Lon-mediated proteolysis of the Escherichia coli UmuD mutagenesis protein: in vitro degradation and identification of residues required for proteolysis.
Most SOS mutagenesis in Escherichia coli is dependent on the UmuD and UmuC proteins. Perhaps as a consequence, the activity of these proteins is exquisitely regulated. The intracellular level of UmuD and UmuC is normally quite low but increases dramatically in lon- strains, suggesting that both proteins are substrates of the Lon protease. We report here that the highly purified UmuD protein is specifically degraded in vitro by Lon in an ATP-dependent manner. To identify the regions of UmuD necessary for Lon-mediated proteolysis, we performed 'alanine-stretch' mutagenesis on umuD and followed the stability of the mutant protein in vivo. Such an approach allowed us to localize the site(s) within UmuD responsible for Lon-mediated proteolysis. The primary signal is located between residues 15 and 18 (FPLF), with an auxiliary site between residues 26 and 29 (FPSP), of the amino terminus of UmuD. Transfer of the amino terminus of UmuD (residues 1-40) to an otherwise stable protein imparts Lon-mediated proteolysis, thereby indicating that the amino terminus of UmuD is sufficient for Lon recognition and the ensuing degradation of the protein.
Gonzalez
M
M
Section on DNA Replication, Repair, and Mutagenesis, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-2725 USA.
Frank
E G
EG
Levine
A S
AS
Woodgate
R
R
D049069:ATP-Dependent Proteases; D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002918:Chymotrypsin / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006207:Half-Life; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D049070:Protease La; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013014:SOS Response, Genetics; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; C099353:phleomycin resistance protein, Streptoalloteichus hindustanus; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases; D004259:DNA-Directed DNA Polymerase; C055218:UmuD protein, E coli; D049069:ATP-Dependent Proteases; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C483567:Lon protein, E coli; D049070:Protease La; D000409:Alanine