The conformation of a signal peptide bound by Escherichia coli preprotein translocase SecA

To understand the structural nature of signal sequence recognition by the preprotein translocase SecA, we have characterized the interactions of a signal peptide corresponding to a LamB signal sequence (modified to enhance aqueous solubility) with SecA by NMR methods. One-dimensional NMR studies showed that the signal peptide binds SecA with a moderately fast exchange rate (Kd approximately 10(-5) m). The line-broadening effects observed from one-dimensional and two-dimensional NMR spectra indicated that the binding mode does not equally immobilize all segments of this peptide. The positively charged arginine residues of the n-region and the hydrophobic residues of the h-region had less mobility than the polar residues of the c-region in the SecA-bound state, suggesting that this peptide has both electrostatic and hydrophobic interactions with the binding pocket of SecA. Transferred nuclear Overhauser experiments revealed that the h-region and part of the c-region of the signal peptide form an alpha-helical conformation upon binding to SecA. One side of the hydrophobic core of the helical h-region appeared to be more strongly bound in the binding pocket, whereas the extreme C terminus of the peptide was not intimately involved. These results argue that the positive charges at the n-region and the hydrophobic helical h-region are the selective features for recognition of signal sequences by SecA and that the signal peptide-binding site on SecA is not fully buried within its structure.     

Residues flanking the COOH-terminal C-region of a model eukaryotic signal peptide influence the site of its cleavage by signal peptidase and the extent of coupling of its co-translational translocation and proteolytic processing in vitro

The polar, COOH-terminal c-region of signal peptides has been considered to be most important for influencing the efficiency and fidelity of signal peptidase cleavage while the hydrophobic core or h-region appears indispensable for initiating translocation. To identify structural features of residues flanking the c-region that influence the fidelity and efficiency of signal peptidase cleavage as well as co-translational translocation, we introduced six amino acid substitutions into the COOH terminus of the hydrophobic core and seven substitutions at the NH2 terminus of the mature region (the +1 position) of a model eukaryotic preprotein-human pre(delta pro)apoA-II. This preprotein contains several potential sites for signal peptidase cleavage. The functional consequences of these mutations were assayed using an in vitro co-translational translocation/processing system and by post-translational cleavage with purified, detergent-solubilized, hen oviduct signal peptidase. The efficiency of translocation could be correlated with the hydrophobic character of the residue introduced at the COOH terminus of the h-region. Some h/c boundary mutants underwent co-translational translocation across the microsomal membrane with only minimal cleavage yet they were cleaved post-translationally by hen oviduct signal peptidase more efficiently than other mutants which exhibited a high degree of coupling of co-translational translocation and cleavage. These data suggest that features at the COOH terminus of the h-domain can influence "presentation" of the cleavage site to signal peptidase. The +1 residue substitutions had minor effects on the extent of co-translational translocation and processing. However, these +1, as well as h/c boundary mutations, had dramatic effects on the site of cleavage chosen by signal peptidase, indicating that residues flanking the c-region of this prototypic eukaryotic signal peptide can affect the fidelity of its proteolytic processing. The site(s) selected by canine microsomal and purified hen oviduct signal peptidase were very similar, suggesting that "intrinsic" structural features of this prepeptide can influence the selectivity of eukaryotic signal peptidase cleavage, independent of the microsomal membrane and associated translocation apparatus.     

The role of the membrane-spanning domain of type I signal peptidases in substrate cleavage site selection

