The internal signal sequence of Escherichia coli leader peptidase is necessary, but not sufficient, for its rapid membrane assembly

Leader peptidase of Escherichia coli, a protein of 323 residues, has three hydrophobic domains. The first, residues 1-22, is the most apolar and is followed by a polar region (23-61) which faces the cytoplasm. The second hydrophobic domain (residues 62-76) spans the membrane. The third hydrophobic domain, which has a minimal apolar character, and the polar, carboxyl-terminal two-thirds of the protein are exposed to the periplasm. Deletion of either the amino terminus (residues 4-50) or the third hydrophobic region (residues 83-98) has almost no effect on the rate of leader peptidase membrane assembly, while the second hydrophobic domain is essential for insertion (Dalbey, R., and Wickner, W. (1987) Science 235, 783-787). To further define the roles of these domains, we have replaced the normal, cleaved leader sequence of pro-OmpA and M13 procoat with regions containing either the first or second apolar domain of leader peptidase. The second apolar domain supports the translocation of OmpA or coat protein across the plasma membrane, establishing its identity as an internal, uncleaved signal sequence. In addition to this sequence, we now find that leader peptidase needs either the amino-terminal domain or the third hydrophobic domain to permit its rapid membrane assembly. These results show that, although a signal sequence is necessary for rapid membrane assembly of leader peptidase, it is not sufficient.     

Sequence analysis of the heat-labile enterotoxin subunit B gene originating in human enterotoxigenic Escherichia coli

In this study, we determined the amino-terminal coding sequence, covering the signal peptide and the amino-terminus of the mature peptide, of the heat-labile enterotoxin subunit B (LT-B) gene originating in human enterotoxigenic Escherichia coli. Neither the signal sequence nor the amino-terminal sequence of the mature LT-B was identical to those sequences from porcine enterotoxigenic E. coli, but there was an extensive homology.     

Deletion of the amino-terminal domain of asialoglycoprotein receptor H1 allows cleavage of the internal signal sequence

Human asialoglycoprotein receptor H1 is a single-spanning membrane protein with an amino-terminal domain of 40 residues exposed to the cytoplasm and the carboxyl-terminal domain translocated to the exoplasmic side of the membrane. It has been shown earlier that the transmembrane segment functions as an internal uncleaved signal sequence for insertion into the endoplasmic reticulum. In a deletion protein lacking almost the entire cytoplasmic domain, the signal sequence is cleaved at the carboxyl-terminal end of the transmembrane segment. All available criteria suggest that the protein is processed by signal peptidase. The cytoplasmic domain of the receptor does not directly inhibit signal cleavage since it does not detectably hinder cleavage of the normally amino-terminal signal sequence of influenza hemagglutinin in fusion proteins. We suggest that by its size or structure it affects the position of the receptor in the membrane and thus the accessibility of the potential cleavage site to signal peptidase.     

A comparative analysis of single- and multiple-residue substitutions in the alkaline phosphatase signal peptide

The alkaline phosphatase signal peptide participates in transport of the enzyme to the periplasmic space of Escherichia coli. The signal sequence, like that of other signal peptides, is composed of a polar amino-terminal segment, a central region rich in hydrophobic residues and a carboxy-terminal region recognized by signal peptidase. We have previously shown that an alkaline phosphatase signal peptide mutant containing a polyleucine core region functions efficiently in transport of the enzyme [D. A. Kendall, S. C. Bock, and E. T. Kaiser (1986) Nature 321, 706-708]. In this study, some of the amino acid changes involved in the polyleucine sequence are examined individually. A Phe to Leu substitution as the sole change results in impaired transport properties in contrast to when it is combined with three other amino acid changes in the polyleucine-containing sequence. A mutant with a Pro to Leu substitution in the hydrophobic core region is comparable to wild type while the same type of substitution (Pro to Leu) in the carboxy-terminal segment results in substantial accumulation of the mutant precursor. Finally, introduction of a basic residue into the hydrophobic segment (Leu to Arg substitution) results in a complete export block. These results exemplify the spectrum of properties produced by individual residue changes and suggest there is some interplay between hydrophobicity and conformation for signal peptide function.     

