integral SRbeta-subunit. In contrast to SRalpha, FtsY is partly membrane associated and partly located in the cytosol. However, the mechanisms by which FtsY associates with the membrane are unclear. No gene encoding an SRbeta homologue has been found in bacterial genomes, and the presence of an FtsY-specific membrane receptor has not been shown so far. We now provide evidence for the direct interaction between FtsY and the SecY translocon. This interaction offers an explanation of how the bacterial SRP cycle is regulated in response to available translocation channels.

Bacterial Proteins↗

PubMed1987

Direct probing of the interaction between the signal sequence of nascent preprolactin and the signal recognition particle by specific cross-linking.

We have studied the interaction between the signal sequence of nascent preprolactin and the signal recognition particle (SRP) during the initial events in protein translocation across the endoplasmic reticulum membrane. A new method of affinity labeling was used, whereby lysine residues, carrying the photoreactive group 4-(3-trifluoromethyldiazirino) benzoic acid in their side chains, are incorporated into a protein by means of modified lysyl-tRNA, and cross-linking to the interacting component is induced by irradiation. SRP interacts through its Mr 54,000 polypeptide component with the signal sequences of nascent preprolactin chains containing about 70 residues, and with decreasing affinity with longer chains as well; it causes inhibition of elongation. Binding of SRP is reversible and requires the nascent chain to be bound to a functional ribosome. SRP cross-linked to the signal sequence still inhibits elongation but does not prevent it completely. We conclude that SRP does not block the exit site of the polypeptide chain on the ribosome. The SRP receptor of the endoplasmic reticulum membrane displaces the signal sequence from SRP and, even if SRP is cross-linked, releases elongation arrest.

Animals↗

PubMed2001

Biogenesis of the signal recognition particle (SRP) involves import of SRP proteins into the nucleolus, assembly with the SRP-RNA, and Xpo1p-mediated export.

The signal recognition particle (SRP) targets nascent secretory proteins to the ER, but how and where the SRP assembles is largely unknown. Here we analyze the biogenesis of yeast SRP, which consists of an RNA molecule (scR1) and six proteins, by localizing all its components. Although scR1 is cytoplasmic in wild-type cells, nuclear localization was observed in cells lacking any one of the four SRP "core proteins" Srp14p, Srp21p, Srp68p, or Srp72p. Consistently, a major nucleolar pool was detected for these proteins. Sec65p, on the other hand, was found in both the nucleoplasm and the nucleolus, whereas Srp54p was predominantly cytoplasmic. Import of the core proteins into the nucleolus requires the ribosomal protein import receptors Pse1p and Kap123p/Yrb4p, which might, thus, constitute a nucleolar import pathway. Nuclear export of scR1 is mediated by the nuclear export signal receptor Xpo1p, is distinct from mRNA transport, and requires, as evidenced by the nucleolar accumulation of scR1 in a dis3/rrp44 exosome component mutant, an intact scR1 3' end. A subset of nucleoporins, including Nsp1p and Nup159p (Rat7p), are also necessary for efficient translocation of scR1 from the nucleus to the cytoplasm. We propose that assembly of the SRP requires import of all SRP core proteins into the nucleolus, where they assemble into a pre-SRP with scR1. This particle can then be targeted to the nuclear pores and is subsequently exported to the cytoplasm in an Xpo1p-dependent way.

Active Transport, Cell Nucleus↗

PubMed2000

Evolutionary conservation of post-transcriptional 3' end adenylation of small RNAs: S. cerevisiae signal recognition particle RNA and U2 small nuclear RNA are post-transcriptionally adenylated.

The 3' terminal nucleotide of several human small RNAs, including Signal Recognition Particle (SRP) RNA, 7SK RNA, U2 small nuclear RNA and ribosomal 5S RNA was previously characterized and a fraction of these RNAs was found to contain a single post-transcriptionally added adenylic acid residue on their 3' ends. Here we report the development of a reverse transcription-polymerase chain reaction (RT-PCR) assay for determining and quantifying the extent of post-transcriptional adenylation of RNAs from different species. Using this assay, we found that a fraction of S. cerevisiae U2 small nuclear RNA and S. cerevisiae SRP RNA contain a post-transcriptionally added adenylic acid residue on their 3' ends. Sequencing analysis confirmed this adenylation to be post-transcriptional. Corresponding small RNAs in Xenopus oocytes also contained this post-transcriptional adenylation on their 3' ends. These data show that post-transcriptional adenylation on the 3' end of several small RNA molecules is conserved through evolution. Xenopus SRP RNA from both cytoplasmic and nuclear compartments contained post-transcriptionally added adenylic acid residue on its 3' end. In addition, the Alu portion of SRP RNA was adenylated, when injected into the cytoplasm of frog oocytes. These data show that this novel adenylating machinery, capable of specifically adding a single adenylic acid to the 3' end of some RNA molecules, is present and functional in both nucleus and cytoplasm.