Type I signal peptidase (SPase I) catalyzes the cleavage of the amino-terminal signal sequences from preproteins destined for cell export. Preproteins contain a signal sequence with a positively charged n-region, a hydrophobic h-region, and a neutral but polar c-region. Despite having no distinct consensus sequence other than a commonly found c-region "Ala-X-Ala" motif preceding the cleavage site, signal sequences are recognized by SPase I with high fidelity. Remarkably, other potential Ala-X-Ala sites are not cleaved within the preprotein. One hypothesis is that the source of this fidelity is due to the anchoring of both the SPase I enzyme (by way of its transmembrane segment) and the preprotein substrate (by the h-region in the signal sequence) in the membrane. This limits the enzyme-substrate interactions such that cleavage occurs at only one site. In this work we have, for the first time, successfully isolated Bacillus subtilis type I signal peptidase (SipS) and a truncated version lacking the transmembrane domain (SipS-P2). With purified full-length as well as truncated constructs of both B. subtilis and Escherichia coli (Lep) SPase I, in vitro specificity studies indicate that the transmembrane domains of either enzyme are not important determinants of in vitro cleavage fidelity, since enzyme constructs lacking them reveal no alternate site processing of pro-OmpA nuclease A substrate. In addition, experiments with mutant pro-OmpA nuclease A substrate constructs indicate that the h-region of the signal peptide is also not critical for substrate specificity. In contrast, certain mutants in the c-region of the signal peptide result in alternate site cleavage by both Lep and SipS enzymes.     

Structural features in the NH2-terminal region of a model eukaryotic signal peptide influence the site of its cleavage by signal peptidase

The 20-amino acid signal peptide of human pre (delta pro)apolipoprotein A-II contains the tripartite domain structure typical of eukaryotic prepeptides, i.e. a positively charged NH2-terminal (n) region, a hydrophobic core (h) region, and a COOH-terminal polar domain (c region). This signal sequence has multiple potential sites for cotranslational processing making it an attractive model for assessing the consequences of systematic structural alterations on the site selected for signal peptidase cleavage. We previously analyzed 40 mutant derivatives of this model preprotein using an in vitro translation/canine microsome processing assay. The results showed that the position of the boundary between the h and c regions and properties of the -1 residue are critical in defining the site of cotranslational cleavage. To investigate whether structural features in the NH2-terminal region of signal peptides play a role in cleavage specificity, we have now inserted various amino acids between the positively charged n region (NH2-Met-Lys) and the h region of a "parental" pre(delta pro)apoA-II mutant that has roughly equal cleavage between Gly18 decreases and Gly20 decreases. Movement of the n/h boundary toward the NH2 terminus results in a dramatic shift in cleavage to Gly18 decreases. Replacement of the Lys2 residue with hydrophilic, negatively charged residues preserves the original sites of cleavage. Replacement with a hydrophobic residue causes cleavage to shift "upstream." Simultaneous alteration of the position of n/h and h/c boundaries has an additive effect on the site of signal peptidase cleavage. None of these mutations produced a marked decrease in the efficiency of in vitro cotranslational translocation or cleavage. However, in sequence contexts having poor signal function, introduction of hydrophobic residues between the n and h regions markedly improved the efficiency of translocation/processing. We conclude that the position of the n/h boundary as well as positioning of the h/c boundary affects the site of cleavage chosen by signal peptidase.     

Cellular import of synthetic peptide using a cell-permeable sequence in plant protoplasts

In this paper, we describe a rapid method to incorporate biologically active synthetic peptide in plant protoplasts. The peptides used contain a hydrophobic membrane permeable sequence as a carrier for the import through the plasma membrane. The membrane permeable sequence corresponds to the h-region, the more hydrophobic domain found in the signal peptide of secreted proteins. To evaluate the feasibility of the method, we synthesized a cell-permeable peptide with an h-region of a plant signal peptide plus residues 410–419 of the human c-myc oncogene product. Detection was performed via fluorescence analysis using specific monoclonal anti-c-myc primary antibody and FITC-conjugated secondary antibody. No saturation of import was observed, suggesting that the mechanisms involved do not require energy. The half-life time of the internalized peptide was estimated and results indicate that peptide concentration into protoplasts was constant for 8 h following incorporation. This method is complementary to microinjection or to the use of membrane permeabilizing reagents to study in vivo protein–protein or DNA–protein interactions. Finally, this method was used to analyse a putative interaction between the conserved cytoplasmic tail of a transmembrane receptor (HaELP, Helianthus annuus EGF receptor like protein) and the cytoskeleton. No interaction was found between these components.     

