Illicit secretion of a cytoplasmic protein into the periplasm of Escherichia coli requires a signal peptide plus a portion of the cognate secreted protein. Demarcation of the critical region of the mature protein

The beta-lactamase signal peptide alone is not sufficient to direct secretion of chicken muscle triosephosphate isomerase, a normally cytoplasmic protein, into the periplasm of Escherichia coli. The signal peptide and at least the first 3 residues of the mature beta-lactamase are required before any secretion of the isomerase can be observed. At this point the level of secretion is very low, but the addition of further residues of the mature beta-lactamase enhances the secretion of the hybrid protein. The maximum level of secretion is achieved when 12 or more residues of the mature beta-lactamase intervene between the signal peptide and the isomerase. It is the proximity of an arginine residue at position 3 of the isomerase that is responsible for the blockade to secretion of these hybrid proteins (see Summers, R.G., Harris, C.R., and Knowles, J.R. (1989) J. Biol. Chem. 264, 20082-20088). With 12 residues of the mature beta-lactamase between the signal peptide and the isomerase, the offending arginine now lies at position 15 of the hybrid. The 14 residues that immediately follow the signal peptide therefore define a region of constrained properties that is critical to the secretability of proteins from E. coli.     

The twin arginine consensus motif of Tat signal peptides is involved in Sec-independent protein targeting in Escherichia coli

In Escherichia coli a subset of periplasmic proteins is exported through the Tat pathway to which substrates are directed by an NH(2)-terminal signal peptide containing a consensus SRRXFLK "twin arginine" motif. The importance of the individual amino acids of the consensus motif for in vivo Tat transport has been assessed by site-directed mutagenesis of the signal peptide of the Tat substrate pre-SufI. Although the invariant arginine residues are crucial for efficient export, we find that slow transport of SufI is still possible if a single arginine is conservatively substituted by a lysine residue. Thus, in at least one signal peptide context there is no absolute dependence of Tat transport on the arginine pair. The consensus phenylalanine residue was found to be a critical determinant for efficient export but could be functionally substituted by leucine, another amino acid with a highly hydrophobic side chain. Unexpectedly, the consensus lysine residue was found to retard Tat transport. These observations and others suggest that the sequence conservation of the Tat consensus motif is a reflection of the functional importance of the consensus residues. Tat signal peptides characteristically have positively charged carboxyl-terminal regions. However, changing the sign of this charge does not affect export of SufI.     

Analysis of signals for secretion in the staphylococcal protein A gene.

Different constructs of the gene encoding staphylococcal protein A were introduced in Staphylococcus aureus and S. xylosus as well as Escherichia coli. The product of the gene without the cell wall anchoring domain was efficiently secreted in all three hosts. N-terminal sequencing of the affinity-purified mature protein revealed a common processing site after the alanine residue at position 36. In contrast, when an internal IgG-binding fragment of protein A (region B) was inserted after the protein A signal sequence, the product was poorly secreted and N-terminal sequencing revealed no processing at the normal site. This demonstrates that the structure of the polypeptide chain beyond the signal peptide cleavage site can affect cleavage. Another construct, containing the N-terminal IgG-binding part of the mature protein A (region E) followed by region B, gave correct processing and efficient secretion. Unexpectedly, the gene product, EB, was not only secreted and correctly processed, but was also excreted to the culture medium of E. coli. Secretion vectors containing the protein A signal sequence were constructed to facilitate secretion of foreign gene products. Insertion of the E. coli gene phoA, lacking its own promoter and signal sequence, led to efficient secretion of alkaline phosphatase both in E. coli and S. aureus.     

A periplasmic intermediate in the extracellular secretion pathway of Pseudomonas aeruginosa exotoxin A.

