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.
Howard
S P
SP
University of Regina, Saskatchewan, Canada. peter.howard@uregina.ca
Lindsay
L
L
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003087:Colicins / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D050356:Lipid Metabolism; D008074:Lipoproteins; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; C058485:CAL protein, Citrobacter freundii; D003087:Colicins; D004269:DNA, Bacterial; D008074:Lipoproteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; C061680:colicin A lysis protein precursor, Bacteria