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.     

Inhibition of secretion of a mutant lipoprotein across the cytoplasmic membrane by the wild-type lipoprotein of the Escherichia coli outer membrane.

A globomycin-resistant mutant of Escherichia coli was found to produce a precursor of the major outer membrane lipoprotein (prolipoprotein), in which the glycine residue at position 14 within the signal peptide was replaced by an aspartic acid residue. The same mutation has been reported by Lin et al. (Proc. Natl. Acad. Sci. U.S.A. 175:4891-4895, 1978). The structural gene of the mutant prolipoprotein was inserted into an inducible expression cloning vehicle. When the mutant prolipoprotein was produced in lipoprotein-minus host cells, 82% of the unprocessed protein was found in the membrane fraction, with the remaining 18% localized in the soluble fraction. However, when the production of the mutant prolipoprotein was induced in the wild-type lpp+ host cells, only 31% of the mutant prolipoprotein was found in the membrane fraction, leaving the remaining 69% in the soluble, cytoplasmic fraction. In addition, the assembly of the wild-type lipoprotein in these cells was not affected, whether the mutant prolipoprotein was produced or not. These results suggest that secretions of both mutant and wild-type prolipoproteins utilize the same component(s) responsible for the initial stages of secretion across the cytoplasmic membrane. However, it appears that the wild-type lipoprotein has a higher affinity for these components than does the mutant lipoprotein.     

Processing of lipid-modified prolipoprotein requires energy and sec gene products in vivo.

The kinetics of processing of glyceride-modified prolipoprotein that accumulated in globomycin-treated Escherichia coli has been found to be affected by sec mutations, i.e., secA, secE, secY, secD, and secF, and by metabolic poisons which affect proton motive force (PMF). The effect of sec mutations on processing of glyceride-modified prolipoprotein in vivo was not due to a secondary effect on PMF. Neither a secF mutation nor metabolic poisons affected the processing of previously accumulated proOmpA protein in vivo, suggesting that the requirements for functional sec gene products and PMF are specific to the processing of lipoprotein precursors by signal peptidase II.     

Neither lipid modification nor processing of prolipoprotein is essential for the formation of murein-bound lipoprotein in Escherichia coli.

The relationship between the modification and processing of prolipoprotein and the formation of murein-bound lipoprotein has been investigated using Escherichia coli mutants altered in the signal sequence of prolipoprotein and an E. coli strain producing OmpF-Lpp hybrid protein. The glyceride-modified prolipoprotein in mutant lppT20 and in globomycin-treated wild-type strain were covalently attached to the peptidoglycan. Likewise, the unmodified prolipoproteins in mutants lppL20, lppV20, and lppG21 were attached to the peptidoglycan. The OmpF-Lpp hybrid protein that is processed but not modified with lipid due to the absence of the cysteine-containing modification site in the hybrid protein was also covalently linked to the peptidoglycan. These results indicate that neither lipid modification nor the processing of prolipoprotein is essential for the formation of murein-bound lipoprotein in E. coli. In contrast, introduction of a charged amino acid residue such as Asp or Arg at the 14th position of prolipoprotein affected not only the lipid modification and processing of the mutant prolipoprotein but also the formation of murein-bound lipoprotein. Replacement of the Gly14 with Glu or Lys partially affected the lipid modification and processing of prolipoprotein; the peptidoglycan of the lppE14 and lppK14 mutants contained a reduced amount of mature lipoprotein but no mutant prolipoprotein. In addition, lpp mutants A20I23I24 and A20I23K24 were found to be defective in both lipid modification/processing of prolipoprotein and the formation of murein-bound lipoprotein. The defective formation of murein-bound lipoprotein in the latter mutants may be related to an alteration in the secondary structure at the modification/processing site of the mutant prolipoproteins.     

Export of the outer membrane lipoprotein is defective in secD, secE, and secF mutants of Escherichia coli.

The export of major outer membrane lipoprotein has been found to be affected in secD, secE, and secF mutants of Escherichia coli, which are defective in protein export in general. After a shift to the nonpermissive temperature, the kinetics of accumulation of prolipoprotein and pre-OmpA protein was indistinguishable from that of pre-OmpA protein accumulation in the secD and secF mutants but different in the secE mutant. The prolipoprotein accumulated in the secD, secE, and secF mutants at the nonpermissive temperature was not modified with glyceride. We conclude from these results and those of previous studies that the export of lipoprotein requires all common sec gene products except the SecB protein, i.e., the SecA, SecD, SecE, SecF, and SecY proteins.     