Adenosine Monophosphate↗

PubMed1994

Mammalian and Escherichia coli signal recognition particles.

Recent evidence from both biochemical and genetic studies indicates that protein targeting to the prokaryotic cytoplasmic membrane and the eukaryotic endoplasmic reticulum membrane may have more in common than previously thought. A ribonucleoprotein particle was identified in Escherichia coli that consists of at least one protein (P48 or Ffh) and one RNA molecule (4.5S RNA), both of which exhibit strong sequence similarity with constituents of the mammalian signal recognition particle (SRP). Like the mammalian SRP, the E. coli SRP binds specifically to the signal sequence of presecretory proteins. Depletion of either P48 or 4.5S RNA affects translation and results in the accumulation of precursors of several secreted proteins. This review discusses the recent studies and speculates on the position of the SRP in the complex network of protein interactions involved in translation and membrane targeting in E. coli.

Animals↗

PubMed1998

Sorting of invertase signal peptide mutants in yeast dependent and independent on the signal-recognition particle.

There is growing evidence that yeast contains two efficient pathways of protein translocation across the endoplasmic reticulum membrane, one dependent on the signal-recognition particle (SRP) and the other independent. Their specificity, however, is largely obscure. For higher eukaryotes it has been shown that a high average hydrophobicity of the core region with a minimal length around six or seven amino acids, as well as a stabilized alpha-helix, are decisive structural features for translocation. Using yeast invertase as a secretory model protein, we have found that mutated signal sequences with Pro or Gly in the core, or having only four hydrophobic amino acids, are not functional in translocation across microsomal membranes of dog pancreas because they do not interact with the SRP. Expression of these mutant variants in Saccharomyces cerevisiae revealed that they are sorted independently of the SRP since translocation was not impaired in an SRP-deficient yeast strain. In contrast to this, wild-type invertase is translocated SRP-dependently in wild-type cells and shows a decreased translocation in SRP-deficient cells. By overexpression of Srp54p, but not of Hsc70p, the translocation defect of wild-type invertase in an SRP54 disruptant is restored. The data indicate that targeting of proteins to the endoplasmic reticulum in Saccharomyces cerevisiae seems to be more flexible than in higher eukaryotes as far as the structural requirements of signal sequences are concerned, and that the route taken is specified by the sequence.

Amino Acid Sequence↗

PubMed1996

Structure of 4.5S RNA in the signal recognition particle of Escherichia coli as studied by enzymatic and chemical probing.

The structure of 4.5S RNA, the Escherichia coli homologue of the signal recognition particle (SRP) RNA, alone and in the SRP complex with protein P48 (Ffh) was probed both enzymatically and chemically. The molecule is largely resistant against single strand-specific nucleases, indicating a highly base paired structure. Reactivity appears mainly in the apical tetraloop and in one of the conserved internal loops. Although some residues are found reactive toward dimethylsulphate and kethoxal in regions predicted to be unpaired by the phylogenetic secondary structure model of 4.5S RNA, generally the reactivity is low, and some residues in internal loops are not reactive at all. RNase V1 cleaves the RNA at multiple sites that coincide with predicted helices, although the cleavages show a pronounced asymmetry. The binding of protein P48 to 4.5S RNA results in a protection of residues in the apical part of the molecule homologous to eukaryotic SRP RNA (domain IV), whereas the cleavages in the conserved apical tetraloop are not protected. Hydroxyl radical treatment reveals an asymmetric pattern of backbone reactivity; in particular, the region encompassing nucleotides 60-82, i.e., the 3' part of the conserved domain IV, is protected. The data suggest that a bend in the domain IV region, most likely at the central asymmetric internal loop, is an important element of the tertiary structure of 4.5S RNA. Hyperchromicity and lead cleavage data are consistent with the model as they reveal the unfolding of a higher-order structure between 30 and 40 degrees C. Protection by protein P48 occurs in this region of the RNA and, more strongly, in the 5' part of domain IV (nt 26-50, most strongly from 35 to 49). It is likely that P48 binds to the outside of the bent form of 4.5S RNA.

Bacterial Proteins↗

PubMed1990

The yeast Yarrowia lipolytica has two, functional, signal recognition particle 7S RNA genes.

Cells containing a deletion of either the SCR1 or SCR2 genes, which code for the 7SL RNA component of the signal recognition particle (SRP) homologue, were found to be viable. Two independent approaches demonstrated that cells containing deletions of both genes were inviable. Therefore, Yarrowia lipolytica contains two (and only two) functional 7SL RNA genes.

Blotting, Southern↗

PubMed2006

Membrane binding of the bacterial signal recognition particle receptor involves two distinct binding sites.

