Novel prlA alleles defective in supporting staphylokinase processing in Escherichia coli

A class of prlA (secY) alleles of Escherichia coli (prlA4-1 and prlA401) which specifically block the export of staphylokinase has been identified (T. Iino and T. Sako, J. Biol. Chem. 263:19077-19082, 1988; T. Sako and T. Iino, J. Bacteriol. 170:5389-5391, 1988). To determine more precisely the region in PrlA (SecY) effective for the blockage of processing of the staphylokinase precursor, additional prlA mutants which failed to support processing of the staphylokinase precursor were isolated. Two of the five mutant alleles isolated (secY121 and secY161) complemented the temperature sensitivity of a secY24 strain and had no detectable effect on the processing of endogenous secretory proteins of E. coli. In addition, a staphylokinase mutant having glycine in place of serine at position 17 in its signal sequence relieved the detrimental effect of these mutations. All of these characteristics indicate that these two alleles resemble the prlA4-1 and prlA401 alleles. On the other hand, the remaining three mutant alleles (secY47, secY105, and secY112) had no significant PrlA activity. The mutations of secY121 and secY161 were mapped very close to those of prlA4-1 and prlA401 in the presumed transmembrane segment 7 of PrlA. These results indicate that transmembrane segment 7 of PrlA plays a crucial role in the recognition of the staphylokinase signal sequence.     

Export defect adjacent to the processing site of staphylococcal nuclease is suppressed by a prlA mutation.

Plasmids have been constructed in which the Escherichia coli alkaline phosphatase promoter and signal sequence have been fused to the staphylococcal nuclease gene to promote the high-level expression and secretion of this gene product in E. coli. We determined that the first amino acid residue after the signal sequence can determine whether this protein was processed and exported to the periplasmic space. Fractionation and protease accessibility studies were used to show that the export-defective, nuclease precursor is internal to the cytoplasmic membrane barrier of the cell. Furthermore, this export defect was suppressed in a strain containing a prlA mutation. These findings are novel in that this region of the polypeptide chain has been implicated in processing but not export and that prlA mutations have not been previously known to suppress such defects.     

Effects of mutations at glycine residues in the hydrophobic region of the Escherichia coli prolipoprotein signal peptide on the secretion across the membrane

Each of the 2 glycine residues in the hydrophobic region of the prolipoprotein signal peptide of Escherichia coli was systematically deleted or substituted with a valine residue by oligonucleotide-directed site-specific mutagenesis. Functional analysis of four such mutants as well as four double mutants, resulting from combinations of any two of the single mutations, revealed that (a) glycine residues at positions 9 and 14 could be replaced individually or at the same time with a valine residue without affecting the secretion of prolipoprotein; (b) the deletion of glycine at position 9 had no effect on the secretion of prolipoprotein whereas, when glycine at position 14 was deleted, the glyceride modification and the processing of the mutant prolipoprotein occurred at a much slower rate at 42 degrees C than those of the wild type prolipoprotein; and (c) the effects of deleting glycine at position 14 could be suppressed by the deletion of glycine at position 9, which resulted in shortening the hydrophobic region of the prolipoprotein signal peptide by 2 amino acid residues. These results indicate that the hydrophobic region of the prolipoprotein signal peptide has remarkable flexibility in terms of the relationship between its primary structure and function in protein secretion.     

Kinetic analysis of lamB mutants suggests the signal sequence plays multiple roles in protein export

We have developed a quantitative assay to measure the rate of processing of precursor LamB into mature protein and have used this assay to characterize 10 previously isolated and 3 new lamB signal sequence mutants. The data suggest that the LamB signal sequence serves a complex function. Our assay has revealed five types of signal sequence defect: 1) a strong kinetic defect resulting from alteration of the secondary structure in the putative alpha-helical region in the hydrophobic core, 2) a strong, or 3) a weak kinetic defect due to placement of a charged residue in the hydrophobic core, 4) decreased synthesis of LamB, and 5) both a decrease in synthesis and a strong kinetic defect. The effect of an extragenic suppressor, prlA4 on the rate of processing pLamB containing signal sequence mutations was also examined and compared to the rates in wild-type strains. It was found that prlA4 increases the rate of processing in some, but not all, mutants having a kinetic defect while having no effect on the decreased synthesis seen in mutants of types 4 and 5.     