Modification and processing of internalized signal sequences of prolipoprotein in Escherichia coli and in Bacillus subtilis

We have cloned the Escherichia coli lipoprotein structural gene (lpp) into a shuttle vector and studied its expression in both E. coli and in Bacillus subtilis. Using in vitro gene fusion techniques, the lpp gene was placed under the control of the promoter for the erythromycin-resistance (ery) gene. This fusion gene directed the synthesis of Braun's prolipoprotein which can be subsequently processed into the mature lipoprotein. In addition to the prolipoprotein, two ery-lpp hybrid proteins containing a 45- and a 22-amino acid extension preceding the NH2 terminus of prolipoprotein, respectively, are also synthesized in E. coli. The synthesis of these three proteins appears to involve the utilization of three distinct translation initiation sites. In B. subtilis, only two proteins are synthesized, the hybrid protein with a 45-amino acid extension and the prolipoprotein. In both E. coli and B. subtilis, the precursor forms of the hybrid proteins are lipid-modified, and they are processed to mature lipoprotein in vivo. These results indicate that internalized signal sequence containing the prolipoprotein modification and processing site (Leu-Ala-Glys-Cys) can function normally and permit the modification of hybrid proteins to lipid-modified precursors which can be subsequently processed by the globomycin-sensitive prolipoprotein signal peptidase.     

An alternate pathway for the processing of the prolipoprotein signal peptide in Escherichia coli

Previous studies showed that when the signal sequence plus 9 amino acid residues from the amino terminus of the major lipoprotein of Escherichia coli was fused to beta-lactamase, the resulting hybrid protein was modified, proteolytically processed, and assembled into the outer membrane as was the wild-type lipoprotein (Ghrayeb, J., and Inouye, M. (1983) J. Biol. Chem. 259, 463-467). We have constructed several hybrid proteins with mutations at the cleavage site of the prolipoprotein signal peptide. These mutations are known to block the lipid modification of the lipoprotein at the cysteine residue, resulting in the accumulation of unprocessed, unmodified prolipoprotein in the outer membrane. The mutations blocked the lipid modification of the hybrid protein. However, in contrast to the mutant lipoproteins, the cleavage of the signal peptides for the mutant hybrid proteins did occur, although less efficiently than the unaltered prolipo-beta-lactamase. The mutant prolipo-beta-lactamase proteins were cleaved at a site 5 amino acid residues downstream of the prolipoprotein signal peptide cleavage site. This new cleavage between alanine and lysine residues was resistant to globomycin, a specific inhibitor for signal peptidase II. This indicates that signal peptidase II, the signal peptidase which cleaves the unaltered prolipo-beta-lactamase, is not responsible for the new cleavage. The results demonstrate that the cleavage of the signal peptide is a flexible process that can occur by an alternative pathway when the normal processing pathway is blocked.     

Effects of replacing serine and threonine residues within the signal peptide on the secretion of the major outer membrane lipoprotein of Escherichia coli

We have investigated the importance of serine and threonine residues within the signal peptide in the secretion and processing of the major outer membrane lipoprotein precursor prolipoprotein in Escherichia coli. This was accomplished by systematically replacing these residues with alanine utilizing oligodeoxyribonucleotide-directed mutagenesis. The results demonstrated that the replacement of serine 15 but not threonine 16 alone caused an initial accumulation of membrane-bound unmodified prolipoprotein. In addition, replacement of both serine 15 and threonine 16 resulted in a greater accumulation of this membrane-bound precursor. The accumulated prolipoprotein could be matured to lipoprotein in a quantitative manner, and this process was inhibited by globomycin and carbonyl cyanide m-chlorophenylhydrazone. These results will be discussed in terms of the contribution that serine and threonine have in determining the overall secondary structure of the signal peptide and its importance in secretion and/or processing.     

Studies on the modification and processing of prolipoprotein in Escherichia coli. Effects of structural alterations in prolipoprotein on its maturation in wild type and lpp mutants

We have previously shown that an Escherichia coli mutant ( mlpA allele) containing a structurally altered murein prolipoprotein due to substitution of Gly14 by Asp14 , is globomycin resistant. In addition, the mutant prolipoprotein is not modified with glyceride and consequently remains uncleaved. Spontaneous revertants possessing a mature lipoprotein of apparent normal structure can be isolated by EDTA selection. Three revertants were chosen in the present study which included the analysis of kinetics of lipoprotein maturation and the determination of globomycin sensitivity. These pseudorevertants in the lpp gene which could be recognized by the anomalous prolipoprotein mobility in sodium dodecyl sulfate gels, exhibited altered globomycin sensitivity in vivo. Our results indicate that alterations in prolipoprotein structure affect the kinetics of prolipoprotein modification and processing reactions, both in vivo and in vitro. Pulse-chase experiments revealed the transient existence of unmodified prolipoprotein and modified prolipoprotein as biosynthetic intermediates of mature lipoprotein. The rate of prolipoprotein modification appeared to be slightly faster than that of processing in the wild type cell. In contrast, modification of prolipoprotein was rate limiting in a pseudorevertant strain 14R21 , and the processing of 14R21 modified prolipoprotein appeared to proceed more rapidly than that of wild type prolipoprotein, both in vitro and in vivo.     

Biogenesis of membrane lipoproteins in Escherichia coli.

Globomycin-resistant mutants of Escherichia coli have been isolated and partially characterized. Approximately 2-5% of these mutants synthesize structurally altered Braun's lipoprotein. The majority of these mutants contain unprocessed and unmodified prolipoprotein. One mutant is found to contain modified, processed, but structurally altered lipoprotein. Mutants containing lipid-deficient prolipoprotein or lipoprotein also show increased resistance to globomycin. These results suggest that the inhibition of processing of modified prolipoprotein by globomycin may require fully modified prolipoprotein as the biochemical target of this novel antibiotic. Our failure to isolate mutant containing cleaved but unmodified lipoprotein among globomycin-resistant mutants is consistent with the possibility that modification of prolipoprotein precedes the removal of signal sequence by a unique signal peptidase. Recent evidence indicates that the minor lipoproteins in the cell envelope of E. coli are also synthesized as lipid-containing prolipoproteins and the processing of these prolipoproteins is inhibited by globomycin. These results suggest the existence of modifying enzymes in E. coli which would transfer glyceryl and fatty acyl moieties to cysteine residues located in the proper sequences of the precursor proteins. This speculation is confirmed by our demonstration that Bacillus licheniformis penicillinase synthesized in E. coli as well as in B. licheniformis is a lipoprotein containing glyceride-cysteine at its NH2-terminus.     

EnvC, a new lipoprotein of the cytoplasmic membrane of Escherichia coli

Abstract A gene product with an apparent molecular mass of approximately 39000 Da can be identified in the cytoplasmic membrane of Escherichia coli upon expression of cloned envC . In this communication we report that the product was labelled with [³H]glycerol and [³H]palmitic acid, and a precursor molecule of increased molecular mass was accumulated when cells were treated with globomycin, a specific inhibitor for the prolipoprotein signal peptidase. The same precursor molecule was encoded by an envC mutant gene, in which the cysteine residue in a pentapeptide sequence, Leu-Ile-Ala-Gly-Cys²⁴ within the amino terminal region of EnvC, was replaced by tryptophane (Trp²⁴). This protein was not labelled with [³H]glycerol. The results demonstrate that the envC gene product represents a new lipoprotein of the cytoplasmic membrane of E. coli .     

Accumulation of prolipoprotein in Escherichia coli mutants defective in protein secretion.

The export of lipoprotein has been found to be affected in both secA and secY mutants of Escherichia coli which are defective in the secretion of a number of outer membrane and periplasmic proteins. The kinetics of accumulation of prolipoprotein upon a temperature shift to 42 degrees C is indistinguishable from that of pre-OmpA protein accumulation in the secA mutant. In both secA and secY mutants, the accumulated prolipoprotein is unmodified with glyceride and localized in the cytoplasmic membrane. We conclude from these results that the early steps in protein export are common to prolipoprotein and non-lipoprotein precursors. The pathways for the export of these two groups of precursor proteins diverge with regard to the modification and processing reactions which are late events in the export process.     

Assembly of outer membrane lipoprotein in an Escherichia coli mutant with a single amino acid replacement within the signal sequence of prolipoprotein.

We have compared the rate of assembly of outer membrane proteins including the lipoprotein in a pair of isogenic mlpA+ (lpp+) and mlpA (lpp) strains by pulse-chase experiments. The rate of assembly of the mutant prolipoprotein into the outer membrane was slightly slower than that of the wild-type lipoprotein. The rate of assembly of protein I and protein H-2 was similar in the wild type and the mutant, whereas the rate of assembly of protein II into the outer membrane was slightly reduced in the mutant strain. The organization of outer membrane was slightly reduced in the mutant strain. The organization of outer membrane proteins in the mutant cells appeared not to be grossly altered, based on the apparent resistance (or susceptibility) of these proteins toward trypsin treatment and their resistance to solubilization by Sarkosyl. Like the wild-type lipoprotein, the mutant prolipoprotein in the outer membrane was resistant to trypsin. On the other hand, the prolipoprotein in the cytoplasmic membrane fraction of the mutant cell envelope was susceptible to trypsin digestion. We conclude from these data that proteolytic cleavage of prolipoprotein is not essential for the translocation and proper assembly of lipoprotein into outer membrane.     

Deletion of internal twenty-one amino acid residues of Escherichia coli prolipoprotein does not affect the formation of the murein-bound lipoprotein.

Mutation pgsA affecting the phosphatidylglycerol phosphate synthesis is lethal for all but certain E. coli strains such as strains deleted for the lpp gene or strains containing unmodifiable prolipoprotein like lppD14. Strain SD312 pgsA3 is tolerant to pgsA mutation, which suggests the lpp alleles in strain SD312 pgsA3 and its parental strain SD12 may be defective. DNA sequence analysis of the lpp genes in Escherichia coli strains SD12 and SD312 pgsA using asymmetric polymerase chain reaction showed that the lpp alleles in these two strains contained a 63 base pair deletion corresponding to the 37th to 57th codons of the wild-type lpp gene. [3H]Palmitate labeling of strains SD12 and SDS312 showed that the mutant lipoprotein in SD12 strain was modified with lipid, while the prolipoprotein in SD312 was not modified. The shortened mature lipoprotein in SD12 and the lipid-modified prolipoprotein in globomycin-treated SD12 were found to be covalently attached to the peptidoglycan, while the unmodified prolipoprotein in SD312 did not form significant amounts of murein-bound lipoprotein.     

Mapping of the lipoprotein signal peptidase gene (lsp)

A pBR322 plasmid which contains a fragment of Escherichia coli DNA encoding the lipoprotein signal peptidase gene was used to transform Hfr polA1 strains. Ampr transformants were used as donors in conjugation experiments, and the location of the plasmid amp gene adjacent to the chromosomal lsp gene was determined to be near the thr ara loci of the E. coli chromosome. P1 transduction experiments established that the location of the lsp gene is closely linked to that of dapB , at 0.5 to 0.6 min on the E. coli genetic map. The position of the lsp gene was further determined to be between ileS and dapB by complementation analysis of an E. coli mutant showing temperature-sensitive prolipoprotein signal peptidase activity.     

The interaction of mammalian medium-chain hydrolase with yeast fatty acid synthetase

The prolipoprotein, a secretory precursor of the outer membrane lipoprotein of Escherichia coli, is known to be accumulated in the cell envelope when cells are grown in the presence of a cyclic antibiotic, globomycin. The prolipoprotein was localized in the cytoplasmic membrane when it was separated from the outer membrane by sucrose-density gradient centrifugation. However, when the envelope fraction was treated with sodium sarcosinate, the prolipoprotein was found almost exclusively in the sarcosinate-insoluble outer membrane fraction. The prolipoprotein separated in the cytoplasmic membrane by sucrose-density gradient centrifugation was soluble in sarcosinate and could not form a complex with the outer membrane once solubilized in sarcosinate. Labeling of the two lysine residues at positions 2 and 5 of the prolipoprotein with [3H]dinitrophenylfluorobenzene was enhanced 26-fold when the cells were disrupted by sonication. On the other hand, a tryptic fragment of the ompA protein, which is known to exist in the periplasmic space, increased its susceptibility to [3H]dinitrophenylfluorobenzene only 5.3-times upon disruption of the cell structure. These results indicate that the prolipoprotein accumulated in the presence of globomycin is translocated across the cytoplasmic membrane and interacts with the outer membrane. At the same time, it is attached to the cytoplasmic membrane with its amino-terminal signal peptide in such a way that the amino-terminal portion of the signal peptide containing two lysine residues is left inside the cytoplasm.     

Lipoprotein-28, a cytoplasmic membrane lipoprotein from Escherichia coli. Cloning, DNA sequence, and expression of its gene

Escherichia coli contains several lipoproteins in addition to the major outer membrane lipoprotein (Ichihara, S., Hussain, M., and Mizushima, S. (1981) J. Biol. Chem. 256, 3125-3129). We cloned the gene for one of these new lipoproteins by using a synthetic 15-mer oligonucleotide probe identical to the DNA sequence at the signal peptide cleavage site of the major lipoprotein. The DNA sequence of the cloned gene revealed an open reading frame encoding a 272-amino acid protein with a signal peptide of 23 amino acid residues. The amino acid sequence of the putative cleavage site region of the signal peptide, -Leu-Leu-Ala-Gly-Cys-, is identical to that of the major lipoprotein. When the cloned gene was expressed in E. coli, a gene product with an apparent molecular weight of approximately 29,000 was identified which agrees well with the calculated molecular weight (27,800). The product was labeled with [3H]glycerol, and a precursor molecule of increased molecular weight was accumulated when cells were treated with globomycin, a specific inhibitor for prolipoprotein signal peptidase. We thus designed the gene product as lipoprotein-28. Unlike the major lipoprotein, lipoprotein-28 was found to be localized in the cytoplasmic membrane. A possible orientation of lipoprotein-28 in the E. coli envelope is discussed.     

Effects of prolipoprotein signal peptide mutations on secretion of hybrid prolipo-beta-lactamase in Escherichia coli

Hybrid proteins were constructed by coupling beta-lactamase to the signal sequence (plus nine amino acids) of selected mutant prolipoproteins of Escherichia coli. The mutant prolipoprotein signal peptides contained lesions in two structural domains of the signal peptide, the basic amino-terminal domain and the hydrophobic core domain. We then compared the processing and localization of the mutant prolipo-beta-lactamases to the processing and localization of the comparable mutant prolipoproteins. We show that a mutant signal sequence with an anionic amino terminus exhibits similar limitations in the processing of prolipo-beta-lactamase as previously observed in prolipoprotein. Deletion of four hydrophobic residues from hydrophobic core results in a signal peptide which slowly translocates a fraction of the total mutant hybrid protein synthesized. This signal peptide was previously shown to translocate lipoprotein efficiently. Alteration of this hydrophobic core, which stimulated synthesis of mutant prolipoproteins, does not stimulate synthesis of prolipo-beta-lactamase. Finally mutations that slowed processing of prolipoprotein by affecting the proposed helical structure of the signal peptide had no significant effect on the processing of prolipo-beta-lactamase. These results suggest that the positively charged amino-terminal domain of the signal peptide has a common role in protein secretion regardless of the secretory protein. On the other hand, other domains of the signal peptide exhibit different phenotypes when the secretory protein is changed.     

A distinct signal peptidase for prolipoprotein in escherichia coli

We have previously demonstrated the modification and processing of Escherichia coli prolipoprotein (Braun's) in vitro (Tokunaga M, Tokunaga H. Wu HC: Proc Natl Acad Sci USA 79:2255, 1982). Using this in vitro assay of prolipoprotein signal peptidase and globomycin selection, we have isolated and partially characterized an E coli mutant which contained a higher level of prolipoprotein signal peptidase activity. In contrast, the procoat protein signal peptidase activity was not increased in this mutant as compared to the wild-type strain. Furthermore, E coli strains containing cloned procoat protein signal peptidase gene were found to contain elevated levels of procoat protein signal peptidase, but normal levels of prolipoprotein signal peptidase. These two signal peptidase activities were also found to exhibit different stabilities during storage at 4°C. Thus biochemical, immunological, and genetic evidence clearly indicate that prolipoprotein signal peptidase is distinct from procoat protein signal peptidase in E coli.     

Translocation of colicin E1 through cytoplasmic membrane of Escherichia coli

The product of the malE—lacZ gene fusion was reported to compete with some proteins including outer membrane lipoprotein in the protein translocation across the Echerichia coli membrane. The fusion product also inhibited colicin E1 export. Furthermore, globomycin, which accumulated prolipoprotein in the membrane, inhibited the translocation of colicin E1 in the wild-type cells, but not in lipoprotein-negative mutant cells. Since colicin E1 contains the internal signal-like sequence [Proc. Natl. Acad. Sci. USA (1982) 79, 2827–2831], these results suggest that colicin E1 is exported by the aid of this sequence at a common site for maltose-binding protein and lipoprotein translocation.