Interactions of signal peptides with signal-recognition particle.

The mechanisms whereby isolated or synthetic signal peptides inhibit processing of newly synthesized prolactin in microsome-supplemented lysates from reticulocytes and wheat-germ were investigated. At a concentration of 5 microM, a consensus signal peptide reverses the elongation arrest imposed by the signal-recognition particle (SRP), and at higher concentrations in addition inhibits elongation of both secretory and non-secretory proteins. A photoreactive form of a synthetic signal peptide cross-links under u.v. illumination to the 54 kDa and 68 kDa subunits of SRP, whereas the major cross-linked protein produced after photoreaction of rough microsomes is of 45 kDa. As SRP-mediated elongation arrest is unlikely to be essential for translocation, it is suggested that signal peptides may interact with components other than SRP in the translation system in vitro.     

An unusual signal peptide extension inhibits the binding of bacterial presecretory proteins to the signal recognition particle, trigger factor, and the SecYEG complex

Considerable evidence indicates that the Escherichia coli signal recognition particle (SRP) selectively targets proteins that contain highly hydrophobic signal peptides to the SecYEG complex cotranslationally. Presecretory proteins that contain only moderately hydrophobic signal peptides typically interact with trigger factor (TF) and are targeted post-translationally. Here we describe a striking exception to this rule that has emerged from the analysis of an unusual 55-amino acid signal peptide associated with the E. coli autotransporter EspP. The EspP signal peptide consists of a C-terminal domain that resembles a classical signal peptide plus an N-terminal extension that is conserved in other autotransporter signal peptides. Although a previous study showed that proteins containing the C-terminal domain of the EspP signal peptide are targeted cotranslationally by SRP, we found that proteins containing the full-length signal peptide were targeted post-translationally via a novel TF-independent mechanism. Mutation of an invariant asparagine residue in the N-terminal extension, however, restored cotranslational targeting. Remarkably, proteins containing extremely hydrophobic derivatives of the EspP signal peptide were also targeted post-translationally. These and other results suggest that the N-terminal extension alters the accessibility of the signal peptide to SRP and TF and promotes post-translational export by reducing the efficiency of the interaction between the signal peptide and the SecYEG complex. Based on data, we propose that the N-terminal extension mediates an interaction with an unidentified cytoplasmic factor or induces the formation of an unusual signal peptide conformation prior to the onset of protein translocation.     

RNA-mediated interaction between the peptide-binding and GTPase domains of the signal recognition particle

The signal recognition particle (SRP) targets nascent proteins to cellular membranes for insertion or secretion by recognizing polypeptides containing an N-terminal signal sequence as they emerge from the ribosome. GTP-dependent binding of SRP to its receptor protein leads to controlled release of the nascent chain into a membrane-spanning translocon pore. Here we show that the association of the SRP with its receptor triggers a marked conformational change in the complex, localizing the SRP RNA and the adjacent signal peptide-binding site at the SRP-receptor heterodimer interface. The orientation of the RNA suggests how peptide binding and GTP hydrolysis can be coupled through direct structural contact during cycles of SRP-directed protein translocation.     

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.     

Production of native, correctly folded bovine pancreatic trypsin inhibitor by Escherichia coli

A gene for bovine pancreatic trypsin inhibitor (BPTI) was fused to the coding sequence for the Escherichia coli alkaline phosphatase signal peptide and expressed in E. coli under the control of the alkaline phosphatase promoter. When induced in phosphate-depleted medium such cells produced a trypsin inhibitor that was indistinguishable from native, properly folded BPTI. In particular, the BPTI produced by E. coli had three disulfide bonds that appeared to be identical to those found in native BPTI, as assayed by sensitivity to iodoacetate, dithiothreitol, and urea. This expression/secretion system will make possible the production of variant BPTI molecules, thus allowing the perturbing effects of amino acid substitutions on BPTI folding, structure, and function to be assessed.     

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.     

Structural determinants for signal sequence function in the mammalian endoplasmic reticulum

Signal sequences function in protein targeting to and translocation across the endoplasmic reticulum membrane. To investigate the structural requirements for signal sequence function, chimeras of the Escherichia coli LamB signal peptide and prolactin were prepared. The LamB signal peptide was chosen by virtue of the extensive biophysical and biological characterization of its activity. In vitro, nascent prolactin chains bearing the LamB signal peptide (LamB) were targeted in a signal recognition particle (SRP)-dependent manner to rough microsomes but remained protease- and salt-sensitive and translocated at low efficiency. Full translocation activity was obtained in a gain of function mutant (LamB*) in which three hydrophobic residues in the LamB hydrophobic core were converted to leucine residues. Cross-linking studies demonstrated that the LamB* signal sequence displayed markedly enhanced interactions with SRP and integral membrane proteins. In contrast, chemically denatured LamB and LamB*-precursors bound with identical efficiencies and in a salt-resistant manner to rough microsomes, suggesting that during de novo synthesis the signal sequence of LamB-bearing precursors assumes a conformation refractory to translocation. These data indicate that a leucine-rich signal sequence is necessary for optimal interaction with SRP and suggest that SRP, by maintaining the signal sequence in a conformation suitable for membrane binding, performs a chaperone function.     