DNA sequence analysis of three missense mutations affecting colicin E3 bactericidal activity

We have determined the nucleotide sequence changes caused by three missense mutations leading to the production of inactive colicin E3 proteins. The ceaC1 mutation, affecting the translocation of colicin E3 through bacterial membranes, is caused by a serine to phenylalanine change at position 37 within the glycine-rich region at the N-terminal part of colicin E3. This confirms previous results suggesting a role for this domain in colicin uptake by sensitive cells. The ceaC2 and ceaC3 mutations, abolishing colicin E3 RNase activity, affect the C-terminal enzymatic domain of the molecule. In the ceaC2 mutant, serine at position 529 was converted to leucine. The ceaC3 mutation replaced a glycine residue at position 524 with an aspartic acid residue. The two mutations ceaC2 and ceaC3 yield information on the amino acid residues involved in the RNase activity of colicin E3.     

DNA sequence analysis of three missense mutations affecting colicin E3 bactericidal activity

Summary
We have determined the nucleotide sequence changes caused by three missense mutations leading to the production of inactive colicin E3 proteins. The ceaC1 mutation, affecting the transiocation of colicin E3 through bacterial membranes, is caused by a serine to phenylalanine change at position 37 within the glycine-rich region at the N-terminal part of colicin E3. This confirms previous results suggesting a role for this domain in colicin uptake by sensitive cells. The ceaC2 and ceaC3 mutations, abolishing colicin E3 RNase activity, affect the C-terminal enzymatic domain of the molecule, in the ceaC2 mutant, serine at position 529 was converted to leucine. The ceaC3 mutation replaced a glycine residue at position 524 with an aspartic acid residue. The two mutations ceaC2 and ceaC3 yieid information on the amino acid residues involved in the RNase activity of colicin E3.     

Export of maltose-binding protein species with altered charge distribution surrounding the signal peptide hydrophobic core in Escherichia coli cells harboring prl suppressor mutations.

It is believed that one or more basic residues at the extreme amino terminus of precursor proteins and the lack of a net positive charge immediately following the signal peptide act as topological determinants that promote the insertion of the signal peptide hydrophobic core into the cytoplasmic membrane of Escherichia coli cells with the correct orientation required to initiate the protein export process. The export efficiency of precursor maltose-binding protein (pre-MBP) was found to decrease progressively as the net charge in the early mature region was increased systematically from 0 to +4. This inhibitory effect could be further exacerbated by reducing the net charge in the signal peptide to below 0. One such MBP species, designated MBP-3/+3 and having a net charge of -3 in the signal peptide and +3 in the early mature region, was totally export defective. Revertants in which MBP-3/+3 export was restored were found to harbor mutations in the prlA (secY) gene, encoding a key component of the E. coli protein export machinery. One such mutation, prlA666, was extensively characterized and shown to be a particularly strong suppressor of a variety of MBP export defects. Export of MBP-3/+3 and other MBP species with charge alterations in the early mature region also was substantially improved in E. coli cells harboring certain other prlA mutations originally selected as extragenic suppressors of signal sequence mutations altering the hydrophobic core of the LamB or MBP signal peptide. In addition, the enzymatic activity of alkaline phosphatase (PhoA) fused to a predicted cytoplasmic domain of an integral membrane protein (UhpT) increased significantly in cells harboring prlA666. These results suggest a role for PrlA/SecY in determining the orientation of signal peptides and possibly other membrane-spanning protein domains in the cytoplasmic membrane.     

Analysis of mutational alterations in the hydrophilic segment of the maltose-binding protein signal peptide.

Oligonucleotide-directed mutagenesis was employed to investigate the role of the hydrophilic segment of the Escherichia coli maltose-binding protein (MBP) signal peptide in the protein export process. The three basic residues residing at the amino terminus of the signal peptide were systematically substituted with neutral or acidic residues, decreasing the net charge in a stepwise fashion from +3 to -3. It was found that a net positive charge was not absolutely required for MBP export to the periplasm. However, export was most rapid and efficient when the signal peptide retained at least a single basic residue and a net charge of +1. The nature of the adjacent hydrophobic core helped to determine the effect of charge changes in the hydrophilic segment on MBP export, which suggested that these two regions of the signal peptide do not have totally distinct functions. Although the stepwise decrease in net charge of the signal peptide also resulted in a progressive decrease in the level of MBP synthesis, the data do not readily support a model in which MBP synthesis and export are obligately coupled events. The export defect resulting from alterations in the hydrophilic segment was partially suppressed in strains harboring certain prl alleles but not in strains harboring prlA alleles that are highly efficient suppressors of signal sequence mutations that alter the hydrophobic core.     

