Temperature-sensitive prolipoprotein signal peptidase in an Escherichia coli mutant: use of the mutant for an efficient and convenient assay system.
Escherichia coli mutant Y815 accumulates the precursor of lipoprotein (prolipoprotein) in its envelope. The accumulated prolipoprotein could be chased to mature lipoprotein at 30 degrees C but not at 60 degrees C (Yamagata, H., Ippolito, C., Inukai, M., & Inouye, M. (1982) J. Bacteriol. 152, 1163). When the envelope fraction prepared from the mutant was mixed with the envelope fraction prepared from wild-type E. coli cells and incubated at 60 degrees C in the presence of Triton X-100, the prolipoprotein in the mutant envelope fraction was cleaved rapidly to mature lipoprotein. The cleavage was dependent on the addition of wild-type envelope fraction and Triton X-100 to the reaction mixture. This indicated that the prolipoprotein accumulated in the mutant envelope is a good substrate for the signal peptidase which cleaves the signal peptide from the prolipoprotein, and hence the accumulation of prolipoprotein was due to lack of the signal peptidase in the mutant. The optimum concentration of Triton X-100 for the cleavage of the prolipoprotein in the above in vitro system was 0.05 to 0.1% (v/v) at a wild-type envelope concentration of 0.35 mg protein/ml. Prolipoprotein accumulated in wild-type cells on treatment with globomycin, a specific inhibitor of the signal peptidase, was also cleaved to mature lipoprotein under the same conditions. Triton X-100 was shown to solubilize the signal peptidase from the envelope fraction. The cleavage of the prolipoprotein was rapid and complete in the in vitro system described here, which provides an efficient and convenient assay system for the solubilized signal peptidase for prolipoprotein.
D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D002474:Cell-Free System; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D008074:Lipoproteins / Q000378:metabolism*; D008565:Membrane Proteins; D009154:Mutation; D010455:Peptides / Q000494:pharmacology; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012697:Serine Endopeptidases; D013696:Temperature
D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D008074:Lipoproteins; C099932:Lpp protein, E coli; D008565:Membrane Proteins; D010455:Peptides; D011498:Protein Precursors; C017339:globomycin; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase