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Results for “Signal Recognition Particle”

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PubMed2000

Characterization of the signal that directs Tom20 to the mitochondrial outer membrane.

Tom20 is a major receptor of the mitochondrial preprotein translocation system and is bound to the outer membrane through the NH(2)-terminal transmembrane domain (TMD) in an Nin-Ccyt orientation. We analyzed the mitochondria-targeting signal of rat Tom20 (rTom20) in COS-7 cells, using green fluorescent protein (GFP) as the reporter by systematically introducing deletions or mutations into the TMD or the flanking regions. Moderate TMD hydrophobicity and a net positive charge within five residues of the COOH-terminal flanking region were both critical for mitochondria targeting. Constructs without net positive charges within the flanking region, as well as those with high TMD hydrophobicity, were targeted to the ER-Golgi compartments. Intracellular localization of rTom20-GFP fusions, determined by fluorescence microscopy, was further verified by cell fractionation. The signal recognition particle (SRP)-induced translation arrest and photo-cross-linking demonstrated that SRP recognized the TMD of rTom20-GFP, but with reduced affinity, while the positive charge at the COOH-terminal flanking segment inhibited the translation arrest. The mitochondria-targeting signal identified in vivo also functioned in the in vitro system. We conclude that NH(2)-terminal TMD with a moderate hydrophobicity and a net positive charge in the COOH-terminal flanking region function as the mitochondria-targeting signal of the outer membrane proteins, evading SRP-dependent ER targeting.

Amino Acid Sequence↗

PubMed2006

Signal sequences directing cotranslational translocation expand the range of proteins amenable to phage display.

Even proteins that fold well in bacteria are frequently displayed poorly on filamentous phages. Low protein presentation on phage might be caused by premature cytoplasmic folding, leading to inefficient translocation into the periplasm. As translocation is an intermediate step in phage assembly, we tested the display levels of a range of proteins using different translocation pathways by employing different signal sequences. Directing proteins to the cotranslational signal recognition particle (SRP) translocation pathway resulted in much higher display levels than directing them to the conventional post-translational Sec translocation pathway. For example, the display levels of designed ankyrin-repeat proteins (DARPins) were improved up to 700-fold by simply exchanging Sec- for SRP-dependent signal sequences. In model experiments this exchange of signal sequences improved phage display from tenfold enrichment to >1,000-fold enrichment per phage display selection round. We named this method 'SRP phage display' and envision broad applicability, especially when displaying cDNA libraries or very stable and fast-folding proteins from libraries of alternative scaffolds.

Gene Expression Regulation, Bacterial↗

PubMed1998

Binding of GTP and GDP induces a significant conformational change in the GTPase domain of Ffh, a bacterial homologue of the SRP 54 kDa subunit.

The bacterial Ffh protein is homologous to the SRP54 subunit of the signal recognition particle. Ffh plays a key role in the targeting of proteins to the membrane and it is composed of a N-terminal domain (N), a middle GTPase (G) domain and a C-terminal M domain which has binding sites for SRP RNA and signal peptide. The GTP binding and hydrolysis of Ffh is critical to its function. We have used protease digestion to probe the conformation of the Mycoplasma mycoides Ffh N+G domain. In the absence of nucleotide the protein was comparatively sensitive to protease cleavage and we identified sites particularly prone to cleavage in a region near the C-terminus of the GTPase domain. However, in the presence of GTPgammaS or GDP this region is stabilized and the protein adopts a more ordered structure. The pattern of cleavage with GTPgammaS was indistinguishable from that when GDP was bound, indicating that the conformation of the nucleotide-free form is distinct from that when either GTPgammaS or GDP is bound to the protein. The possible functional role of this significant conformational change is discussed.

Amino Acid Sequence↗

PubMedDate not supplied

A signal sequence receptor in the endoplasmic reticulum membrane.

Protein translocation across the endoplasmic reticulum (ER) membrane is triggered at several stages by information contained in the signal sequence. Initially, the signal sequence of a nascent secretory protein upon emergence from the ribosome is recognized by a polypeptide of relative molecular mass 54,000 (Mr54K) which is part of the signal recognition particle (SRP). Binding of SRP may induce a site-specific elongation arrest of translation in vitro. Attachment of the arrested translation complex to the ER membrane is mediated by the SRP-receptor (docking protein) and is accompanied by displacement of the SRP from both the ribosome and the signal sequence. We have investigated the fate of the signal sequence following the disengagement of SRP and its receptor by a crosslinking approach. We report here that the signal sequence of nascent preprolactin, after its release from the SRP, interacts with a newly discovered component, a signal sequence receptor (SSR), which is an integral, glycosylated protein of the rough ER membrane (Mr approximately 35K).

