Effects of Inorganic Polyphosphate on the Proteolytic and DNA-binding Activities of Lon in Escherichia coli

Abstract

Lon belongs to a unique group of proteases that bind to DNA and is involved in the regulation of several important cellular functions, including adaptation to nutritional downshift. Previously, we revealed that inorganic polyphosphate (polyP) increases in Escherichia coli in response to amino acid starvation and that it stimulates the degradation of free ribosomal proteins by Lon. In this work, we examined the effects of polyP on the proteolytic and DNA-binding activities of Lon. An order-of-addition experiment suggested that polyP first binds to Lon, which stimulates Lon-mediated degradation of ribosomal proteins. A polyP-binding assay using Lon deletion mutants showed that the polyP-binding site of Lon is localized in the ATPase domain. Because the same ATPase domain also contains the DNA-binding site, polyP can compete with DNA for binding to Lon. In fact, an equimolar amount of polyP almost completely inhibited DNA-Lon complex formation, suggesting that Lon binds to polyP with a higher affinity than it binds to DNA. Collectively, our results showed that polyP may control the cellular activity of Lon not only as a protease but also as a DNA-binding protein.

A Polyphosphate-Lon Protease Complex in the Adaptation ofEscherichia colito Amino Acid Starvation

“…We found that Lon efficiently degrades L24 when Lon and polyP are mixed together prior to the addition of L24, whereas there is little degradation of L24 when it is first mixed with polyP before the addition of Lon. 21) This result indicates that polyP-Lon complex formation is important for the stimulation of protein degradation and that polyP does not act as a tag for Lonmediated degradation. PolyP functions as an adaptor molecule to stimulate a Lon-substrate complex.…”

“…19,20) In contrast to the ribosomal proteins tested, the degradation of a maltose-binding protein (MBP)-SulA fusion was slightly inhibited in the presence of 1.4 mM polyP. 21) Therefore, polyP does not always stimulate Lon-mediated protein degradation.…”

“…21) But not all polyP-binding proteins are degraded by Lon in the presence of polyP. For example, PPK, L15, and L18 bind to polyP, but they are not degraded by Lon in its presence.…”

“…Therefore, it is likely that polyP binding is necessary but not sufficient for stimulation of Lonmediated proteolysis. 21) Is it that polyP first binds to Lon and then the polyPLon complex degrades the ribosomal proteins? Alternatively, is it that polyP binds to the ribosomal proteins and then Lon recognizes the polyP-ribosomal protein complex for degradation?…”

Roles for the Human ATP-dependent Lon Protease in Mitochondrial DNA Maintenance

“…DNA binding by the ATP-dependent Lon (La) protease is evolutionarily conserved from bacteria to man, suggesting that it is an essential property of the protein (1)(2)(3)(4)(5)(6)(7)(8). Purified bacterial Lon has been shown in some experiments to bind doublestranded DNA without apparent sequence specificity when assayed with relatively large DNA molecules (e.g.…”