Pseudomonas aeruginosa exotoxin A is synthesized with a secretion signal peptide typical of proteins whose final destination is the periplasm. However, exotoxin A is released from the cell without a detectable periplasmic pool, suggesting that additional determinants in this protein are important for recognition by a specialized machinery of extracellular secretion. The role of the N terminus of the mature exotoxin A in this recognition was investigated. A series of exotoxin A proteins with amino acid substitutions for the glutamic acid pair at the +2 and +3 positions were constructed by mutagenesis of the exotoxin A gene. These N-terminal acidic residues of the mature exotoxin A protein were found to be important not only for efficient processing of the precursor protein but also for extracellular localization of the toxin. The mutated exotoxin A proteins, in which a glutamic acid at the +2 position was replaced by a lysine or a double substitution of lysine and glutamine for the pair of adjacent glutamic acids, accumulated in precursor forms in the mixed cytoplasmic and membrane fractions, which was not seen with the wild-type exotoxin A. The processing of the precursor form of one exotoxin A mutant, in which the glutamic acid at the +2 position was replaced with a glutamine, was not affected. Moreover, a substantial fraction of the mature forms of all three mutants of exotoxin A accumulated in the periplasm, while wild-type exotoxin A could be detected only extracellularly. The periplasmic pools of these variants of exotoxin A could therefore represent the intermediate state during extracellular secretion. The signal for extracellular localization may be located in a small region near the amino terminus of the mature protein or could consist of several regions that are brought together after the polypeptide has folded. Alternatively, the acidic residues may be important for ensuring a conformation essential for exotoxin A to traverse the outer membrane.     

Proteolytic processing of the alpha-chain of the lysosomal enzyme, beta-hexosaminidase, in normal human fibroblasts

The two subunits of beta-hexosaminidase undergo many post-translational modifications characteristic of lysosomal proteins, including limited proteolysis. To identify proteolytic cleavage sites in the alpha-chain, we have biosynthetically radiolabeled the transient forms, isolated these by immunoprecipitation, gel electrophoresis, and electroelution, and subjected them to automated Edman degradation. The position of the NH2-terminal amino acid was inferred from the elution cycle of the radioactive amino acid and the primary sequence encoded in the alpha-chain cDNA. The amino terminus of the precursor obtained by in vitro translation of SP6 alpha-chain mRNA in the presence of microsomes was leucine 23. The same amino terminus was found in precursor alpha-chain synthesized by normal human fibroblasts (IMR90) in a 1- or 3-h pulse or secreted by these cells in the presence of NH4Cl. The alpha-chain isolated after a 3-h pulse followed by a 5-h chase (intermediate form) included a mixture of molecular species of which the amino terminus was arginine 87 (most abundant), histidine 88, or leucine 90. After a 20-h chase (mature form) the latter species predominated. This mature form of the alpha-chain remained fully reactive with antibody raised against the carboxyl-terminal 15 amino acids, indicating little if any proteolysis at the carboxyl terminus. Thus synthesis and maturation of the alpha-chain of beta-hexosaminidase includes two major proteolytic cleavages: the first, between alanine 22 and leucine 23, removes the signal peptide to generate the precursor form, whereas the second occurs between the dibasic amino acids, lysine 86 and arginine 87. The second cleavage is followed by trimming of 3 additional amino acids to give the mature form of the alpha-chain.     

Mapping of export signals of Pseudomonas aeruginosa pilin with alkaline phosphatase fusions.

Pili of Pseudomonas aeruginosa are assembled from monomers of the structural subunit, pilin, after secretion of this protein across the bacterial membrane. These subunits are initally synthesized as precursors (prepilin) with a six-amino-acid leader peptide that is cleaved off during or after membrane traversal, followed by methylation of the amino-terminal phenylalanine residue. This report demonstrates that additional sequences from the N terminus of the mature protein are necessary for membrane translocation. Gene fusions were made between amino-terminal coding sequences of the cloned pilin gene (pilA) and the structural gene for Escherichia coli alkaline phosphatase (phoA) devoid of a signal sequence. Fusions between at least 45 amino acid residues of the mature pilin and alkaline phosphatase resulted in translocation of the fusion proteins across the cytoplasmic membranes of both P. aeruginosa and E. coli strains carrying recombinant plasmids, as measured by alkaline phosphatase activity and Western blotting. Fusion proteins constructed with the first 10 amino acids of prepilin (including the 6-amino-acid leader peptide) were not secreted, although they were detected in the cytoplasm. Therefore, unlike that of the majority of secreted proteins that are synthesized with transient signal sequences, the membrane traversal of pilin across the bacterial membrane requires the transient six-amino-acid leader peptide as well as sequences contained in the N-terminal region of the mature pilin protein.     