Functional limits of conformation, hydrophobicity, and steric constraints in prokaryotic signal peptide cleavage regions. Wild type transport by a simple polymeric signal sequence

These experiments examine the role of conformation, hydrophobicity, and steric constraints in the function of the prokaryotic signal peptide cleavage region. The experimental strategy involves replacement of the wild type Escherichia coli alkaline phosphatase signal peptide cleavage region with a series of idealized model sequences designed to epitomize the particular structural and physical variables under study. By analyzing model sequences whose conformations have been determined by physical studies, we have demonstrated that efficient transport does not depend on the structural preference of the cleavage region. Although previous studies based on Chou-Fasman analysis have suggested that the cleavage region forms a beta-turn which is required for transport, our results demonstrate that either a beta-turn- or alpha-helix-fostering sequence in the cleavage region functions indistinguishably from wild type. Furthermore, the presence of a proline residue between the core and cleavage region, although common in natural sequences, is not essential for export. Cleavage regions of varying hydrophobicities can support translocation across the inner membrane, but the placement of bulky residues at positions -1 and -3 upstream of the cleavage site abolishes processing and transport to the periplasm. By reducing the signal peptide to simplified, idealized segments, this study has identified a largely polymeric sequence, MKQST(L10)-(A6), that functions equivalently to the wild type alkaline phosphatase signal peptide. This work starts to provide a basis for the design of a universal prokaryotic signal peptide that incorporates all the critical physical and structural characteristics required for transport function.     

Export of unprocessed precursor maltose-binding protein to the periplasm of Escherichia coli cells.

The Escherichia coli maltose-binding protein (MBP) R2 signal peptide is a truncated version of the wild-type structure that still facilitates very efficient export of MBP to the periplasm. Among single amino acid substitutions in the R2 signal peptide resulting in an export-defective precursor MBP (pMBP) were two that replaced residues in the consensus Ala-X-Ala sequence (residues -3 to -1) that immediately precedes the cleavage site. It was suggested that the functional hydrophobic core and signal peptidase recognition sequence of this signal peptide substantially overlap and that these two alterations affect both pMBP translocation and processing. In this study, the export of pMBP by the mutants, designated CC15 and CC17, with these two alterations was investigated further. The pMBP of mutant CC17 has an Arg substituted for Leu at the -2 position. It was found that CC17 cells exported only a very small amount of MBP, but that which was exported appeared to be correctly processed. This result was consistent with other studies that have concluded that virtually any amino acid can occupy the -2 position. For mutant CC15, which exhibits a fully Mal+ phenotype, an Asp is substituted for the Ala at the -3 position. CC15 cells were found to export large quantities of unprocessed, soluble pMBP to the periplasm, although such export was achieved in a relatively slow, posttranslational manner. This result was also consistent with other studies that suggested that charged residues are normally excluded from the -3 position of the cleavage site. Using in vitro oligonucleotide-directed mutagenesis, we constructed a new signal sequence mutant in which Asp was substituted for Arg at the -3 position of an otherwise wild-type MBP signal peptide. This alteration had no apparent effect on pMBP translocation across the cytoplasmic membrane, but processing by signal peptidase was inhibited. This pMBP species with its full-length hydrophobic core remained anchored to the membrane, where it could still participate in maltose uptake. The implications of these results for models of protein export are discussed.     

A putative signal peptidase recognition site and sequence in eukaryotic and prokaryotic signal peptides