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.     

Isolation and analysis of novel mutants of Escherichia coli prlA (secY).

Plasmid libraries of prlA mutants containing single-base-pair changes throughout the gene were generated by in vitro random mutagenesis. The prlA mutations capable of suppressing the secretion defect of LamB caused by mutations in the LamB signal peptide were selected and analyzed. Together with additional mutations generated by site-directed mutagenesis, a number of novel prlA mutations and/or suppressors were identified. These mutations provide the starting points for studying the relationship of structure and function of PrlA in its interaction with LamB and/or other component(s) in the Escherichia coli protein secretion-translocation complex.     

In Vivo Analysis of Sequence Requirements for Processing and Degradation of the Colicin A Lysis Protein Signal Peptide

The lipid modification and processing of a number of colicin lysis proteins take place exceedingly slowly and result in the release of a stable signal peptide. It is possible that this peptide or the presence of lipid-modified precursors which result from the slow processing plays a role in the release of colicins and in the quasilysis that occurs in induced colicinogenic cultures. We used in vitro mutagenesis and pulse-chase radiolabeling and immunoprecipitation to examine the reasons for the slow processing and signal peptide degradation reactions for the colicin A lysis protein (Cal). In one mutant, isoleucine 13 was replaced with serine, and in another, alanine 18, the last residue of the signal peptide, was replaced with glycine. In each case, the mutation caused a striking increase in the rate of maturation of the precursor, and in the case of the serine 13 derivative, the mutation also destabilized the signal peptide. A precursor containing both of these mutations was completely matured and its signal sequence degraded within seconds of its synthesis. The release of colicin A and the quasilysis of producing cultures were unchanged for each of these mutants, indicating that neither the stable signal peptide nor lipid-modified processing intermediates of Cal are required for either of these events in wild-type cells.     

Use of site-directed mutagenesis to define the limits of sequence variation tolerated for processing of the M13 procoat protein by the Escherichia coli leader peptidase.

Leader peptidase cleaves the leader sequence from the amino terminus of newly made membrane and secreted proteins after they have translocated across the membrane. Analysis of a large number of leader sequences has shown that there is a characteristic pattern of small apolar residues at -1 and -3 (with respect to the cleavage site) and a helix-breaking residue adjacent to the central apolar core in the region -4 to -6. The conserved sequence pattern of small amino acids at -1 and -3 around the cleavage site most likely represents the substrate specificity of leader peptidase. We have tested this by generating 60 different mutations in the +1 to -6 domain of the M13 procoat protein. These mutants were analyzed for in vivo and in vitro processing, as well as for protein insertion into the cytoplasmic membrane. We find that in vivo leader peptidase was able to process procoat with an alanine, a serine, a glycine, or a proline residue at -1 and with a serine, a glycine, a threonine, a valine, or a leucine residue at -3. All other alterations at these sites were not processed, in accordance with predictions based on the conserved features of leader peptides. Except for proline and threonine at +1, all other residues at this position were processed by leader peptidase. None of the mutations at -2, -4, or -5 of procoat (apart from proline at -4) completely abolished leader peptidase cleavage in vivo although there were large effects on the kinetics of processing.(ABSTRACT TRUNCATED AT 250 WORDS)     

Processing of Escherichia coli alkaline phosphatase. Sequence requirements and possible conformations of the -6 to -4 region of the signal peptide

Analysis of the precursors of bacterial exported proteins revealed that those having bulky hydrophobic residues at position -5 have a high incidence of Pro residues at positions -6 and -4, Val at position -3, and Ser at positions -4 and -2. This led to a hypothesis that the previously observed inhibition of processing by bulky residues at position -5 can be suppressed by introduction of Pro, Ser, or Val in the corresponding nearby positions. Subsequent mutational analysis of Escherichia coli alkaline phosphatase showed that, as it was predicted, Pro on either side of bulky hydrophobic -5 Leu, Ile, or Tyr completely restores efficiency of the maturation. Introduction of Val at position -3 also partially suppresses the inhibition imposed by -5 Leu, while a Ser residue at position -4 or -2 does not restore processing. In addition, effective maturation of a mutant with Pro residues at positions from -6 throughout -4 proved that polyproline conformation of this region is permissive for processing. To understand the effects of the mutations, we modeled a peptide substrate into the active site of the signal peptidase using the known position of the beta-lactam inhibitor. The inhibitory effect of the -5 residue and its suppression by either Pro -6 or Pro -4 can be explained if we assume that Pro-containing -6 to -4 regions adopt a polyproline conformation whereas the region without Pro residues has a beta-conformation. These results permit us to specify sequence requirements at -6, -5, and -4 positions for efficient processing and to improve the prediction of yet unknown cleavage sites.     

Molecular cloning of dog mast cell tryptase and a related protease: structural evidence of a unique mode of serine protease activation

Mast cell tryptase is a secretory granule associated serine protease with trypsin-like specificity released extracellularly during mast cell degranulation. To determine the full primary structure of the catalytic domain and precursor forms of tryptase and to gain insight into its mode of activation, we cloned cDNAs coding for the complete amino acid sequence of dog mast cell tryptase and a second, possibly related, serine protease. Using RNA from dog mastocytoma cells, we constructed a cDNA library in lambda gt 10. Screening of the library with an oligonucleotide probe based on the N-terminal sequence of tryptase purified from the same cell source allowed us to isolate and sequence overlapping clones coding for dog mast cell tryptase. The tryptase sequence includes the essential residues of the catalytic triad and an aspartic acid at the base of the putative substrate binding pocket that confers P1 Arg and Lys specificity on tryptic serine proteases. The apparent N-terminal signal/activation peptide terminates in a glycine. A glycine in this position has not been observed previously in serine proteases and suggests a novel mode of activation. Additional screening of the library with a trypsinogen cDNA led to the isolation and sequencing of a full-length clone apparently coding for the complete sequence of a second tryptic serine protease (DMP) which is only 53.4% identical with the dog tryptase sequence but which contains an apparent signal/activation peptide also terminating in a glycine. Thus, the proteases encoded by these cloned cDNAs may share a common mode of activation from N-terminally extended precursors.     

Site-directed mutagenesis of alanine-382 of human antithrombin III

Antithrombin III Hamilton is a structural variant of antithrombin III (AT-III) with normal heparin affinity but impaired serine protease inhibitory activity. The molecular defect of AT-III-Hamilton is a substitution of threonine for alanine at amino acid residue 382. Recently it has been shown that both plasma-derived and cell-free-derived AT-III-Hamilton polypeptides act as substrates rather than inhibitors of thrombin and factor Xa. In the present study, the cell-free expression phagemid vector pGEM-3Zf(+)-AT-III1-432 was mutated at amino acid residue 382 of AT-III to generate 7 cell-free-derived variants. All these cell-free-derived AT-III variants were able to bind heparin as effectively as cell-free-derived normal AT-III. In terms of alpha-thrombin inhibitory activity each variant reacted differently. Variants could be grouped into 3 categories with respect to thrombin-AT-III complex formation: (1) near normal activity (glycine, isoleucine, leucine, valine); (2) low activity (threonine, glutamine); (3) no detectable activity (lysine). These data suggest that mutations at position 382 of AT-III may have a variable effect on protease inhibitory activity, depending on either the stability of the P12-P9 region of the exposed loop of AT-III, or the inability of the amino acid residue at position 382 to interact with a conserved hydrophobic pocket consisting of phenylalanine (at positions 77, 221 and 422) and isoleucine (position 412) residues.     

Effect of amino acid substitutions at the signal peptide cleavage site of the Escherichia coli major outer membrane lipoprotein

The requirement for the glycine residue at the COOH terminus of the signal peptide of the precursor of the major Escherichia coli outer membrane lipoprotein was examined. Using oligonucleotide-directed site-specific mutagenesis, this residue was replaced by residues of increasing side chain size. Substitution by serine had no effect on the modification or processing of the prolipoprotein. Substitution by valine or leucine resulted in the accumulation of the unmodified precursor, whereas threonine substitution resulted in slow lipid modification and no detectable processing of the lipid modified precursor. The results indicate that serine is the upper limit on size for the residue at the cleavage site. Larger residues at this position prevent the action of both the glyceride transferase and signal peptidase II enzymes, indicating that the cleavage site residue plays a role in events prior to proteolytic cleavage. The upper limit on size of the cleavage site residue is similar to that found for exported proteins cleaved by signal peptidase I, as well as eucaryotic exported proteins. The possibility that the cleavage site residue may have a role other than active site recognition by the signal peptidase is discussed.     