Processing and methylation of PulG, a pilin-like component of the general secretory pathway of Klebsiella oxytoca

The signal sequence of the Klebsiella oxytoca pulG gene product, which is required for extracellular secretion of the enzyme pullulanase, is similar in many respects to the corresponding segment of the precursors of type IV (me-Phe) pilins. The significance of this similarity is confirmed by the observation that the pulO gene product processes prePulG at the consensus type IV prepilin peptidase cleavage site at the amino-terminal end of the PulG signal sequence. Like most type IV pilins, processed PuiG was found to have a methylated amino-terminal phenylaianine residue. Site-directed mutagenesis was used to replace amino acids in prePulG that correspond to residues shown by others to be essential for processing, methylation and assembly of type IV pilins. The glycine residue on the amino-terminal side of the prePulG cleavage site is absolutely required for processing and for pullulanase secretion. The glutamate residue at position 11 (+5) is also required for pullulanase secretion but not for processing or methylation. This result contrasts with that reported for corresponding variants of Pseudomonas aeruginosa type IV prepilin, which were processed but only inefficiently IV-methylated. Cleavage of prePulG and pullulanase secretion were both unaffected by replacement of the phenylalanine residue on the car-boxy-terminal side of the cleavage site by leucine, isoleucine or valine, by a conservative substitution within the hydrophobic core of the prePulG signal sequence, or by a glutamine to proline substitution within the processed segment. However, replacement of the same glutamine residue by arginine abolished secretion without affecting either processing or methylation.     

Requirements for substrate recognition by bacterial leader peptidase.

Many secreted and membrane proteins have amino-terminal leader peptides which are essential for their insertion across the membrane bilayer. These precursor proteins, whether from prokaryotic or eukaryotic sources, can be processed to their mature forms in vitro by bacterial leader peptidase. While different leader peptides have shared features, they do not share a unique sequence at the cleavage site. To examine the requirements for substrate recognition by leader peptidase, we have truncated M13 procoat, a membrane protein precursor, from both the amino- and carboxy-terminal ends with specific proteases or chemical cleavage agents. The fragments isolated from these reactions were assayed as substrates for leader peptidase. A 16 amino acid residue peptide which spans the leader peptidase cleavage site is accurately cleaved. Neither the basic amino-terminal region nor most of the hydrophobic central region of the leader peptide are essential for accurate cleavage.     

The role of the polar, carboxyl-terminal domain of Escherichia coli leader peptidase in its translocation across the plasma membrane

Leader peptidase, an integral membrane protein of Escherichia coli, is made without a cleavable leader sequence. It has 323 amino acid residues and spans the plasma membrane with a small amino-terminal domain exposed to the cytoplasm and a large, carboxyl-terminal domain exposed to the periplasm. We have investigated which regions of leader peptidase are necessary for its assembly across the membrane. Deletions were made in the carboxyl-terminal domain of leader peptidase, removing residues 141-222, 142-323, or 222-323. Protease accessibility was used to determine whether the polar, carboxyl-terminal domains of these truncated leader peptidases were translocated across the membrane. The removal of either residues 222-323 (the extreme carboxyl terminus) or residues 141-222 does not prevent leader peptidase membrane assembly. However, leader peptidase lacking both regions, i.e. amino acid residues 142-323, cannot translocate the remaining portion of its carboxyl terminus across the membrane. Our data suggest that the polar, periplasmic domain of leader peptidase contains information which is needed for membrane assembly.     

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.     

