A small hydrophobic domain anchors leader peptidase to the cytoplasmic membrane of Escherichia coli

Leader peptidase is an enzyme of the Escherichia coli cytoplasmic membrane which removes amino-terminal leader sequences from many secreted and membrane proteins. Three potential membrane-spanning segments exist in the first 98 amino acids of leader peptidase. We have characterized the topology of leader peptidase based on its sensitivity to protease digestion. Proteinase K and trypsin treatment of right-side-out inner membrane vesicles and spheroplasts yields protected fragments of approximately 80 and 105 amino acid residues, respectively. We have shown that both fragments are derived from the amino terminus of the protein and that the smaller protected peptide can be derived from the larger. Removal of the third potential membrane-spanning segment (residues 82-98) does not affect the size of the proteinase K-protected fragment but does reduce the size of the trypsin-protected peptide. Because the proteinase K-protected fragment is about 9000 daltons, is derived from the amino terminus of leader peptidase, and its size is not affected when amino acids 82-98 are removed from the protein, it must extend from the amino terminus to approximately residue 80. Likewise, the trypsin-protected fragment must extend from the amino terminus to about residue 105. These data suggest a model for the orientation of leader peptidase in which the second hydrophobic stretch (residues 62-76) spans the cytoplasmic membrane and the third hydrophobic stretch resides in the periplasmic space.     

Mapping of catalytically important domains in Escherichia coli leader peptidase.

Leader peptidase (Lep) is a central component of the secretory machinery of Escherichia coli, where it serves to remove signal peptides from secretory proteins. It spans the inner membrane twice with a large C-terminal domain protruding into the periplasmic space. To investigate the importance of the different structural domains for the catalytic activity, we have studied the effects of a large panel of Lep mutants on the processing of signal peptides, both in vivo and in vitro. Our data suggest that the first transmembrane and cytoplasmic regions are not directly involved in catalysis, but that the second transmembrane region and the region immediately following it may be in contact with the signal peptide and/or located spatially close to the active site of Lep.     

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.     

Characterization of the internal signal-anchor domain of Escherichia coli leader peptidase

Leader peptidase, an integral transmembrane protein of Escherichia coli, is synthesized without a cleavable amino-terminal leader peptide. Of the five domains that participate in the membrane assembly of this protein, one is an internal "signal" region. We have used oligonucleotide-directed mutagenesis to examine the properties of the internal signal that are crucial for leader peptidase assembly. For this purpose, the net charge at the amino terminus of the internal signal was changed from +2 to +1 and -1 and, at the carboxyl terminus of the signal, from 0 to -1 or +1. These mutations had no effect on the membrane assembly of leader peptidase, suggesting that the charges have little role in the signal function. The apolar core of this signal was disrupted by substitution of basic amino acids for apolar residues. Substitution of an arginyl residue at position 70, or two arginyl residues at position 67 and 69, prevented membrane assembly. However, substitution of an arginyl residue at position 66 or either arginyl or lysyl residue at position 68 was without effect. Thus, while the apolar character of the internal signal is important, the precise position of a charged residue determines its effect on assembly.     

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.     

The effects of mutations in the carboxyl-terminal region on the catalytic activity of Escherichia coli signal peptidase I

Escherichia coli signal peptidase I (SPase I) is a membrane-bound serine endopeptidase that catalyses the cleavage of signal peptides from the pre-forms of membrane or secretory proteins. Our previous studies using chemical modification and site-directed mutagenesis suggested that Trp(300) and Arg(77), Arg(222), Arg(315) and Arg(318) are important for the proper and stable conformation of the active site of SPase I. Interestingly, many of these residues reside in the C-terminal region of the enzyme. As a continuation of these studies, we investigated in the present study the effects of mutations in the C-terminal region including amino acid residues at positions from 319 to 323 by deletions and site-directed mutagenesis. As a result, the deletion of the C-terminal His(323) was shown to scarcely affect the enzyme activity of SPase I, whereas the deletion of Gly(321)-His(323) or Ile(319)-His(323) as well as the point mutation of Ile(322) to alanine was shown to decrease significantly both the activity in vitro and in vivo without a big gross conformational change in the enzyme. These results suggest a significant contribution of Ile(322) to the construction and maintenance of the proper and critical local conformation backing up the active site of SPase I.     