Requirement of pro-sequence for the production of active subtilisin E in Escherichia coli

Subtilisin E, an alkaline serine protease of Bacillus subtilis 168, is first produced as a precursor, pre-pro-subtilisin, which consists of a signal peptide for protein secretion (pre-sequence) and a peptide extension of 77 amino acid residues (pro-sequence) between the signal peptide and mature subtilisin. When the entire coding region for pre-pro-subtilisin E was cloned into an Escherichia coli expression vector, active mature subtilisin E was secreted into the periplasmic space. When the pre-sequence was replaced with the E. coli OmpA signal peptide, active subtilisin E was also produced. When the OmpA signal peptide was directly fused to the mature subtilisin sequence, no protease activity was detected, although this product had the identical primary structure as subtilisin E as a result of cleavage of the OmpA signal peptide and was produced at a level of approximately 10% of total cellular protein. When the OmpA signal peptide was fused to the 15th or 44th amino acid residue from the amino terminus of the pro-sequence, active subtilisin was also not produced. These results indicate that the pro-sequence of pre-pro-subtilisin plays an important role in the formation of enzymatically active subtilisin. It is proposed that the pro-sequence is essential for guiding appropriate folding of the enzymatically active conformation of subtilisin E.     

Monkey erythropoietin gene: cloning, expression and comparison with the human erythropoietin gene

The erythropoietin (Epo) gene from Cynomolgus monkeys has been isolated from a kidney cDNA library using mixed 20-mer oligodeoxynucleotide probes. The gene encodes a 168 amino acid (aa) mature protein with a calculated Mr of 18,490 and a presumptive signal peptide of 24 aa. The Epo gene, when transfected into Chinese hamster ovary (CHO) cells, produces a glycosylated protein with an apparent Mr of 34,000. The expressed product is biologically active in vivo. The monkey gene exhibits 92% and 94% homology to the human gene at the aa and nucleotide sequence levels, respectively. When compared with the human Epo, monkey Epo has an additional 3-aa residue at the N terminus of the mature protein and a deletion of an internal lysine residue.     

The human CLN2 protein/tripeptidyl-peptidase I is a serine protease that autoactivates at acidic pH

The CLN2 gene mutated in the fatal hereditary neurodegenerative disease late infantile neuronal ceroid lipofuscinosis encodes a lysosomal protease with tripeptidyl-peptidase I activity. To understand the enzymological properties of the protein, we purified and characterized C-terminal hexahistidine-tagged human CLN2p/tripeptidyl-peptidase I produced from insect cells transfected with a baculovirus vector. The N terminus of the secreted 66-kDa protein corresponds to residue 20 of the primary CLN2 gene translation product, indicating removal of a 19-residue signal peptide. The purified protein is enzymatically inactive; however, upon acidification, it is proteolytically processed and concomitantly acquires enzymatic activity. The N terminus of the final 46-kDa processed form (Leu196) corresponds to that of mature CLN2p/tripeptidyl-peptidase I purified from human brain. The activity of the mature enzyme is irreversibly inhibited by the serine esterase inhibitor diisopropyl fluorophosphate, which specifically and stoichiometrically reacts with CLN2p/tripeptidyl-peptidase I at Ser475, demonstrating that this residue represents the active site nucleophile. Expression of wild type and mutant proteins in CHO cells indicate that Ser475, Asp360, Asp517, but not His236 are essential for activity. These data indicate that the CLN2 gene product is synthesized as an inactive proenzyme that is autocatalytically converted to an active serine protease.     

Changes in a mammalian signal sequence required for efficient protein secretion by yeasts

A plasmid-encoded gene for a hybrid pre-protein containing most of the bovine prolactin signal peptide (SpPRL) fused to the mature sequence of yeast invertase (IVT) was expressed and the product was processed and secreted by yeast. However, the level of IVT activity was reduced about six-fold when compared to that obtained with the wild type (wt) invertase signal peptide (SpIVT). When the 5'-untranslated sequence of the hybrid mRNA was truncated by 29 nucleotides, a 2.5-fold increase in secreted IVT was observed. Replacement of the PRL codons with preferred yeast codons did not result in any improvement in the production of secreted IVT. An increase in IVT activity to the level observed with the wt SpIVT was obtained by replacement of the Gly residue located between the N terminus and the central lipophilic region of the SpPRL by Ala. Since this amino acid replacement results in a higher probability of the SpPRL assuming an alpha-helical conformation, it suggests that the secondary structure of this region is important in recognition by the yeast secretory apparatus.     

