Early steps initiating a degradation pathway in Escherichia coli. Characterization of the first intermediate.
Our previous studies demonstrated that a site-specific cleavage event initiates the degradation of large premature termination polypeptides of beta-galactosidase in Escherichia coli. We have isolated the first cleavage intermediate, the "B" polypeptide, by elution from sodium dodecyl sulfate-polyacrylamide gels. The NH2 terminus of this protein, determined by automated Edman degradation, was that of the wild-type molecule and thus established that the first cleavage event was at the COOH-terminal end. The sequence of the COOH-terminal end of the B polypeptide was determined by using the enzyme carboxypeptidase Y. Direct assignment of COOH-terminal residues was made by using o-phthaldialdehyde derivatization and the stoichiometry confirmed by a double-label analysis. The COOH-terminal end of the B polypeptide is at position 837 in the beta-galactosidase sequence. If a single endoproteolytic cleavage event was responsible, the cleavage would have occurred between 2 threonine residues (at positions 837 and 838) that are located within a hydrophobic domain. We have observed other covalent modifications that precede the appearance of the B polypeptide, but these do not appear to participate in signaling the first cleavage event. The structure of the COOH-terminal end of B suggests a high degree of specificity by the initial cleavage enzyme. We propose that this unique site serves as a specific signal and that exposure of this site to the specific cleavage enzyme controls the event initiating the degradation pathway.
D000595:Amino Acid Sequence; D002268:Carboxypeptidases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D005696:Galactosidases / Q000096:biosynthesis*; D010443:Peptide Chain Termination, Translational; D010455:Peptides / Q000378:metabolism; D001616:beta-Galactosidase / Q000096:biosynthesis* / Q000378:metabolism
D010455:Peptides; D005696:Galactosidases; D001616:beta-Galactosidase; D002268:Carboxypeptidases; C022633:serine carboxypeptidase