Requirement for Signal Peptide Cleavage of
            <i>Escherichia coli</i>
            Prolipoprotein

Inouye, Sumiko; Hsu, Chen-Ming; Itakura, Keiichi; Inouye, Masayori

doi:10.1126/science.6344218

Cited by 58 publications

(23 citation statements)

References 12 publications

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“…Cal protein is translated from the same mRNA as colicin A (23), but it has to interact with modifying enzymes and signal peptidase II which are membrane bound. It has been reported that mutations altering amino acids in the vicinity of (12,18,19,30) and prepenicillinase (14,38) affect the modification, processing, and secretion of these proteins in both E. coli and Bacillus subtilis. Furthermore, various studies have suggested that the overall conformation of preproteins can exert a considerable effect on the rate of modification and processing (for a review, see reference 38).…”

Section: Discussionmentioning

confidence: 99%

“…Inouye et al (19) have shown that replacing the consensus sequence Leu-Ala-Gly-Cys with Leu-Ala-Ala-Cys does not affect the synthesis, processing, or export of the lipoprotein. It has been previously demonstrated that the Cal protein undergoes a particularly slow processing accompanied by accumulation of the signal peptide and that the mature form is partly released to the spent medium with colicin A (7).…”

mentioning

confidence: 99%

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Lipoprotein nature of the colicin A lysis protein: effect of amino acid substitutions at the site of modification and processing

Cavard¹,

Baty²,

Howard³

et al. 1987

J Bacteriol

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The colicin A lysis protein (Cal) is required for the release of colicin A to the medium by producing bacteria. This protein is produced in a precursor form that contains a cysteine at the cleavage site (-Leu-Ala-Ala-Cys). The precursor must be modified by the addition of lipid before it can be processed. The maturation is prevented by globomycin, an inhibitor of signal peptidase II. Using oligonucleotide-directed mutagenesis, the alanine and cysteine residues in the -1 and + 1 positions of the cleavage site were replaced by proline and threonine residues, respectively, in two different constructs. Both substitutions prevented the normal modification and cleavage of the protein. The marked activation of the outer membrane detergent-resistant phospholipase A observed with wild-type Cal was not observed with the Cal mutants. Both Cal mutants were also defective for the secretion of colicin A. In one mutant, the signal peptide appeared to be cleaved off by an alternative pathway involving signal peptidase I. Electron microscope studies with immunogold labeling of colicin A on cryosections of pidA and cal mutant cells indicated that the colicin remains in the cytoplasm and is not transferred to the periplasmic space. These results demonstrate that Cal must be modified and processed to activate the detergent-resistant phospholipase A and to promote release of colicin A.Proteins of low molecular weight called lysis protein are required for the release of colicins or cloacin from Escherichia coli producing cells (7,13,21,31,33). The genes that encode these proteins are located downstream of the immunity genes on the colicin plasmid. They are organized in operons with the corresponding colicin structural genes (8,13,23,34). Their expression is thus under the same regulation as that of the colicin structural genes, but they seem to be produced in lower amounts than the colicins owing to the presence of a transcription terminator after the colicin structural gene (10,13,26).The structures of the five known lysis proteins feature a high degree of homology (7,8,13,35). They all contain a signal peptide with a sequence at the cleavage site resembling the consensus lipoprotein modification sequence described by Wu et al. (38). The polypeptide chain of the mature form is small: 33 amino acids for the colicin A lysis (Cal) protein and 28 amino acids for colicin El, E2, and E3 and cloacin DF13 lysis proteins.The consensus sequence for lipoproteins, Leu-Ala-GlyCys (12,20,38), constitutes the recognition site for lipid modification enzymes. A glyceride thioether is formed on the cysteine residue, after which lipoprotein is processed by signal peptidase II between the glycine and cysteine residues. This signal peptidase is specifically inhibited by globomycin, a cyclic peptide antibiotic (15, 35). After processing, a fatty acid is covalently attached to the N-terminal cysteine.The Cal protein, encoded by the cal gene, has the sequence Leu-Ala-Ala-Cys at the end of its signal sequence, that is, the lipoprotein consensus sequence in ...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Lipoprotein nature of the colicin A lysis protein: effect of amino acid substitutions at the site of modification and processing

