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PMID: 9869642 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Lon-mediated proteolysis of the Escherichia coli UmuD mutagenesis protein: in vitro degradation and identification of residues required for proteolysis.

Genes & development ·Vol. 12 ·No. 24 ·1998-12-15 ·Pages 3889-99

Gonzalez M, Frank EG, Levine AS, Woodgate R

Abstract

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.

MeSH Terms
ATP-Dependent Proteases Adenosine Triphosphate/metabolism Alanine/genetics,metabolism Amino Acid Sequence Bacterial Proteins/chemistry,genetics,isolation & purification,metabolism Chymotrypsin/metabolism DNA-Directed DNA Polymerase Escherichia coli/enzymology,metabolism Escherichia coli Proteins Half-Life Heat-Shock Proteins/genetics,metabolism Molecular Sequence Data Mutagenesis, Site-Directed Protease La Protein Conformation Protein Processing, Post-Translational Rec A Recombinases/genetics,metabolism Recombinant Fusion Proteins/metabolism SOS Response, Genetics Sequence Homology, Amino Acid Serine Endopeptidases/genetics,metabolism Substrate Specificity
Chemicals
Bacterial Proteins Escherichia coli Proteins Heat-Shock Proteins Recombinant Fusion Proteins phleomycin resistance protein, Streptoalloteichus hindustanus Adenosine Triphosphate Rec A Recombinases DNA-Directed DNA Polymerase UmuD protein, E coli ATP-Dependent Proteases Serine Endopeptidases Chymotrypsin Lon protein, E coli Protease La Alanine
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Gonzalez 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
Levine A S
Woodgate R
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Article Info
Journal
Genes & development
Abbr.
Genes Dev
ISSN
0890-9369
DOI
10.1101/gad.12.24.3889
Published
1998-12-15
Pages
3889-99
Language
English
Region
United States
NLM ID
8711660
PMCID
PMC317269
Subset
IM
External Links
PubMed source DOI: 10.1101/gad.12.24.3889 PMC Full Text View journal details
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