The reaction specificities of the thylakoidal processing peptidase and Escherichia coli leader peptidase are identical.

Proteins which are transported across the bacterial plasma membrane, endoplasmic reticulum and thylakoid membrane are usually synthesized as larger precursors containing amino-terminal targeting signals. Removal of the signals is carried out by specific, membrane-bound processing peptidases. In this report we show that the reaction specificities of these three peptidases are essentially identical. Precursors of two higher plant thylakoid lumen proteins are efficiently processed by purified Escherichia coli leader peptidase. Processing of one precursor, that of the 23 kd photosystem II protein, by both the thylakoidal and E. coli enzymes generates the correct mature amino terminus. Similarly, leader (signal) peptides of both eukaryotic and prokaryotic origin are cleaved by partially purified thylakoidal processing peptidase. No evidence of incorrect processing was obtained. Both leader peptidase and thylakoidal peptidase are inhibited by a synthetic leader peptide.     

Identification of arginine residues important for the activity of Escherichia coli signal peptidase I

Escherichia coil signal peptidase I (leader peptidase, SPase I) is an integral membrane serine protease that catalyzes the cleavage of signal (leader) peptides from pre-forms of membrane or secretory proteins. We previously demonstrated that E. coil SPase I was significantly inactivated by reaction with phenylglyoxal with concomitant modification of three to four of the total 17 arginine residues in the enzyme. This result indicated that several arginine residues are important for the optimal activity of the enzyme. In the present study, we have constructed 17 mutants of the enzyme by site-directed mutagenesis to investigate the role of individual arginine residues in the enzyme. Mutation of Arg127, Arg146, Arg198, Arg199, Arg226, Arg236, Arg275, Arg282, and Arg295 scarcely affected the enzyme activity in vivo and in vitro. However, the enzymatic activity toward a synthetic substrate was significantly decreased by replacements of Arg77, Arg222, Arg315, or Arg318 with alanine/lysine. The kcat values of the R77A, R77K, R222A, R222K, R315A, R318A, and R318K mutant enzymes were about 5.5-fold smaller than that of the wild-type enzyme, whereas the Km values of these mutant enzymes were almost identical with that of the wild-type. Moreover, the complementing abilities in E. Arg222, Arg315, coil IT41 were lost completely when Arg77, or Arg318 was replaced with alanine/lysine. The circular dichroism spectra and other enzymatic properties of these mutants were comparable to those of the wild-type enzyme, indicating no global conformational changes. However, the thermostability of R222A, R222K, R315A, and R318K was significantly lower compared to the wild type. Therefore, Arg77, Arg222, Arg315, and Arg318 are thought to be important for maintaining the proper and stable conformation of SPase I.     

Inhibition of purified Escherichia coli leader peptidase by the leader (signal) peptide of bacteriophage M13 procoat.

The leader peptide of bacteriophage M13 procoat inhibited the cleavage of M13 procoat or pre-maltose-binding protein by purified Escherichia coli leader peptidase. This finding confirms inferences that the leader is the primary site of enzyme recognition and suggests a rationale for the rapid hydrolysis of leader peptides in vivo.     

Genetic characterization of a gene for prolipoprotein signal peptidase in Escherichia coli

A mutation (lspA, prolipoprotein signal peptidase) rendering the prolipoprotein signal peptidase temperature-sensitive in Escherichia coli has been analyzed. The mutation was mapped in the dnaJ-rpsT-ileS-dapB region by interrupted mating with various Hfr strains and P1 phage transduction. lambda transducing phage lambda ddapB2 that carries the rpsT-ileS-dapB region was shown to complement the lspA mutation. Plasmid pLC3-13 which had been isolated from Clarke and Carbon's collection as a plasmid carrying the lspA locus was shown to carry the dnaJ and rpsT loci. Complementation analysis with plasmids carrying various DNA fragments derived from pLC3-13 showed that the lspA locus is between the rpsT and ileS loci. The wildtype allele was dominant over the lspA allele.     

Identification of potential active-site residues in the Escherichia coli leader peptidase.

Leader peptidase of Escherichia coli cleaves the leader sequence from the amino terminus of membrane and secreted proteins after these proteins insert across the membrane. Despite considerable research, the mechanism of catalysis of leader peptidase remains unknown. This peptidase cannot be classified using protease inhibitors to the serine, cysteine, aspartic acid, or metallo- classes of proteases (Zwizinski, C., Date, T., and Wickner, W. (1981) J. Biol. Chem. 256, 3593-3597). Using site-directed mutagenesis, we have attempted to place leader peptidase in one of these groups. We found that leader peptidase, lacking all of the cysteine residues, can cleave the leader peptide from procoat, the precursor to bacteriophage M13 coat protein. Replacement of each histidine residue with an alanyl residue was without effect on catalysis. Among all the serine and aspartic acid residues, serine 90 and serine 185 as well as aspartic acid 99, 153, 273, and 276 are necessary to cleave procoat in a detergent extract. However, only serine 90 and aspartic acid 153 were required for processing using a highly sensitive in vivo assay. In addition to the residues directly affecting catalysis, aspartic acid 99 plays a role in maintaining the structure of leader peptidase. Replacement of this residue with alanine results in a very unstable leader peptidase protein. This study thus defines two critical residues, serine 90 and aspartic acid 153, that may be directly involved in catalysis and provides evidence that leader peptidase belongs to a novel class of serine proteases.     

TatA and TatB generate a hydrophobic mismatch important for the function and assembly of the Tat translocon in Escherichia coli

The twin-arginine translocation (Tat) system serves to translocate folded proteins across energy-transducing membranes in bacteria, archaea, plastids, and some mitochondria. In Escherichia coli, TatA, TatB, and TatC constitute functional translocons. TatA and TatB both possess an N-terminal transmembrane helix (TMH) followed by an amphipathic helix. The TMHs of TatA and TatB generate a hydrophobic mismatch with the membrane, as the helices comprise only 12 consecutive hydrophobic residues; however, the purpose of this mismatch is unclear. Here, we shortened or extended this stretch of hydrophobic residues in either TatA, TatB, or both and analyzed effects on translocon function and assembly. We found the WT length helices functioned best, but some variation was clearly tolerated. Defects in function were exacerbated by simultaneous mutations in TatA and TatB, indicating partial compensation of mutations in each by the other. Furthermore, length variation in TatB destabilized TatBC-containing complexes, revealing that the 12-residue-length is important but not essential for this interaction and translocon assembly. To also address potential effects of helix length on TatA interactions, we characterized these interactions by molecular dynamics simulations, after having characterized the TatA assemblies by metal-tagging transmission electron microscopy. In these simulations, we found that interacting short TMHs of larger TatA assemblies were thinning the membrane and—together with laterally-aligned tilted amphipathic helices—generated a deep V-shaped membrane groove. We propose the 12 consecutive hydrophobic residues may thus serve to destabilize the membrane during Tat transport, and their conservation could represent a delicate compromise between functionality and minimization of proton leakage.     

Expression study with the Escherichia coli lep gene for leader peptidase in phototrophic purple bacteria

Synthesis and assembly of leader peptidase of Escherichia coli (signal peptidase I), was studied by heterologous expression of its lep gene in three species of phototrophic purple bacteria. Cell extracts of the recipient species showed neither cross reaction with antibodies against E. coli leader peptidase nor cleavage of the model substrate M13-procoat in vitro. The lep gene was transferred via conjugation using the plasmid expression vector for phototrophic bacteria pJAJ9. Plasmidborne leader peptidase enzyme was identified by immunochemical means. However, extracts of transconjugant cells showed no cleavage function. Trypsin digestion studies revealed that the enzyme was not properly integrated across the host membranes. The data suggest that cleaving enzymes for protein export and/or their assembly pathway in purple bacteria differ from the E. coli type.Abbreviations DMSO dimethylsulfoxide - EDTA ethylenediamine tetraacetic acid - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate     

Conditionally lethal amber mutations in the leader peptidase gene of Escherichia coli.