Proteome-based identification of signal peptides for improved secretion of recombinant cyclomaltodextrin glucanotransferase in Escherichia coli

Secretion of recombinant proteins in heterologous host has drawn attention for its simpler purification and downstream processes. Searching for secretion aid molecules to improve protein secretion can be done through synthetic biology, screening of genome data and proteome-based approach. In the present study, the extracellular proteome on starch-containing medium of Bacillus lehensis G1 was analyzed to identify naturally secreted proteins with signal peptide. A total of 87 protein spots were identified by mass spectrometry, which were categorized mostly in the metabolism of carbohydrates and related molecules (20%). Over-expression and secretion studies were performed for all the 14 selected signal peptides fused to a reporter protein, cyclomaltodextrin glucanotransferase (CGTase). All clones were found to allow CGTase to be excreted into the medium, as observed and measured from the iodine plate assay and enzyme activity assay. Compared to native signal peptide (G1) of CGTase, signal peptide of GlcNAc-binding protein A (GAP) significantly improved CGTase activities by 735% and 205% in extracellular and periplasmic compartment, respectively, with an increase of only ∼1.7 fold the amount of β-galactosidase (cell lysis) in the medium. GAP has the highest secretion rate of 45.6 U/ml/h among all clones, where physicochemical characteristics of signal peptide play significant role.     

Role of the propilin leader peptide in the maturation of F pilin.

F-pilin maturation and translocation result in the cleavage of a 51-amino-acid leader sequence from propilin and require LepB and TraQ but not the SecA-SecY secretion pathway. The unusual propilin leader peptide and the dependence of its cleavage on TraQ suggested that TraQ recognition may be specific for the leader peptide. An in vitro propilin cleavage assay yielded propilin (13 kDa), the pilin polypeptide (7 kDa), and a 5.5-kDa protein as the traA products. The 5.5-kDa protein comigrates with the full-length 51-amino-acid leader peptide, and [14C]proline labeling confirmed its identity since the only proline residues of propilin are found within the leader peptide. The in vitro and in vivo propilin-processing reactions proceed similarly in a single polypeptide cleavage step. Furthermore, TraQ dependence is a property of F-pilin maturation specifically rather than a property of the leader peptide. A propilin derivative with an amino-terminal signal sequence generated by deleting codons 2 to 28 required TraQ for processing in vivo. On the other hand, a chimeric protein with the propilin wild-type leader peptide fused to the mature portion of beta-lactamase was processed in a TraQ-independent manner. Thus, despite its unusual length, the propilin leader peptide seems to perform a function similar to that of the typical amino-terminal signal sequence. This work suggests that TraQ is not necessary for the proteolysis of propilin and therefore is likely to act as a chaperone-like protein that promotes the translocation of propilin.     

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

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

A part of the transmembrane domain of pro-TNF can function as a cleavable signal sequence that generates a biologically active secretory form of TNF.

To determine the minimum requirement in the 76-residue leader sequence of pro-tumor necrosis factor (TNF) for membrane translocation across the endoplasmic reticulum (ER) and for the maturation of pro-TNF, we constructed pro-TNF mutants in which a part of the transmembrane domain of pro-TNF was directly linked to the N-terminus of the mature domain, and evaluated their translocational behavior across the ER-membrane and their secretion from the transfected cells. The in vitro translation/translocation assay involving a canine pancreatic microsomal membrane system including a mutant, Delta-75-47, -32-1, revealed that the N-terminal half of the transmembrane domain of pro-TNF consisting of 14 residues functioned as a cleavable signal sequence; it generated a cleaved form of TNF having a molecular mass similar to that of mature TNF. Analysis of the cleavage site by site-directed mutagenesis indicated that the site was inside the leader sequence of this mutant. When the mutant, Delta-75-47, -32-1, was expressed in COS-1 cells, efficient secretion of a biologically active soluble TNF was observed. Further deletion of the hydrophobic domain from this mutant inhibited the translocation, indicating that some extent of hydrophobicity is indispensable for the membrane translocation of the mature domain of TNF. Thus, the N-terminal half of the transmembrane domain of pro-TNF could function as a cleavable signal sequence when linked to the mature domain of TNF, and secretion of a biologically active secretory form of TNF could be achieved with this 14-residue hydrophobic segment. In intact pro-TNF, however, this 14-residue sequence could not function as a cleavable signal sequence during intracellular processing, indicating that the remainder of the 76-residue leader sequence of pro-TNF inhibits the signal peptide cleavage and thus enables the leader sequence to function as a type II signal-anchor sequence that generates a transmembrane form of TNF.     