Presecretory signal peptides of 39 proteins from diverse prokaryotic and eukaryotic sources have been compared. Although varying in length and amino acid composition, the labile peptides share a hydrophobic core of approximately 12 amino acids. A positively charged residue (Lys or Arg) usually precedes the hydrophobic core. Core termination is defined by the occurrence of a charged residue, a sequence of residues which may induce a beta-turn in a polypeptide, or an interruption in potential alpha-helix or beta-extended strand structure. The hydrophobic cores contain, by weight average, 37% Leu: 15% Ala: 10% Val: 10% Phe: 7% Ile plus 21% other hydrophobic amino acids arranged in a non-random sequence. Following the hydrophobic cores (aligned by their last residue) a highly non-random and localized distribution of Ala is apparent within the initial eight positions following the core: (formula; see text) Coincident with this observation, Ala-X-Ala is the most frequent sequence preceding signal peptidase cleavage. We propose the existence of a signal peptidase recognition sequence A-X-B with the preferred cleavage site located after the sixth amino acid following the core sequence. Twenty-two of the above 27 underlined Ala residues would participate as A or B in peptidase cleavage. Position A includes the larger aliphatic amino acids, Leu, Val and Ile, as well as the residues already found at B (principally Ala, Gly and Ser). Since a preferred cleavage site can be discerned from carboxyl and not amino terminal alignment of the hydrophobic cores it is proposed that the carboxyl ends are oriented inward toward the lumen of the endoplasmic reticulum where cleavage is thought to occur. This orientation coupled with the predicted beta-turn typically found between the core and the cleavage site implies reverse hairpin insertion of the signal sequence. The structural features which we describe should help identify signal peptides and cleavage sites in presumptive amino acid sequences derived from DNA sequences.     

Establishment of a signal peptide with cross-species compatibility for functional antibody expression in both Escherichia coli and Chinese hamster ovary cells

Signal peptides are short peptides located at the N-terminus of secreted proteins. They characteristically have three domains; a basic region at the N-terminus (n-region), a central hydrophobic core (h-region) and a carboxy-terminal cleavage region (c-region). Although hundreds of different signal peptides have been identified, it has not been completely understood how their features enable signal peptides to influence protein expression. Antibody-derived signal peptides are often used to prepare recombinant antibodies expressed by eukaryotic cells, especially Chinese hamster ovary (CHO) cells. However, when prokaryotic Escherichia coli (E. coli) are utilized in drug discovery processes, such as for phage display selection or antibody humanization, signal peptides have been selected separately due to the differences in the expression systems between the species. In this study, we successfully established a signal peptide that enables a functional antibody to be expressed in both prokaryotic and eukaryotic cells by focusing on the importance of having an Ala residue in the c-region of the signal sequence. We found that changing Ser to Ala at only two positions significantly augmented the anti-HER2 antigen binding fragment (Fab) expression in E. coli. In addition, this altered signal peptide also retained the ability to express functional anti-HER2 antibody in CHO cells. Taken together, the present findings indicate that the signal peptide can promote functional antibody expression in both prokaryotic E. coli and eukaryotic CHO cells. This finding will contribute to the understanding of signal peptides and accelerate therapeutic antibody research.     

Alterations to the signal peptide of an outer membrane protein (OmpA) of Escherichia coli K-12 can promote either the cotranslational or the posttranslational mode of processing

The signal sequence of the precursor of the Escherichia coli outer membrane protein OmpA was altered by oligonucleotide insertions into the corresponding gene. In one case, OmpA-S1, the hydrophobic core of the signal peptide, is reduced from 12 to 10 residues, and one positive charge is added near the NH2-terminus. In another case, OmpA-P1, the hydrophobic core is extended from 12 to 16 residues. The pro-OmpA protein is normally processed partially co- and partially posttranslationally. Processing of the pro-OmpA-S1 protein was entirely posttranslational and that of the pro-OmpA-P1 protein strictly cotranslational. Evidence is presented which strongly suggests that posttranslational processing reflects posttranslational translocation across the plasma membrane. The generation times of cells expressing pro-OmpA-P1 or pro-OmpA-S1 were identical and the pro-OmpA-S1 polypeptide could be chased into the mature protein in the absence of protein synthesis. Hence, it does not matter which mode of processing, or rather translocation, is used. The same oligonucleotides were inserted into the ompA gene of plasmid pRD87; a plasmid which leads to overproduction of the protein and to massive accumulation of both the mature protein and the precursor. In the OmpA signal sequence encoded by pRD87-P2 the hydrophobic core is extended from 12 to 20 residues. This peptide was also rapidly removed. Therefore, regardless of whether the hydrophobic core contains 12, 16, or 20 lipophilic residues, not only does the signal sequence always function correctly to mediate export, but in each case, the cleavage site is always accessible to the signal peptidase.     