Cotranslational protein targeting in bacteria is mediated by the signal recognition particle (SRP) and FtsY, the bacterial SRP receptor (SR). FtsY is homologous to the SRalpha subunit of eukaryotes, which is tethered to the membrane via its interaction with the membrane-integral SRbeta subunit. Despite the lack of a membrane-anchoring subunit, 30% of FtsY in Escherichia coli are found stably associated with the cytoplasmic membrane. However, the mechanisms that are involved in this membrane association are only poorly understood. Our data indicate that membrane association of FtsY involves two distinct binding sites and that binding to both sites is stabilized by blocking its GTPase activity. Binding to the first site requires only the NG-domain of FtsY and confers protease protection to FtsY. Importantly, the SecY translocon provides the second binding site, to which FtsY binds to form a carbonate-resistant 400-kD FtsY-SecY translocon complex. This interaction is stabilized by the N-terminal A-domain of FtsY, which probably serves as a transient lipid anchor.

Bacterial Proteins↗

PubMed1990

Isolation of a yeast gene, SRH1, that encodes a homologue of the 54K subunit of mammalian signal recognition particle.

A 1.7 kilobase HindIII fragment of Saccharomyces cerevisiae DNA was cloned by cross-hybridization with the Escherichia coli secY gene. The complete nucleotide sequence of the 2.6 kb fragment of the yeast genomic DNA containing the cross-hybridizing HindIII fragment was determined. The sequence showed no apparent similarity with that of the E. coli secY gene with the exception of a completely matched sequence of 21 bp, but it contained a 1,623 nucleotide open reading frame coding for a protein of 541 amino acids with a calculated Mr of 59,600. The N-terminal portion of 303 residues of the predicted sequence was homologous to the cytosolic domain of the alpha-subunit of the signal recognition particle receptor (SR alpha), including consensus sequence elements for a GTP binding site, whereas the C-terminal portion of 238 residues had an unusual methionine-rich domain containing several repetitive sequences. An mRNA of 2.0 kb was detected on Northern blotting analysis. The predicted sequence was 48% identical with the reported sequences of the 54K subunit of the mammalian signal recognition particle (SRP54) (Romisch K. et al. (1989) Nature 340, 478-483; Bernstein, H.D. et al. (1989) Nature 340, 482-486). We designated this gene as SRH1 (SRP54 homologue). Gene disruption experiments showed that the SRH1 gene product is essential for cell growth.

Amino Acid Sequence↗

PubMed1994

The heterodimeric subunit SRP9/14 of the signal recognition particle functions as permuted single polypeptide chain.

The targeting of nascent polypeptide chains to the endoplasmic reticulum is mediated by a cytoplasmic ribonucleoprotein, the signal recognition particle (SRP). The 9 kD (SRP9) and the 14 kD (SRP14) subunits of SRP are required to confer elongation arrest activity to the particle. SRP9 and SRP14 form a heterodimer which specifically binds to SRP RNA. We have constructed cDNAs that encode single polypeptide chains comprising SRP9 and SRP14 sequences in the two possible permutations linked by a 17 amino acid peptide. We found that both fusion proteins specifically bound to SRP RNA as monomeric molecules folded into a heterodimer-like structure. Our results corroborate the previous hypothesis that the authentic heterodimer binds to SRP RNA in equimolar ratio. In addition, both fusion proteins conferred elongation arrest activity to SRP(-9/14), which lacks this function, and one fusion protein could functionally replace the heterodimer in the translocation assay. Thus, the normal N-and C-termini of both proteins have no essential role in folding, RNA-binding and in mediating the biological activities. The possibility to express the heterodimeric complex as a single polypeptide chain facilitates the analysis of its functions and its structure in vivo and in vitro.

Amino Acid Sequence↗

PubMed1991

Binding sites of the 9- and 14-kilodalton heterodimeric protein subunit of the signal recognition particle (SRP) are contained exclusively in the Alu domain of SRP RNA and contain a sequence motif that is conserved in evolution.

The mammalian signal recognition particle (SRP) is a small cytoplasmic ribonucleoprotein required for the cotranslational targeting of secretory proteins to the endoplasmic reticulum membrane. The heterodimeric protein subunit SRP9/14 was previously shown to be essential for SRP to cause pausing in the elongation of secretory protein translation. RNase protection and filter binding experiments have shown that binding of SRP9/14 to SRP RNA depends solely on sequences located in a domain of SRP RNA that is strongly homologous to the Alu family of repetitive DNA sequences. In addition, the use of hydroxyl radicals, as RNA-cleaving reagents, has revealed four distinct regions in this domain that are in close contact with SRP9/14. Surprisingly, the nucleotide sequence in one of these contact sites, predicted to be mostly single stranded, was found to be extremely conserved in SRP RNAs of evolutionarily distant organisms ranging from eubacteria and archaebacteria to yeasts and higher eucaryotic cells. This finding suggests that SRP9/14 homologs may also exist in these organisms, where they possibly contribute to the regulation of protein synthesis similar to that observed for mammalian SRP in vitro.

Animals↗