Defective Escherichia coli signal peptides function in yeast

To investigate structural characteristics important for eukaryotic signal peptide function in vivo, a hybrid gene with interchangeable signal peptides was cloned into yeast. The hybrid gene encoded nine residues from the amino terminus of the major Escherichia coli lipoprotein, attached to the amino terminus of the entire mature E. coli beta-lactamase sequence. To this sequence were attached sequences encoding the nonmutant E. coli lipoprotein signal peptide, or lipoprotein signal peptide mutants lacking an amino-terminal cationic charge, with shortened hydrophobic core, with altered potential helicity, or with an altered signal-peptide cleavage site. These signal-peptide mutants exhibited altered processing and secretion in E. coli. Using the GAL10 promoter, production of all hybrid proteins was induced to constitute 4-5% of the total yeast protein. Hybrid proteins with mutant signal peptides that show altered processing and secretion in E. coli, were processed and translocated to a similar degree as the non-mutant hybrid protein in yeast (approximately 36% of the total hybrid protein). Both non-mutant and mutant signal peptides appeared to be removed at the same unique site between cysteine 21 and serine 22, one residue from the E. coli signal peptidase II processing site. The mature lipo-beta-lactamase was translocated across the cytoplasmic membrane into the yeast periplasm. Thus the protein secretion apparatus in yeast recognizes the lipoprotein signal sequence in vivo but displays a specificity towards altered signal sequences which differs from that of E. coli.     

Amino acid substitutions in pilin of Pseudomonas aeruginosa. Effect on leader peptide cleavage, amino-terminal methylation, and pilus assembly.

A total of 37 separate mutants containing single and multiple amino acid substitutions in the leader and amino-terminal conserved region of the Type IV pilin from Pseudomonas aeruginosa were generated by oligonucleotide-directed mutagenesis. The effect of these substitutions on the secretion, processing, and assembly of the pilin monomers into mature pili was examined. The majority of substitutions in the highly conserved amino-terminal region of the pilin monomer had no effect on piliation. Likewise, substitution of several of the residues within the six amino acid leader sequence did not affect secretion and leader cleavage (processing), including replacement of one or both of the positively charged lysine residues with uncharged or negatively charged amino acids. One characteristic of the Type IV pili is the presence of an amino-terminal phenylalanine after leader peptide cleavage which is N-methylated prior to assembly of pilin monomers into pili. Substitution of the amino-terminal phenylalanine with a number of other amino acids, including polar, hydrophobic, and charged residues, did not affect proper leader cleavage and subsequent assembly into pili. Amino-terminal sequencing showed that the majority of substitute residues were also methylated. Substitution of the glycine residue at the -1 position to the cleavage site resulted in the inability to cleave the prepilin monomers and blocked the subsequent assembly of monomers into pili. These results indicate that despite the high degree of conservation in the amino-terminal sequences of the Type IV pili, N-methylphenylalanine at the +1 position relative to the leader peptide cleavage site is not strictly required for pilin assembly. N-Methylation of the amino acids substituted for phenylalanine was shown to have taken place in four of the five mutants tested, but it remains unclear as to whether pilin assembly is dependent on this modification. Recognition and proper cleavage of the prepilin by the leader peptidase appears to be dependent only on the glycine residue at the -1 position. Cell fractionation experiments demonstrated that pilin isolated from mutants deficient in prepilin processing and/or assembly was found in both inner and outer membrane fractions, indistinguishable from the results seen with the wild type.     

Both a short hydrophobic domain and a carboxyl-terminal hydrophilic region are important for signal function in the Escherichia coli leader peptidase

Leader peptidase, typical of inner membrane proteins of Escherichia coli, does not have an amino-terminal leader sequence. This protein contains an internal signal peptide, residues 51-83, which is essential for assembly and remains as a membrane anchor domain. We have employed site-directed mutagenesis techniques to either delete residues within this domain or substitute a charged amino acid for one of these residues to determine the important properties of the internal signal. The deletion analysis showed that a very small apolar domain, residues 70-76, is essential for assembly, whereas residues that flank it are dispensable for its function. However, point mutations with charged amino acid residues within the polar sequence (residues 77-82) slow or abolish leader peptidase membrane assembly. Thus, a polar region, Arg-Ser-Phe-Ile-Tyr-Glu, is important for the signal peptide function of leader peptidase, unlike other signals identified thus far.     

Signal peptidase can cleave inside a polytopic membrane protein

The signal peptides of most proteins targeted to the endoplasmic reticulum are specifically cleaved by signal peptidase. Although potential cleavage sites occur frequently in polytopic proteins after membrane-spanning segments, processing is restricted to the first hydrophobic domain, suggesting that signal peptidase might not have access to subsequently translocated, internal domains. To test this hypothesis, we replaced the third transmembrane segment of an artificial threefold membrane-spanning protein by a sequence which is normally an amino-terminal signal. Upon in vitro translation and insertion into microsomes, efficient cleavage at this sequence was observed, thus demonstrating the ability of signal peptidase to cleave within polytopic membrane proteins.     

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.     

Limited proteolysis as a probe of the conformation and nucleic acid binding regions of nucleolin.

Nucleolin, also called protein C23, is a RNA-associated protein implicated in the early stages of ribosome assembly. To study the general conformation and map the nucleic acid binding regions, rat nucleolin was subjected to limited proteolysis using trypsin and chymotrypsin in the presence or absence of poly(G). The cleavage sites were classified according to their locations in the three putative domains: the highly polar amino-terminal domain, the central nucleic acid binding domain, which contains four 90-residue repeats, and the carboxyl-terminal domain, which is rich is glycine, dimethylarginine, and phenylalanine. The most labile sites were found in basic segments of the amino-terminal domain. This region was stabilized by Mg2+. At low enzyme concentrations, cleavage by trypsin or chymotrypsin in the amino-terminal domain was enhanced by poly(G). Trypsin produced a relatively stable 48-kDa fragment containing the central and carboxyl-terminal domains. The enhanced cleavage suggests that binding of nucleic acid by the central domain alters the conformation of the amino-terminal domain, exposing sites to proteolytic cleavage. At moderate enzyme concentrations, the 48-kDa fragment was protected by poly(G) against tryptic digestion. At the highest enzyme concentrations, both enzymes cleaved near the boundaries between repeats 2, 3, and 4 with some sites protected by poly(G), suggesting that the repeats themselves form compact units. The carboxyl-terminal domain was resistant to trypsin but was cleaved by chymotrypsin either in the presence or in the absence of poly(G), indicating exposure of some phenylalanines in this region. These studies provide a general picture of the topology of nucleolin and suggest that the nucleic acid binding region communicates with the amino-terminal domain.     

The membrane-spanning segment of invariant chain (I gamma) contains a potentially cleavable signal sequence

The human invariant chain (I gamma) of class II histocompatibility antigens spans the membrane of the endoplasmic reticulum once. It exposes a small amino-terminal domain on the cytoplasmic side and a carboxy-terminal, glycosylated domain on the exoplasmic side of the membrane. When the exoplasmic domain of I gamma is replaced by the cytoplasmic protein chloramphenicol acetyltransferase (CAT), CAT becomes the exoplasmic, glycosylated domain of the resulting membrane protein I gamma CAT. Deletion of the hydrophilic cytoplasmic domain from I gamma CAT gives rise to a secreted protein from which an amino-terminal segment is cleaved, most likely by signal peptidase. We conclude that the membrane-spanning region of I gamma contains a signal sequence in its amino-terminal half and that hydrophilic residues at the amino-terminal end of a signal sequence can determine cleavage by signal peptidase.     

Positive charges in the cytoplasmic domain of Escherichia coli leader peptidase prevent an apolar domain from functioning as a signal.

Leader peptidase, an integral transmembrane protein of Escherichia coli, requires two apolar topogenic elements for its membrane assembly: a 'hydrophobic helper' and an internal signal. The highly basic cytoplasmic region between these domains is a translocation poison sequence, which we have shown blocks the function of a preceding signal sequence. We have used oligonucleotide-directed mutagenesis to remove positively charged residues within this polar domain to determine if it is the basic character in this region that has the negative effect on translocation. Our results show that mutations that remove two or more of the positively charged residues within the polar region no longer block membrane assembly of leader peptidase. In addition, when the translocation poison domain (residues 30-52) is replaced with six lysine residues, the preceding apolar domain cannot function as an export signal, whereas it can with six glutamic acids. Thus, positively charged residues within membrane proteins may have a major role in determining the function of hydrophobic domains in membrane assembly.     