Translation initiation in the HEXB gene encoding the beta-subunit of human beta-hexosaminidase

The human lysosomal enzyme beta-hexosaminidase (EC 3.2.1.52) is a glycoprotein composed of dimers of alpha- and/or beta-subunits. The subunits of the enzymes are synthesized in the rough endoplasmic reticulum and transported through the Golgi apparatus to the lysosome. As such, each subunit contains an amino-terminal signal peptide that directs the nascent polypeptide into the lumen of the endoplasmic reticulum. The signal peptide cleavage site of the beta-polypeptide is known, but its NH2 terminus has not been determined due to the presence of three candidate initiation codons upstream of the cleavage site. In this study, we identified the mRNA cap site, confirming the presence of all three AUGs in the majority of HEXB mRNA. To identify the site of translation initiation, we mutated the three ATGs by deletion and site-directed mutagenesis and showed that all three AUG codons can be used for translation initiation after expression in COS cells. Furthermore, in each case, a fully processed, i.e. mature lysosomal, and enzymatically active beta-hexosaminidase was produced indicating that a functional signal peptide was synthesized. However, expression of a frameshift mutation in the normal construct, created by insertion of a single nucleotide between the first and second ATG, resulted in no significant enzyme activity or beta-subunit protein. We conclude, therefore, that the first in-frame ATG is used exclusively in vivo, in keeping with the scanning model of eukaryotic translation initiation. Interestingly, substitution of all three ATGs with CTG resulted in a significant amount of mature beta-hexosaminidase, showing that under these conditions, initiation could occur from non-AUG codons. Translation initiation from the first AUG gives the prepro-beta-polypeptide a signal peptide of 42 amino acids that has an unusually long hydrophobic core more typical of membrane spanning domains. Such a large hydrophobic core has not been found in other cleavable signal peptides.     

Effects of signal sequence on phage-displayed disulfide-stabilized single chain antibody variable fragment (sc-dsFv) libraries

Phage-displayed single chain variable fragment (scFv) libraries are powerful tools in antibody engineering. Disulfide-stabilized scFv (sc-dsFv) with an interface disulfide bond is structure-wise more stable than the corresponding scFv. A set of recently discovered signal sequences replacing the wild type (pelB) signal peptidase cleavage site in the c-region has been shown to be effective in rescuing the expression of sc-dsFv libraries on the phage surface. However, the effects of the other regions of the signal sequence on the expression of the sc-dsFv libraries and on the formation of the interface disulfide bond in the phage-displayed sc-dsFv have not been clear. In this work, selected novel signal sequence variants in the h-region were shown to be equally effective in promoting sc-dsFv library expression on the phage surface; the expression level and complexity of the sc-dsFv libraries were comparable to the corresponding scFv libraries produced with the wild-type (pelB) signal sequence. The interface disulfide bond in the phage-displayed sc-dsFv was proven to form to a large extent in the library variant ensemble generated with signal sequence variants in both the h-region and the c-region. The sc-dsFv engineering platform established in this work can be applied to many of the known scFv molecules which are in need of a more stable version for the applications under harsh conditions or for longer shelf-life.     