Substitution of Ser61----Gly61 in human apolipoprotein C-II does not alter its activation of lipoprotein lipase

Lipoprotein lipase (LpL) activity is enhanced by apolipoprotein C-II (apoC-II), a 79 amino acid residue peptide. The minimal apoC-II sequence required for activation of LpL resides between residues 56-79. To determine the possible role of an acyl-apoC-II intermediate involving Ser61 in enzyme catalysis, a synthetic peptide of apoC-II containing residues 56-79 was synthesized and compared to the corresponding peptide with serine at position 61 being substituted with glycine. With two different LpL assay systems, both peptides enhanced enzyme activity. Since glycine does not contain a hydroxyl group, these results rule out the possibility that an acyl-apoC-II intermediate with Ser61 is required for enzyme activation.     

Testing the '+2 rule' for lipoprotein sorting in the Escherichia coli cell envelope with a new genetic selection

We report a novel strategy for selecting mutations that mislocalize lipoproteins within the Escherichia coli cell envelope and describe the mutants obtained. A strain carrying a deletion of the chromosomal malE gene, coding for the periplasmic maltose-binding protein (MalE), cannot use maltose unless a wild-type copy of malE is present in trans. Replacement of the natural signal peptide of preMalE by the signal peptide and the first four amino acids of a cytoplasmic membrane-anchored lipoprotein resulted in N-terminal fatty acylation of MalE (lipoMalE) and anchoring to the periplasmic face of the cytoplasmic membrane, where it could still function. When the aspartate at position +2 of this protein was replaced by a serine, lipoMalE was sorted to the outer membrane, where it could not function. Chemical mutagenesis followed by selection for maltose-using mutants resulted in the identification of two classes of mutations. The single class I mutant carried a plasmid-borne mutation that replaced the serine at position +2 by phenylalanine. Systematic substitutions of the amino acid at position +2 revealed that, besides phenylalanine, tryptophan, tyrosine, glycine and proline could all replace classical cytoplasmic membrane lipoprotein sorting signal (aspartate +2). Analysis of known and putative lipoproteins encoded by the E. coli K-12 genome indicated that these amino acids are rarely found at position +2. In the class II mutants, a chromosomal mutation caused small and variable amounts of lipoMalE to remain associated with the cytoplasmic membrane. Similar amounts of another, endogenous outer membrane lipoprotein, NlpD, were also present in the cytoplasmic membrane in these mutants, indicating a minor, general defect in the sorting of outer membrane lipoproteins. Four representative class II mutants analysed were shown not to carry mutations in the lolA or lolB genes, known to be involved in the sorting of lipoproteins to the outer membrane.     

Role of amino-terminal positive charge on signal peptide in staphylokinase export across the cytoplasmic membrane of Escherichia coli

Staphylokinase mutants having amino acid substitutions within the amino-terminal charged segment of the signal peptide have been produced by in vitro oligonucleotide-directed mutagenesis. When the processing of the gene products was analyzed in Escherichia coli cells, the rate of processing of the mutant staphylokinase precursor decreased as the net charge became more negative. A net positive charge, but not specific amino acid residues, was required on the amino-terminal segment for efficient processing. Staphylokinase precursor having a net negative charge accumulated in the cytoplasm, tending to bind to the cytoplasmic membrane as determined by subcellular fractionation and immunoelectron microscopy. Although a mutant carrying an amino acid substitution in the hydrophobic segment and wild-type staphylokinases had an interfering effect on the processing of other normal secreted proteins, this effect was lost when they also contained charge-altering substitutions in the amino-terminal region. From these results, we concluded that a positive charge on the amino-terminal segment of the staphylokinase signal peptide is required for entrance into the protein export process.     

A previously unidentified gene in the spc operon of Escherichia coli K12 specifies a component of the protein export machinery

The gene prlA codes for a factor that appears to function in the export of proteins in Escherichia coli. This conclusion is based on the finding that mutations altering the prlA gene product restore export of envelope proteins with defective signal sequences. Previous results showed that the prlA gene lies in an operon (spc) known to code for ten different ribosomal proteins. Our studies show that the prlA gene lies promoter-distal to the last known ribosomal protein gene in this operon. Evidence from gene fusions constructed in vitro suggests that prlA codes for a protein containing at least 300 amino acids. Thus a heretofore unidentified protein specified by a gene within the spc operon appears to be a component of the cellular protein export machinery.