Reinitiation of translocation in the Semliki Forest virus structural polyprotein: identification of the signal for the E1 glycoprotein.

The biosynthesis of the Semliki Forest virus (SFV) structural proteins provides an interesting model system to study the reinitiation of translocation of membrane proteins into the endoplasmic reticulum membrane. The two transmembrane spike proteins, p62 and E1, are derived from a single polypeptide precursor. Once the first protein, p62, has been anchored and its cytoplasmic tail has been synthesized, translocation must be reinitiated to account for the insertion of the E1 protein. We have used deletion mutagenesis of the SFV cDNA to investigate the requirements for this event and map in detail the location of the signal. We have shown by deleting the region encoding the p62 signal and expressing the modified cDNA in COS cells that the p62 protein is not involved in the translocation of the E1 protein. The E1 signal was precisely mapped by progressive truncations of the 6 K peptide (located between p62 and E1 in the SFV polyprotein) and subsequent analysis in cell-free systems. A segment within the last 26 residues of the 6 K peptide was shown to be essential for translocation. This segment was then fused to the N-terminus of the chimpanzee alpha-globin and was shown to be sufficient for translocation. The E1 signal was cleaved efficiently even when attached to the alpha-globin protein. The activity of the signal was found to be SRP dependent in a wheat-germ cell-free system. We conclude that prior attachment of the ribosome to the membrane via the p62 signal peptide is not necessary for E1 translocation and that the reinitiation of translocation is mediated by an independent internal signal likely to be SRP dependent.     

Advances in the structure and functions of signal recognition particle in protein targeting

The signal recognition particle (SRP) is a unique moiety in living cells, which has been conserved during evolution for protein targeting and translocation across membranes in collaboration with its receptor (SR). The structural and functional features of its components, (six polypeptides and RNA) are being rapidly elucidated. We have endeavored in this review to epitomize most recent advances in this field. Its two domains (S and Alu) play important roles in signal recognition, elongation arrest and protein targeting of the polypeptide being synthesized in the cytoplasm. SRP14 and SRP9 help in the elongation arrest by interacting with signal peptide. GTPase activity of SRP54 releases SRP from SR. In addition, alpha and beta subunits of SR also possess GTPase activities and the three GTPases help in docking of nascent peptide chain-ribosome complex to the translocation site. Further strides in proteomics employing mass spectrometry and X-ray crystallography are expected to throw more light on the molecular events occurring during protein targeting and translocation.     

Internally transposed signal sequence of carp preproinsulin retains its functions with the signal recognition particle

It is shown that the signal sequence of carp preproinsulin is functional with the dog pancreatic signal recognition particle (SRP) both when present at its normal location at the amino-terminus of the protein or when engineered to an internal location. Inhibition of translation by SRP in the absence of microsomal membranes, reconstitution by SRP of the translocation competence of high-salt inactivated microsomes and signal peptide cleavage all occur with the signal sequence being preceded by a highly charged peptide segment of 39 amino acid residues (the distance from the amino-terminus to the cleavage site of the signal peptidase is increased to 56 residues).     

Efficient secretion of small proteins in mammalian cells relies on Sec62-dependent posttranslational translocation

Mammalian cells secrete a large number of small proteins, but their mode of translocation into the endoplasmic reticulum is not fully understood. Cotranslational translocation was expected to be inefficient due to the small time window for signal sequence recognition by the signal recognition particle (SRP). Impairing the SRP pathway and reducing cellular levels of the translocon component Sec62 by RNA interference, we found an alternate, Sec62-dependent translocation path in mammalian cells required for the efficient translocation of small proteins with N-terminal signal sequences. The Sec62-dependent translocation occurs posttranslationally via the Sec61 translocon and requires ATP. We classified preproteins into three groups: 1) those that comprise ≤100 amino acids are strongly dependent on Sec62 for efficient translocation; 2) those in the size range of 120-160 amino acids use the SRP pathway, albeit inefficiently, and therefore rely on Sec62 for efficient translocation; and 3) those larger than 160 amino acids depend on the SRP pathway to preserve a transient translocation competence independent of Sec62. Thus, unlike in yeast, the Sec62-dependent translocation pathway in mammalian cells serves mainly as a fail-safe mechanism to ensure efficient secretion of small proteins and provides cells with an opportunity to regulate secretion of small proteins independent of the SRP pathway.     