The lep gene of Escherichia coli encodes the leader peptidase which cleaves amino-terminal leader sequences of secreted proteins. To facilitate the study of structure-function relationships of the leader peptidase, 22 amber mutations in lep were isolated by localized mutagenesis. These amber mutants grew at 32 degrees C but not at 42 degrees C in the presence of a temperature-sensitive amber suppressor. Most of them were lethal under sup0 conditions. However, one amber mutant, the lep-9 mutant, exhibited temperature-sensitive growth in the sup0 strain, indicating that the amber fragment is active at 32 degrees C but not at 42 degrees C. Protein precursors of the maltose-binding protein and OmpA accumulate strikingly in the lep-9 mutant.     

Cloning and expression of a gene coding for the prolipoprotein signal peptidase of Escherichia coli

An Escherichia coli mutant, Y815, has a temperature-sensitive prolipoprotein signal peptidase. IPTG-induced synthesis of the major outer membrane prolipoprotein (PLP) results in the inhibition of cell growth because of accumulation of PLP in its envelope [J. Bacteriol. (1982) 152, 1163-1168]. The 2000 E. coli strains of Clarke and Carbon's collection were screened for the presence of a plasmid complementing the IPTG-sensitivity of the growth of Y815. One plasmid, pLC3-13, complemented the IPTG-sensitivity. The envelope fraction prepared from Y815 transformed by pLC3-13 showed high activity of the PLP signal peptidase in vitro at high temperature. A 4 kb AccI fragment subcloned onto plasmid pHY001 was shown to carry the gene for the PLP signal peptidase.     

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.     

Recognition of the carboxyl-terminal signal for GPI modification requires translocation of its hydrophobic domain across the ER membrane

A carboxyl-terminal hydrophobic domain is an essential component of the processed signal for attachment of the glycosyl-phosphatidylinositol (GPI) membrane anchor to proteins and it is linked to the site (omega) of GPI modification by a spacer domain. This study was designed to test the hypothesis that the hydrophobic domain interacts with the lipid bilayer of the endoplasmic reticulum (ER) membrane to optimally position the omega site for GPI modification. The hydrophobic domain of the GPI signal in the human folate receptor (FR) type alpha was substituted with the carboxyl-terminal segment of the low-density lipoprotein receptor (LDLR), including its membrane spanning region, without altering either the spacer or the omega site. The FR-alpha/LDLR chimera was not GPI modified but was attached to the plasma membrane by a polypeptide anchor. When the carboxyl-terminal half of the hydrophobic transmembrane polypeptide in the FR-alpha/LDLR chimera was altered by introduction of negatively charged (Asp) residues, or when the cytosolic domain in the chimera was deleted, the mutated proteins became GPI-anchored. On the other hand, attachment of a carboxyl-terminal segment of LDLR including the entire cytosolic domain to FR-alpha converted it into a transmembrane protein. The results indicate that in the FR-alpha/LDLR chimera the inability of the cellular machinery for GPI modification to recognize the hydrophobic domain is not due to the intrinsic nature of the peptide, but is rather due to the retention of the peptide within the lipid bilayer. It follows that the hydrophobic domain in the signal for GPI modification must traverse the ER membrane prior to recognition of the omega site by the GPI-protein transamidase. The results thus establish a critical topographical requirement for recognition of the GPI signal in the ER.     

Prolipoprotein signal peptidase of Escherichia coli requires a cysteine residue at the cleavage site

A signal peptidase specifically required for the secretion of the lipoprotein of the Escherichia coli outer membrane cleaves off the signal peptide at the bond between a glycine and a cysteine residue. This cysteine residue was altered to a glycine residue by guided site-specific mutagenesis using a synthetic oligonucleotide and a plasmid carrying an inducible lipoprotein gene. The induction of mutant lipoprotein production was lethal to the cells. A large amount of the prolipoprotein was accumulated in the outer membrane fraction. No protein of the size of the mature lipoprotein was detected. These results indicate that the prolipoprotein signal peptidase requires a glyceride modified cysteine residue at the cleavage site.