Lipoprotein nature of the colicin A lysis protein: effect of amino acid substitutions at the site of modification and processing.
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 cystein 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 pldA 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.
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002451:Cell Compartmentation; D003087:Colicins / Q000378:metabolism*; D004789:Enzyme Activation; D000431:Ethanol / Q000494:pharmacology; D008074:Lipoproteins / Q000378:metabolism*; D010169:Palmitic Acids / Q000378:metabolism; D010455:Peptides / Q000494:pharmacology; D010740:Phospholipases / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021382:Protein Sorting Signals / Q000235:genetics; D013329:Structure-Activity Relationship
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D003087:Colicins; D008074:Lipoproteins; D010169:Palmitic Acids; D010455:Peptides; D021382:Protein Sorting Signals; D000431:Ethanol; C017339:globomycin; D010740:Phospholipases