title: Substrate sequestration by a proteolytically inactive Lon mutant.
abstract: Lon protein of Escherichia coli is an ATP-dependent protease responsible for the rapid turnover of both abnormal and naturally unstable proteins, including SulA, a cell division inhibitor made after DNA damage, and RcsA, a positive regulator of transcription. Lon is a multimer of identical 94-kDa subunits, each containing a consensus ATPase motif and a serine active site. We found that overexpressing Lon, which is mutated for the serine active site (LonS679A) and is therefore devoid of proteolytic activity, unexpectedly led to complementation of the UV sensitivity and capsule overproduction of a lon deletion mutant. SulA was not degraded by LonS679A, but rather was completely protected by the Lon mutant from degradation by other cellular proteases. We interpret these results to mean that the mutant LonS679A binds but does not degrade Lon substrates, resulting in sequestration of the substrate proteins and interference with their activities, resulting in apparent complementation. Lon that carried a mutation in the consensus ATPase site, either with or without the active site serine, was no longer able to complement a Deltalon mutant. These in vivo results suggest that the pathway of degradation by Lon couples ATP-dependent unfolding with movement of the substrate into protected chambers within Lon, where it is held until degradation proceeds. In the absence of degradation the substrate remains sequestered. Comparison of our results with those from a number of other systems suggest that proteins related to the regulatory portions of energy-dependent proteases act as energy-dependent sequestration proteins.
authors.affiliation: Laboratory of Molecular Biology, National Cancer Institute, Bethesda, MD 20892-4255, USA.
authors.forename: L
authors.initials: L
authors.lastname: Van Melderen
authors.forename: S
authors.initials: S
authors.lastname: Gottesman
mesh_terms: D049069:ATP-Dependent Proteases; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001089:Arabinose / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D002455:Cell Division; D016384:Consensus Sequence; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000528:radiation effects; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D049070:Protease La; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012694:Serine; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014466:Ultraviolet Rays
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; C053139:sulA protein, E coli; D012694:Serine; D000255:Adenosine Triphosphate; D001089:Arabinose; D049069:ATP-Dependent Proteases; D012697:Serine Endopeptidases; C483567:Lon protein, E coli; D049070:Protease La; D000251:Adenosine Triphosphatases