Structural requirement of C‐terminal region of chicken lysozyme signal peptide

Abstract

The mechanisms of signal peptide cleavage has not been fully elucidated yet. In previous investigation, we have examined the effect of chicken lysozyme signal peptide mutations on the secretion of human lysozyme. During this study, we determined that the hydrophobic bulky amino acid Val at position ‐1 inhibited the function of signal peptide. To determine why the ‐1Val suppressed the function of signal peptide, turn‐promoting amino acids Pro and Gly were introduced after ‐lVal to prevent the signal peptide from forming α‐helix and induce β‐turn around the cleavage site. This mutation resulted in no processing of signal peptide and no secretion of human lysozyme. However, the replacement of ‐1Val with Ala permitted a functional signal. Based on these results, three dimensional models around the cleavage site of each signal peptide were made, which show that bulky side chain at ‐1 residue of signal peptide limits the reaction space for signal peptidase and suppresses cleavage by steric hindrance. We suggest that the bulky side chain at ‐1 residue suppresses the signal peptide cleavage by its local steric hindrance and not by a change in whole structure around the cleavage site. On the other hand, introduction of Pro at position +1 did not inhibit signal cleavage completely resulting in poor secretion and processing efficiency although Pro in position +1 has been recently reported to block cleavage of the prokaryotic signal peptide. The mechanism of cleavage of prokaryotic signal may be different than that of eukaryotic signal.     

Structure analysis of the membrane-bound PhoD signal peptide of the Tat translocase shows an N-terminal amphiphilic helix

Tat signal peptides provide the key signature for proteins that get exported by the bacterial twin arginine translocase. We have characterized the structure of the PhoD signal peptide from Bacillus subtilis in suitable membrane-mimicking environments. High-resolution (13)C/(15)N NMR analysis in detergent micelles revealed a helical stretch in the signal peptide between positions 5 and 15, in good agreement with secondary structure prediction and circular dichroism results. This helix was found to be aligned parallel to the membrane surface according to oriented circular dichroism experiments carried out with planar lipid bilayers. The N-terminal α-helix exhibits a pronounced amphiphilic character, in contrast to the general view in the literature. So far, signal sequences had been supposed to consist of a positively charged N-terminal domain, followed by an α-helical hydrophobic segment, plus a C-terminal domain carrying the peptidase cleavage site. Based on our new structural insights, we propose a model for the folding and membrane interactions of the Tat signal sequence from PhoD.     

Intramembrane proteolysis and endoplasmic reticulum retention of hepatitis C virus core protein

Hepatitis C virus (HCV) core protein is suggested to localize to the endoplasmic reticulum (ER) through a C-terminal hydrophobic region that acts as a membrane anchor for core protein and as a signal sequence for E1 protein. The signal sequence of core protein is further processed by signal peptide peptidase (SPP). We examined the regions of core protein responsible for ER retention and processing by SPP. Analysis of the intracellular localization of deletion mutants of HCV core protein revealed that not only the C-terminal signal-anchor sequence but also an upstream hydrophobic region from amino acid 128 to 151 is required for ER retention of core protein. Precise mutation analyses indicated that replacement of Leu(139), Val(140), and Leu(144) of core protein by Ala inhibited processing by SPP, but cleavage at the core-E1 junction by signal peptidase was maintained. Additionally, the processed E1 protein was translocated into the ER and glycosylated with high-mannose oligosaccharides. Core protein derived from the mutants was translocated into the nucleus in spite of the presence of the unprocessed C-terminal signal-anchor sequence. Although the direct association of core protein with a wild-type SPP was not observed, expression of a loss-of-function SPP mutant inhibited cleavage of the signal sequence by SPP and coimmunoprecipitation with unprocessed core protein. These results indicate that Leu(139), Val(140), and Leu(144) in core protein play crucial roles in the ER retention and SPP cleavage of HCV core protein.     

Alterations in the transport and processing of Rous sarcoma virus envelope glycoproteins mutated in the signal and anchor regions

The env gene of Rous sarcoma virus codes for two glycoproteins which are located on the surface of infectious virions. Subcloning of these coding sequences in the place of the late region of SV40 DNA has allowed the expression of a normally glycosylated, functionally active glycoprotein complex on the surface of monkey cells. Through the use of site-directed mutagenesis, the role of specific amino acids in the signal peptide, signal peptidase cleavage site, and membrane anchor region have been investigated. Amino-terminal mutations have shown that deletion of the signal peptidase cleavage site along with one or two amino acids of the hydrophobic signal peptide results in the synthesis of an unglycosylated, uncleaved, and presumably cytoplasmically located precursor. Nevertheless, changing the signal peptidase cleavage site from ala/asp to ala/asn does not block the translocation of the glycoprotein across the membrane or the action of the peptidase. At the other end of the molecule, carboxy-terminal mutations have shown that the deletion of the hydrophobic membrane anchor region is not sufficient for the secretion of the truncated glycoprotein. Interpretations of these results based on recent models for protein transport and secretion are discussed.     