Human alpha-fetoprotein primary structure: a mass spectrometric study.

The amino acid sequence of human alpha-fetoprotein, a 67-kDa protein present in mammalian embryonic serum, was verified by fast atom bombardment mass spectrometric (FAB/MS) analyses of three different enzymatic digests of the protein. Human alpha-fetoprotein obtained from a large-scale cell culture was digested with trypsin and V-8 protease either separately on two different samples or combined on the same one. The V-8 protease digest of the protein was partially fractionated by HPLC; the other samples were directly analyzed by FAB/MS without previous purification steps. About 90% of the alpha-fetoprotein amino acid sequence was verified by mass spectrometric analysis; this also confirmed that the cell-derived protein is identical with the hepatoma-derived protein. FAB analysis revealed that the N terminus of the mature protein is arginine rather than threonine, with the threonine occupying the second position. Therefore, the processing site of the alpha-fetoprotein signal peptide during maturation of the protein occurs at the N-terminal side of the arginine residue formerly indicated as residue-1. Thus mature alpha-fetoprotein contains 591 amino acids rather than 590.     

Subset of hybrid eukaryotic proteins is exported by the type I secretion system of Erwinia chrysanthemi

Erwinia chrysanthemi exports degradative enzymes by using a type I protein secretion system. The proteases secreted by this system lack an N-terminal signal peptide but contain a C-terminal secretion signal. To explore the substrate specificity of this system, we have expressed the E. chrysanthemi transporter system (prtDEF genes) in Escherichia coli and tested the ability of this ABC transporter to export hybrid proteins carrying C-terminal fragments of E. chrysanthemi protease B. The C terminus contains six glycine-rich repeated motifs, followed by two repeats of the sequences DFLV and DIIV. Two types of hybrid proteins were assayed for transport, proteins with the 93-residue-protease-B C terminus containing one glycine-rich repeat and both hydrophobic terminal repeats and proteins with the 181-residue C terminus containing all repeat motifs. Although the shorter C terminus is unable to export the hybrids, the longer C terminus can promote the secretion of hybrid proteins with N termini as large as 424 amino acids, showing that the glycine-rich motifs are required for the efficient secretion of these hybrids. However, the secretion of hybrids occurs only if these proteins do not carry disulfide bonds in their mature structures. These latter results suggest that disulfide bond formation can occur prior to or during the secretion. Disulfide bonds may prevent type I secretion of hybrids. One simple hypothesis to explain these results is that the type I channel is too narrow to permit the export of proteins with secondary structures stabilized by disulfide bonds.     

Fluorescence assay of SIRT protein deacetylases using an acetylated peptide substrate and a secondary trypsin reaction

A novel fluorescent substrate was devised for the sirtuin (SIRT) class of human protein deacetylases comprised of a peptide sequence containing a single acetyl-lysine residue, with a fluorescent group (tetramethylrhodamine-6-carboxylic acid, 6-TAMRA) near the carboxyl terminus and a nonfluorescent quenching group (QSY-7) near the amino terminus. The peptide sequence is modeled after the p53 acetylation site but is unreactive toward trypsin because all other lysine and arginine residues have been replaced by serine. However, the SIRT-deacetylated peptide is readily cleaved by trypsin, resulting in a maximal 30-fold enhancement of the 6-TAMRA fluorescence. Nicotinamide at millimolar concentrations stops the deacetylation but does not inhibit trypsin, and a microtiter plate assay of the SIRTs has been devised using the fluorescent substrate and these reagents. Using this method, the kinetics of the reaction of the cosubstrate nicotinamide adenine dinucleotide and the competitive inhibitor nicotinamide with SIRT1 and SIRT2 has been analyzed. Several nicotinamide analogs have also been tested as inhibitors and found to have much lower affinity for these enzymes than does the parent compound.     

A positive residue in the hydrophobic core of the Escherichia coli lipoprotein signal peptide suppresses the secretion defect caused by an acidic amino terminus.

The signal peptide of secretory proteins requires a basic amino terminus followed by a stretch of hydrophobic residues to effect efficient translocation of precursor proteins. Replacement of the positively charged amino-terminal residues of prolipoprotein by acidic amino acids decreased the rate of precursor translocation (Inouye, S., Soberon, X., Franceschini, T., Nakamura, K., Itakura, K., and Inouye, M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3438-3441; Vlasuk, G. P., Inouye, S., Ito, H., Itakura, K., and Inouye, M. (1983) J. Biol. Chem. 258, 7141-7148). We demonstrate here that an arginine residue, but not an aspartate, when localized at position 9 of the hydrophobic region of the lipoprotein signal peptide, is able to suppress intramolecularly the processing defect caused by an acidic amino terminus. Furthermore, when present at position 14 of the signal peptide, this positive residue, but not aspartate, was able to support efficient translocation of unmodified prolipoprotein. This demonstrates that a positive residue can restore the function of a severely defective signal peptide and need not be localized at the amino terminus to do so. Both aspartate and arginine substitution at position 14 of the lipoprotein signal peptide stimulated prolipoprotein synthesis. This effect was position-specific, did not require precursor translocation, and was dominant to the inhibition of synthesis caused by an acidic amino terminus.     

Critical molecular regions for elicitation of the sweetness of the sweet-tasting protein, thaumatin I

Thaumatin is an intensely sweet-tasting protein. To identify the critical amino acid residue(s) responsible for elicitation of the sweetness of thaumatin, we prepared mutant thaumatin proteins, using Pichia pastoris, in which alanine residues were substituted for lysine or arginine residues, and the sweetness of each mutant protein was evaluated by sensory analysis in humans. Four lysine residues (K49, K67, K106 and K163) and three arginine residues (R76, R79 and R82) played significant roles in thaumatin sweetness. Of these residues, K67 and R82 were particularly important for eliciting the sweetness. We also prepared two further mutant thaumatin I proteins: one in which an arginine residue was substituted for a lysine residue, R82K, and one in which a lysine residue was substituted for an arginine residue, K67R. The threshold value for sweetness was higher for R82K than for thaumatin I, indicating that not only the positive charge but also the structure of the side chain of the arginine residue at position 82 influences the sweetness of thaumatin, whereas only the positive charge of the K67 side chain affects sweetness.     

Alteration of N-terminal residues of mature human lysozyme affects its secretion in yeast and translocation into canine microsomal vesicles

Signal sequences play a central role in the initial membrane translocation of secretory proteins. Their functions depend on factors such as hydrophobicity and conformation of the signal sequences themselves. However, some characteristics of mature proteins, especially those of the N-terminal region, might also affect the function of the signal sequences. To examine this possibility, several mutants of human lysozyme modified in the N-terminal region of the mature protein were constructed, and their secretion in yeast as well as in vitro translocation into canine pancreatic microsomes were analyzed using an idealized signal sequence L8 (MR(L)8PLAALG). Our results show the following. (1) Change in the charge at the N-terminal residue of the mature protein does not affect secretion drastically. (2) Substitution of a proline residue at the N terminus prevents cleavage of the signal sequence, although translocation itself is not impaired. (3) Excessive positive charges in the N-terminal region delay translocation of the precursor protein across the membrane. (4) Polar and negatively charged residues introduced into the N-terminal region affect the secretion of the mature protein by preventing its correct folding.     

Secretion of N-glycosylated interleukin-1 beta in Saccharomyces cerevisiae using a leader peptide from Candida albicans. Effect of N-linked glycosylation on biological activity

Human interleukin-1 beta (IL-1 beta) is expressed in activated monocytes as a 31-kDa precursor protein which is processed and secreted as a mature, unglycosylated 17-kDa carboxyl-terminal fragment, despite the fact that it contains a potential N-linked glycosylation site near the NH2 terminus (-Asn7-Cys8-Thr9-). cDNA coding for authentic mature IL-1 beta was fused to the signal sequence from the Candida albicans glucoamylase gene, two amino acids downstream from the signal processing site. Upon expression in Saccharomyces cerevisiae, approximately equimolar amounts of N-glycosylated (22 kDa) and unglycosylated (17 kDa) IL-1 beta protein were secreted. The N-glycosylated yeast recombinant IL-1 beta exhibited a 5-7-fold lower specific activity compared to the unglycosylated species. The mechanism responsible for inefficient glycosylation was also studied. We found no differences in secretion kinetics or processing between the two extracellular forms of IL-1 beta. The 17-kDa protein, which was found to lack core sugars, does not result from deglycosylation of the 22-kDa protein in vivo and does not result from saturation of the glycosylation enzymatic machinery through overexpression. Alteration of the uncommon Cys8 residue in the -Asn-X-Ser/Thr-glycosylation site to Ser also had no effect. However, increasing the distance between Asn7 and the signal processing site increased the extent of core N-linked glycosylation, suggesting a reduction in glycosylation efficiency near the NH2 terminus.     