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.     

Amino acid sequence of heat-labile enterotoxin from chicken enterotoxigenic Escherichia coli is identical to that of human strain H 10407

Abstract The DNA sequence of heat-labile enterotoxin from the chicken enterotoxigenic Escherichia coli 21d strain was determined by direct dideoxy sequencing of polymerase chain reaction (PCR)-amplified DNA and was compared with those of heat-labile enterotoxins from porcine and human enterotoxigenic E. coli strains EWD 299 and H 10407. The structural genes of the A and B subunits of chicken heat-labile enterotoxin were identical to those of human heat-labile enterotoxin from the human H 10407 strain. Moreover, 67 base pairs of the upstream and 60 base pairs of the downstream region of the chicken heat-labile enterotoxin gene were also identical to those of the human heat-labile enterotoxin from strain H 10407. However, the patterns of plasmids from the 21d and H 10407 strains were different. The 21d strain had no band corresponding to the 42-MDa plasmid of the H10407 strain encoding the heat-labile enterotoxin gene but it had a smaller plasmid. These data suggest that although the DNA sequence of chicken heat-labile enterotoxin is identical to that of human heat-labile enterotoxin, the plasmid encoding the chicken heat-labile enterotoxin gene in the chicken might be different from that encoding the human heat-labile enterotoxin gene in the H10407 strain.     

Functional analysis of the signal-sequence processing site of yeast acid phosphatase

A systematic study of the signal peptidase cleavage site of the main cell-wall-repressible Saccharomyces cerevisiae acid phosphatase encoded by the PHO5 gene is presented. The last amino acid of the signal sequence, the chromosomally encoded alanine of the wild-type gene, was changed by any of 19 other amino acids in the chromosomal DNA by using in vitro mutagenesis in Escherichia coli and the technique of gene replacement. Processing and secretion are normal when the amino acid at this position is a small neutral amino acid, i.e. alanine, glycine, cysteine, serine or threonine. Processing glycosylation, and secretion of regulated acid phosphatase are distinctly affected with other amino acid substitutions and core-glycosylated protein accumulates in the cell. Surprisingly, PHO5 protein is still secreted to the cell wall and into the growth medium but at a lower rate and without cleavage of the signal sequence. The same features are exhibited by a mutated acid phosphatase with a deletion of four amino acids at the end of the signal peptide (-7 to -4 relative to the processing site) thus preserving the important -3 to -1 region.     

Propeptide of human protein C is necessary for gamma-carboxylation

Protein C is one of a family of vitamin K dependent proteins, including blood coagulation factors and bone proteins, that contains gamma-carboxyglutamic acid. Sequence analysis of the cDNAs for these proteins has revealed the presence of a prepro leader sequence that contains a pre sequence or hydrophobic signal sequence and a propeptide containing a number of highly conserved amino acids. The pre region is removed from the growing polypeptide chain by signal peptidase, while the pro region is subsequently removed from the protein prior to secretion. In the present study, deletion mutants have been constructed in the propeptide region of the cDNA for human protein C, and the cDNAs were then expressed in mammalian cell culture. These deletions included the removal of 4, 9, 12, 15, 16, or 17 amino acids comprising the carboxyl end of the leader sequence of 42 amino acids. The mutant proteins were then examined by Western blotting, barium citrate adsorption and precipitation, amino acid sequence analysis, and biological activity and compared with the native protein present in normal plasma. These experiments have shown that protein C is readily synthesized in mammalian cell cultures, processed, and secreted as a two-chain molecule with biological activity. Furthermore, the pre portion or signal sequence in human protein C is 18 amino acids in length, and the pro portion of the leader sequence is 24 amino acids in length. Also, during biosynthesis and secretion, the amino-terminal region of the propeptide (residues from about -12 through -17) is important for gamma-carboxylation of protein C, while the present data and those of others indicate that the carboxyl-terminal portion of the propeptide (residues -1 through -4) is important for the removal of the pro leader sequence by proteolytic processing.