Animals↗

PubMed1991

Requirements for the membrane insertion of signal-anchor type proteins.

Proteins which are inserted and anchored in the membrane of the ER by an uncleaved signal-anchor sequence can assume two final orientations. Type I signal-anchor proteins translocate the NH2 terminus across the membrane while type II signal-anchor proteins translocate the COOH terminus. We investigated the requirements for cytosolic protein components and nucleotides for the membrane targeting and insertion of single-spanning type I signal-anchor proteins. Besides the ribosome, signal recognition particle (SRP), GTP, and rough microsomes (RMs) no other components were found to be required. The GTP analogue GMPPNP could substitute for GTP in supporting the membrane insertion of IMC-CAT. By using a photocrosslinking assay we show that for secreted, type I and type II signal-anchor proteins the presence of both GTP and RMs is required for the release of the nascent chain from the 54-kD subunit of SRP. For two of the proteins studied the release of the nascent chain from SRP54 was accompanied by a new interaction with components of the ER. We conclude that the GTP-dependent release of the nascent chain from SRP54 occurs in an identical manner for each of the proteins studied.

Animals↗

PubMed2001

A universal mode of helix packing in RNA.

RNA molecules fold into specific three-dimensional shapes to perform structural and catalytic functions. Large RNAs can form compact globular structures, but the chemical basis for close helical packing within these molecules has been unclear. Analysis of transfer, catalysis, in vitro-selected and ribosomal RNAs reveal that helical packing predominantly involves the interaction of single-stranded adenosines with a helix minor groove. Using the Tetrahymena thermophila group I ribozyme, we show here that the near-perfect shape complementarity between the adenine base and the minor groove allows for optimal van der Waals contacts, extensive hydrogen bonding and hydrophobic surface burial, creating a highly energetically favorable interaction. Adenosine is recognized in a chemically similar fashion by a combination of protein and RNA components in the ribonucleoprotein core of the signal recognition particle. These results provide a thermodynamic explanation for the noted abundance of conserved adenosines within the unpaired regions of RNA secondary structures.

Adenosine↗

PubMed2001

Inhibition of protein translocation across the endoplasmic reticulum membrane by sterols.

Cholesterol and related sterols are known to modulate the physical properties of biological membranes and can affect the activities of membrane-bound protein complexes. Here, we report that an early step in protein translocation across the endoplasmic reticulum (ER) membrane is reversibly inhibited by cholesterol levels significantly lower than those found in the plasma membrane. By UV-induced chemical cross-linking we further show that high cholesterol levels prevent cross-linking between ribosome-nascent chain complexes and components of the Sec61 translocon, but have no effect on cross-linking to the signal recognition particle. The inhibiting effect on translocation is different between different sterols. Our data suggest that the protein translocation machinery may be sensitive to changes in cholesterol levels in the ER membrane.

Biological Transport↗

PubMed2000

SRP-dependent co-translational targeting and SecA-dependent translocation analyzed as individual steps in the export of a bacterial protein.

Recently it has been recognized that the signal recognition particle (SRP) of Escherichia coli represents a specific targeting device for hydrophobic inner membrane proteins. It has remained unclear, however, whether the bacterial SRP functions in concert with SecA, which is required for the translocation of secretory proteins across the inner membrane. Here, we have analyzed a hybrid protein constructed by fusing the signal anchor sequence of an SRP-dependent inner membrane protein (MtlA) to the mature part of an exclusively SecA-requiring secretory protein (OmpA). We show that the signal anchor sequence of MtlA confers the novel properties onto nascent chains of OmpA of being co-translationally recognized and targeted to SecY by SRP. Once targeted to SecY, ribosome-associated nascent chains of the hybrid protein, however, remain untranslocated unless SecA is present. These results indicate that SRP and SecA cooperate in a sequential, non-overlapping manner in the topogenesis of those membrane proteins which, in addition to a signal anchor sequence, harbor a substantial hydrophilic domain to be translocated into the periplasm.

Adenosine Triphosphatases↗

PubMed1987

Import of honeybee prepromelittin into the endoplasmic reticulum: structural basis for independence of SRP and docking protein.

