A small hydrophobic domain anchors leader peptidase to the cytoplasmic membrane of Escherichia coli.
Leader peptidase is an enzyme of the Escherichia coli cytoplasmic membrane which removes amino-terminal leader sequences from many secreted and membrane proteins. Three potential membrane-spanning segments exist in the first 98 amino acids of leader peptidase. We have characterized the topology of leader peptidase based on its sensitivity to protease digestion. Proteinase K and trypsin treatment of right-side-out inner membrane vesicles and spheroplasts yields protected fragments of approximately 80 and 105 amino acid residues, respectively. We have shown that both fragments are derived from the amino terminus of the protein and that the smaller protected peptide can be derived from the larger. Removal of the third potential membrane-spanning segment (residues 82-98) does not affect the size of the proteinase K-protected fragment but does reduce the size of the trypsin-protected peptide. Because the proteinase K-protected fragment is about 9000 daltons, is derived from the amino terminus of leader peptidase, and its size is not affected when amino acids 82-98 are removed from the protein, it must extend from the amino terminus to approximately residue 80. Likewise, the trypsin-protected fragment must extend from the amino terminus to about residue 105. These data suggest a model for the orientation of leader peptidase in which the second hydrophobic stretch (residues 62-76) spans the cytoplasmic membrane and the third hydrophobic stretch resides in the periplasmic space.
D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D004262:DNA Restriction Enzymes; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D008565:Membrane Proteins; D010447:Peptide Hydrolases; D010957:Plasmids; D012697:Serine Endopeptidases
D008565:Membrane Proteins; D004262:DNA Restriction Enzymes; D010450:Endopeptidases; D010447:Peptide Hydrolases; D012697:Serine Endopeptidases; C026515:type I signal peptidase