Using string kernel to predict signal peptide cleavage site based on subsite coupling model

Summary. Owing to the importance of signal peptides for studying the molecular mechanisms of genetic diseases, reprogramming cells for gene therapy, and finding new drugs for healing a specific defect, it is in great demand to develop a fast and accurate method to identify the signal peptides. Introduction of the so-called {−3,−1, +1} coupling model (Chou, K. C.: Protein Engineering, 2001, 14–2, 75–79) has made it possible to take into account the coupling effect among some key subsites and hence can significantly enhance the prediction quality of peptide cleavage site. Based on the subsite coupling model, a kind of string kernels for protein sequence is introduced. Integrating the biologically relevant prior knowledge, the constructed string kernels can thus be used by any kernel-based method. A Support vector machines (SVM) is thus built to predict the cleavage site of signal peptides from the protein sequences. The current approach is compared with the classical weight matrix method. At small false positive ratios, our method outperforms the classical weight matrix method, indicating the current approach may at least serve as a powerful complemental tool to other existing methods for predicting the signal peptide cleavage site. The software that generated the results reported in this paper is available upon requirement, and will appear at http://www.pami.sjtu.edu.cn/wm. An erratum to this article is available at .     

Degradation of a signal peptide by protease IV and oligopeptidase A.

The degradation of the prolipoprotein signal peptide in vitro by membranes, cytoplasmic fraction, and two purified major signal peptide peptidases from Escherichia coli was followed by reverse-phase liquid chromatography (RPLC). The cytoplasmic fraction hydrolyzed the signal peptide completely into amino acids. In contrast, many peptide fragments accumulated as final products during the cleavage by a membrane fraction. Most of the peptides were similar to the peptides formed during the cleavage of the signal peptide by the purified membrane-bound signal peptide peptidase, protease IV. Peptide fragments generated during the cleavage of the signal peptide by protease IV and a cytoplasmic enzyme, oligopeptidase A, were identified from their amino acid compositions, their retention times during RPLC, and knowledge of the amino acid sequence of the signal peptide. Both enzymes were endopeptidases, as neither dipeptides nor free amino acids were formed during the cleavage reactions. Protease IV cleaved the signal peptide predominantly in the hydrophobic segment (residues 7 to 14). Protease IV required substrates with hydrophobic amino acids at the primary and the adjacent substrate-binding sites, with a minimum of three amino acids on either side of the scissile bond. Oligopeptidase A cleaved peptides (minimally five residues) that had either alanine or glycine at the P'1 (primary binding site) or at the P1 (preceding P'1) site of the substrate. These results support the hypothesis that protease IV is the major signal peptide peptidase in membranes that initiates the degradation of the signal peptide by making endoproteolytic cuts; oligopeptidase A and other cytoplasmic enzymes further degrade the partially degraded portions of the signal peptide that may be diffused or transported back into the cytoplasm from the membranes.     

Characterization of the promoter, signal sequence, and amino terminus of a secreted beta-galactosidase from "Streptomyces lividans"

The gene for a secreted 130-kilodalton beta-galactosidase from "Streptomyces lividans" has been cloned, its promoter, signal sequence, and amino terminal region have been localized, and their nucleotide sequence has been determined. The signal sequence extends over 56 amino acids and shows the characteristic-features of signal sequences, including a hydrophilic amino terminus followed by a hydrophobic core near the signal cleavage site. The secretion of beta-galactosidase depends on the presence of the signal sequence. beta-Galactosidase is the major protein in culture supernatants and extracts of strains expressing the cloned beta-galactosidase gene and represents a valuable tool in the study of protein secretion in Streptomyces spp.