Honeybee prepromelittin is correctly processed and imported by dog pancreas microsomes. Insertion of prepromelittin into microsomal membranes, as assayed by signal sequence removal, does not depend on signal recognition particle (SRP) and docking protein. We addressed the question as to how prepromelittin bypasses the SRP/docking protein system. Hybrid proteins between prepromelittin, or carboxy-terminally truncated derivatives, and the cytoplasmic protein dihydrofolate reductase from mouse were constructed. These hybrid proteins were analysed for membrane insertion and sequestration into microsomes. The results suggest the following: (i) The signal sequence of prepromelittin is capable of interacting with the SRP/docking protein system, but this interaction is not mandatory for membrane insertion; this is related to the small size of prepromelittin. (ii) In prepromelittin a cluster of negatively charged amino acids must be balanced by a cluster of positively charged amino acids in order to allow membrane insertion. (iii) In general, a signal sequence can be sufficient to mediate membrane insertion independently of SRP and docking protein in the case of short precursor proteins; however, the presence and distribution of charged amino acids within the mature part of these precursors can play distinct roles.

Animals↗

PubMed2002

Construction of the 'minimal' SRP that interacts with the translating ribosome but not with specific membrane receptors in Escherichia coli.

Escherichia coli signal recognition particle (SRP) consists of 4.5S RNA and Ffh protein. In contrast to eukaryotes, it remains unclear whether translation arrest takes place in prokaryotic cells. To study this problem we constructed a fusion of the M domain of Ffh protein with a cleavable affinity tag. This mutant Ffh, in a complex with 4.5S RNA, can bind signal peptide at the translating ribosome but is unable to bind the membrane. This SRP-ribosome complex should accumulate in the cell if translation is arrested. To test this, the complex was purified from the cells by ultracentrifugation and affinity chromatography. The composition of the complex was analyzed and found to consist of ribosomal RNAs and proteins, the Ffh M domain and 4.5S RNA. The accumulation of this complex in the cell in significant amounts indicated that SRP-mediated translation arrest did occur in bacterial cells.

Escherichia coli↗

PubMed2002

Structural basis for mobility in the 1.1 A crystal structure of the NG domain of Thermus aquaticus Ffh.

The NG domain of the prokaryotic signal recognition protein Ffh is a two-domain GTPase that comprises part of the prokaryotic signal recognition particle (SRP) that functions in co-translational targeting of proteins to the membrane. The interface between the N and G domains includes two highly conserved sequence motifs and is adjacent in sequence and structure to one of the conserved GTPase signature motifs. Previous structural studies have shown that the relative orientation of the two domains is dynamic. The N domain of Ffh has been proposed to function in regulating the nucleotide-binding interactions of the G domain. However, biochemical studies suggest a more complex role for the domain in integrating communication between signal sequence recognition and interaction with receptor. Here, we report the structure of the apo NG GTPase of Ffh from Thermus aquaticus refined at 1.10 A resolution. Although the G domain is very well ordered in this structure, the N domain is less well ordered, reflecting the dynamic relationship between the two domains previously inferred. We demonstrate that the anisotropic displacement parameters directly visualize the underlying mobility between the two domains, and present a detailed structural analysis of the packing of the residues, including the critical alpha4 helix, that comprise the interface. Our data allows us to propose a structural explanation for the functional significance of sequence elements conserved at the N/G interface.

Bacterial Proteins↗

PubMed2002

RNA interference of signal peptide-binding protein SRP54 elicits deleterious effects and protein sorting defects in trypanosomes.

Trypanosomes are protozoan parasites that have a major impact on health. This family diverged very early from the eukaryotic lineage and possesses unique RNA processing mechanisms such as trans-splicing and RNA editing. The trypanosome signal recognition particle (SRP) has a unique composition compared with all known SRP complexes, because it contains two RNA molecules, the 7SL RNA and a tRNA-like molecule. RNA interference was utilized to elucidate the essentiality of the SRP pathway and its role in protein translocation in Trypanosoma brucei. The production of double stranded RNA specific for the signal peptide-binding protein SRP54 induced the degradation of the mRNA and a loss of the SRP54 protein. SRP54 depletion elicited inhibition in growth and cytokinesis, suggesting that the SRP pathway is essential. The translocation of four signal peptide-containing proteins was examined. Surprisingly, the proteins were translocated to the endoplasmic reticulum and properly processed. However, the surface EP procyclin, the lysosomal protein p67, and the flagellar pocket protein CRAM were mislocalized and accumulated in megavesicles, most likely because of a secondary effect on protein sorting. The translocation of these proteins to the endoplasmic reticulum under SRP54 depletion suggests that an alternative pathway for protein translocation exists in trypanosomes.

Amino Acid Sequence↗

PubMed2004

Heterodimeric GTPase core of the SRP targeting complex.