Role of the mature protein sequence of maltose-binding protein in its secretion across the E. coli cytoplasmic membrane

Secretion of amber fragments of an E. coli periplasmic protein, the maltose-binding protein, was studied to determine if the mature portion of the protein is required for its export across the cytoplasmic membrane. A fragment lacking 25–35 amino acid residues at the C terminus is secreted at normal levels, suggesting that this sequence is not required for secretion. This is in contrast to the results obtained with the periplasmic protein β-lactamase. In studying another fragment of one-third the molecular weight of the intact protein, we found that the majority of the fragment is not recovered from the periplasmic fraction. However, a small amount of secretion of this polypeptide was observed. This fragment is synthesized as a larger molecular weight form when cells are induced for the synthesis of a maltose-binding protein-β-galactosidase hybrid protein, which was previously shown to block the proper localization and processing of envelope proteins. This result is consistent with the idea that the larger form is a precursor with an unprocessed signal sequence, whereas in the absence of the hybrid protein the fragment is a processed mature form. Thus secretion of the smaller fragment may be occurring up to the point where the signal sequence is removed. That this fragment has passed through the cytoplasmic membrane is further supported by its accessibility to externally added trypsin. We suggest that the fragment may be secreted to the periplasm, but cannot assume a water-soluble conformation; the majority of the polypeptide may be associated with the external surface of the cytoplasmic membrane. Thus the mature sequence of maltose-binding protein, at least its C-terminal two thirds, may not be required for its export across the cytoplasmic membrane.     

A proline residue near the amino terminus of the mature domain of secretory proteins lowers the level of the proton motive force required for translocation.

A large variety of proOmpF-Lpps, hybrid secretory proteins composed of the signal region of proOmpF and the mature part of the major lipoprotein, either possessing or not possessing a proline residue near the amino terminus of their mature domains, were constructed at a DNA level, and the rates of their in vitro translocation were determined in the presence and absence of the proton motive force (delta muH+). A proline residue at the signal peptide cleavage site (position +1) blocked the cleavage reaction but not the translocation reaction. All the proOmpF-Lpps examined exhibited approximately the same translocation rate in the presence of delta muH+ irrespective of the presence or absence of a proline residue near the amino terminus. In the absence of the delta muH+, which was achieved by either depletion of the respiratory substrate or the use of urea-treated membrane vesicles permeable to protons, proOmpF-Lpps possessing a proline residue near the amino terminus of the mature domain were translocated whereas those possessing no proline residue in this region were not translocated at all or only very weakly. The position of the proline residue was then moved stepwise away from the amino terminus of the mature domain. The further the position was moved away, the slower was the rate of translocation in the absence of delta muH+. The removal of the proline residue at position +2 of the mature domain of proOmpA also made the delta mu(H+)-independent translocation appreciably slower. It is suggested that the conformational flexibility endowed by the proline residue on the junction region between the signal peptide and the mature domain allows the translocation in the absence of delta muH+ and that this junction region must take on a particular conformation for initiation of the translocation reaction.     

Intragenic suppressor mutations that restore export of maltose binding protein with a truncated signal peptide

A deletion mutation, malE delta 12-18, removes seven residues from the hydrophobic core of the maltose binding protein (MBP) signal peptide and thus prevents secretion of this protein to the periplasm of E. coli. Intragenic suppressor mutations of malE delta 12-18 have been obtained, some highly efficient in their ability to restore proper MBP export. Twelve independently isolated suppressors represent six unique mutational events. Five result in alterations within the MBP signal peptide; one changes the amino acid at residue 19 of the mature MBP. Analysis of these suppressors indicates that the length of the hydrophobic core is a major determinant of signal peptide function. The experiments further suggest that the hydrophobic core region serves primarily a structural role in mediating protein secretion, and that other sequences outside of this region may be responsible for providing the initial recognition of the MBP nascent chain as a secreted protein.