The signal recognition particle-targeting pathway does not necessarily deliver proteins to the sec-translocase in Escherichia coli.

ProW is an Escherichia coli inner membrane protein that consists of a 100-residue-long periplasmic N-terminal tail (N-tail) followed by seven closely spaced transmembrane segments. N-tail translocation presumably proceeds in a C-to-N-terminal direction and represents a poorly understood aspect of membrane protein biogenesis. Here, using an in vivo depletion approach, we show that N-tail translocation in a ProW derivative comprising the N-tail and the first transmembrane segment fused to the globular P2 domain of leader peptidase depends both on the bacterial signal recognition particle (SRP) and the Sec-translocase. Surprisingly, however, a deletion construct with only one transmembrane segment downstream of the N-tail can assemble properly even under severe depletion of SecE, a central component of the Sec-translocase, but not under SRP-depletion conditions. To our knowledge, this is the first demonstration that the SRP-targeting pathway does not necessarily deliver SRP-dependent inner membrane proteins to the Sec-translocase. The data further suggest that N-tail translocation can proceed in the absence of a functional Sec-translocase.     

SRP keeps polypeptides translocation-competent by slowing translation to match limiting ER-targeting sites

SRP is essential for targeting nascent chains to the endoplasmic reticulum, and it delays nascent chain elongation in cell-free translation systems. However, the significance of this function has remained unclear. We show that efficient protein translocation into the ER is incompatible with normal cellular translation rates due to rate-limiting concentrations of SRP receptor (SR). We complemented mammalian cells depleted of SRP14 by expressing mutant versions of the protein lacking the elongation arrest function. The absence of a delay caused inefficient targeting of preproteins leading to defects in secretion, depletion of proteins in the endogenous membranes, and reduced cell growth. The detrimental effects were reversed by either reducing the cellular protein synthesis rate or increasing SR expression. SRP therefore ensures that nascent chains remain translocation competent during the targeting time window dictated by SR. Since SRP-signal sequence affinities vary, the delay may also regulate which proteins are preferentially targeted.     

Signal peptide hydrophobicity is critical for early stages in protein export by Bacillus subtilis

Signal peptides that direct protein export in Bacillus subtilis are overall more hydrophobic than signal peptides in Escherichia coli. To study the importance of signal peptide hydrophobicity for protein export in both organisms, the alpha-amylase AmyQ was provided with leucine-rich (high hydrophobicity) or alanine-rich (low hydrophobicity) signal peptides. AmyQ export was most efficiently directed by the authentic signal peptide, both in E. coli and B. subtilis. The leucine-rich signal peptide directed AmyQ export less efficiently in both organisms, as judged from pulse-chase labelling experiments. Remarkably, the alanine-rich signal peptide was functional in protein translocation only in E. coli. Cross-linking of in vitro synthesized ribosome nascent chain complexes (RNCs) to cytoplasmic proteins showed that signal peptide hydrophobicity is a critical determinant for signal peptide binding to the Ffh component of the signal recognition particle (SRP) or to trigger factor, not only in E. coli, but also in B. subtilis. The results show that B. subtilis SRP can discriminate between signal peptides with relatively high hydrophobicities. Interestingly, the B. subtilis protein export machinery seems to be poorly adapted to handle alanine-rich signal peptides with a low hydrophobicity. Thus, signal peptide hydrophobicity appears to be more critical for the efficiency of early stages in protein export in B. subtilis than in E. coli.     

Gateway cloning is compatible with protein secretion from Pichia pastoris

Secretion of a recombinant protein from the yeast Pichia pastoris requires the presence of a signal peptide at the amino terminus. Maintaining the full amino acid sequence of the signal peptide is thought to be important for proper signal processing and protein secretion. We show that at least for one protein, a synthetic human interferon, the presence of a Gateway recombination site within the signal peptide is fully compatible with high levels of protein secretion. The amino termini of the secreted interferon proteins cloned with Gateway and cloned with restriction enzymes and ligase are identical, and the proteins were highly active in biological assays. Compatibility with Gateway cloning simplifies construction of plasmids directing secretion of recombinant proteins from P. pastoris.