Two structurally homologous guanosine triphosphatase (GTPase) domains interact directly during signal recognition particle (SRP)-mediated cotranslational targeting of proteins to the membrane. The 2.05 angstrom structure of a complex of the NG GTPase domains of Ffh and FtsY reveals a remarkably symmetric heterodimer sequestering a composite active site that contains two bound nucleotides. The structure explains the coordinate activation of the two GTPases. Conformational changes coupled to formation of their extensive interface may function allosterically to signal formation of the targeting complex to the signal-sequence binding site and the translocon. We propose that the complex represents a molecular "latch" and that its disengagement is regulated by completion of assembly of the GTPase active site.

Amino Acid Motifs↗

PubMed1999

Archaea sister group of Bacteria? Indications from tree reconstruction artifacts in ancient phylogenies.

The 54-kDa signal recognition particle and the receptor SR alpha, two proteins involved in the cotranslational translocation of proteins, are paralogs. They originate from a gene duplication that occurred prior to the last universal common ancestor, allowing one to root the universal tree of life. Phylogenetic analysis using standard methods supports the generally accepted cluster of Archaea and Eucarya. However, a new method increasing the signal-to-noise ratio strongly suggests that this result is due to a long-branch attraction artifact, with the Bacteria evolving fastest. In fact, the Archaea/Eucarya sisterhood is recovered only by the fast-evolving positions. In contrast, the most slowly evolving positions, which are the most likely to retain the ancient phylogenetic signal, support the monophyly of prokaryotes. Such a eukaryotic rooting provides a simple explanation for the high similarity of Archaea and Bacteria observed in complete-genome analysis, and should prompt a reconsideration of current views on the origin of eukaryotes.

Archaea↗

PubMed1991

SRP-RNA sequence alignment and secondary structure.

The secondary structures of the RNAs from the signal recognition particle, termed SRP-RNA, were derived buy comparative analyses of an alignment of 39 sequences. The models are minimal in that only base pairs are included for which there is comparative evidence. The structures represent refinements of earlier versions and include a new short helix.

Archaea↗

PubMed1998

A general mechanism for regulation of access to the translocon: competition for a membrane attachment site on ribosomes.

For proteins to enter the secretory pathway, the membrane attachment site (M-site) on ribosomes must bind cotranslationally to the Sec61 complex present in the endoplasmic reticulum membrane. The signal recognition particle (SRP) and its receptor (SR) are required for targeting, and the nascent polypeptide associated complex (NAC) prevents inappropriate targeting of nonsecretory nascent chains. In the absence of NAC, any ribosome, regardless of the polypeptide being synthesized, binds to the endoplasmic reticulum membrane, and even nonsecretory proteins are translocated across the endoplasmic reticulum membrane. By occupying the M-site, NAC prevents all ribosome binding unless a signal peptide and SRP are present. The mechanism by which SRP overcomes the NAC block is unknown. We show that signal peptide-bound SRP occupies the M-site and therefore keeps it free of NAC. To expose the M-site and permit ribosome binding, SR can pull SRP away from the M-site without prior release of SRP from the signal peptide.

Animals↗

PubMed1995

Early events in preprotein recognition in E. coli: interaction of SRP and trigger factor with nascent polypeptides.

In Escherichia coli, components of a signal recognition particle (SRP) and its receptor have been identified which appear to be essential for efficient translocation of several proteins. In this study we use cross-linking to demonstrate that E. coli SRP interacts with a variety of nascent presecretory proteins and integral inner membrane proteins. Evidence is presented that the interaction is correlated with the hydrophobicity of the core region of the signal sequence and thereby with its ability to promote transport in vivo. A second E. coli component, which is identified as trigger factor, can be efficiently cross-linked to all tested nascent chains derived from both secreted and cytosolic proteins. We propose that SRP and trigger factor act as secretion-specific and general molecular chaperone respectively, early in protein synthesis.

Amino Acid Isomerases↗

PubMed1998

Sec/SRP-independent insertion of two thylakoid membrane proteins bearing cleavable signal peptides.

Two imported thylakoid membrane proteins, PSII-X and PSII-W, are synthesised with cleavable N-terminal signal peptides that closely resemble those of Sec-dependent lumenal proteins. In this report we have reconstituted the insertion of pre-PSII-X and pre-PSII-W into isolated thylakoids. We show that insertion does not require either nucleoside triphosphates or stromal extracts, both of which are required for Sec- and signal recognition particle (SRP)-dependent targeting mechanisms. Insertion is furthermore unaffected by protease treatments that destroy the known protein translocation apparatus in the thylakoid membrane. We conclude that these membrane proteins are inserted by an unusual Sec/SRP-independent mechanism that probably resembles that used by CFoII, and we discuss possible parallels with the biogenesis of phage M13 procoat.

Chloroplasts↗