Cavard¹,

Baty²,

Howard³

et al. 1987

J Bacteriol

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“…This sequence conforms favorably to the consensus sequence for lipid modification of gram-negative bacterial lipoproteins (4,10,26 (4,26). If so, the Cys-20 residue of the 17K antigen would be modified through a thio-ester linkage with glycerol, which would subsequently be modified by the addition of fatty acids.…”

Section: Discussionmentioning

confidence: 99%

“…The deduced amino acid sequence of the 17K antigen contained the residues Leu-Gln-Ala-Cys at positions 17 to 20. This sequence is strikingly similar to the consensus sequence Leu-Ala(Gly)-Gly(Ala)-Cys for lipid modification in E. coli (4,10,26) harboring the plasmid vector pKK223-3 was used as the source of radiolabeled protein (Fig. 5A, lane C).…”

mentioning

confidence: 99%

“…Lipid modification of the murein lipoprotein of E. coli has been shown to occur at the cysteine residue within the consensus sequence Leu-Ala(Gly)-Gly(Ala)-Cys (4,10,26). To determine whether the 17K antigen was modified by a similar mechanism, we changed the cysteine codon (TGT) within the presumed lipid modification site by in vitro mutangenesis to a glycine codon (GGC) and measured the effect of this alteration on the modification of the translational product.…”

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confidence: 99%

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Expression of the gene encoding the 17-kilodalton antigen from Rickettsia rickettsii: transcription and posttranslational modification

1988

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Recently, we reported the molecular cloning and nucleotide sequence analysis of a gene from Rickeffsia rckettsii that codes for a 17-kilodalton antigen (17K antigen) and is preceded by sequences closely resembling the -10 and -35 consensus sequences for recognition by Escherichia coli RNA polymerase (Anderson et al., J. Bacteriol. 169:2385-2390. Experimnents described in this report indicate that the start sites for initiating transcription of the 17K antigen gene are identical in the E. coli clone and in intact R. rickettsii. In each case, initiation was shown to begin 9 bases downstream of the presumed Pribnow box sequence (TATACT). A 169-base-pair fragment containing the promoter sequence initiated transcription in both directions when cloned into an E. coli promoter probe vector. The rickettsial fragment was found to contain sequences identical to the -10 region (but not the -35 region) of the E. coli promoter consensus sequence directed away from the 17K antigen gene. The amino-terminal portion (residues 17 to 20) of the deduced amino acid sequence for the 17K antigen contained the tetrapeptide Leu-Gln-Ala-Cys, a sequence that conforms favorably to those described for lipid modification and cleavage by lipoprotein signal peptidase II. The 17K antigen produced by the E. coli clone was shown to be labeled with [3H]

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Isolation and characterization of genes encoding two chitinase enzymes from Serratia marcescens

et al. 1986

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Analysis of clones isolated from a cosmid DNA library indicates that the Serratia marcescens chromosome contains at least two genes, chiA and chiB, which encode distinct secreted forms of the enzyme chitinase. These genes have been characterized by inspection of chitinase activity and secreted proteins in Escherichia coli strains containing subclones of these cosmids. The two chitinase genes show no detectable homology to each other. DNA sequence analysis of one of the genes predicts an amino acid sequence with an N‐terminal signal peptide typical of genes encoding secreted bacterial proteins. This gene was mutagenized by cloning a neomycin phosphotransferase gene within its coding region, and the insertion mutation was recombined into the parental S. marcescens strain. The resulting chiA mutant transconjugant showed reduced chitinase production, reduced inhibition of fungal spore germination and reduced biological control of a fungal plant pathogen.

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Requirement for Signal Peptide Cleavage of Escherichia coli Prolipoprotein

Cited by 58 publications

References 12 publications

Lipoprotein nature of the colicin A lysis protein: effect of amino acid substitutions at the site of modification and processing

Lipoprotein nature of the colicin A lysis protein: effect of amino acid substitutions at the site of modification and processing

Expression of the gene encoding the 17-kilodalton antigen from Rickettsia rickettsii: transcription and posttranslational modification

Isolation and characterization of genes encoding two chitinase enzymes from Serratia marcescens

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