Site‐directed mutagenesis of La protease

Amerik, Alexander Y.; Antonov, V.K.; Gorbalenya, Alexander E.; Kotova, S. A.; Rotanova, T. V.; Shimbarevich, Elena V.

doi:10.1016/0014-5793(91)80053-6

Cited by 91 publications

(25 citation statements)

References 15 publications

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“…The first region (amino acids 526-597 in the human Lon sequence) has =z47% complete identity and contains the consensus sequence for ATPbinding sites (28). The second region (amino acids 844-870) shows approximately 73% complete identity and includes the serine residue at the putative proteolytic active site (29).…”

Section: Resultsmentioning

confidence: 99%

A human mitochondrial ATP-dependent protease that is highly homologous to bacterial Lon protease.

Wang

Gottesman²,

Willingham³

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

178

124

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We have cloned a human ATP-dependent protease that is highly homologous to members of the bacterial Lon protease family. The cloned gene encodes a protein of 963 amino acids with a calculated molecular mass of 106 kDa, slightly higher than that observed by Western blotting the protein from human tissues and ceil lines (100 kDa). A single species of mRNA was found for this Lon protease in all human tissues examined. The protease is encoded in the nucleus, and the amino-terminal portion of the protein sequence contains a potential mitochondrial targeting presequence. Immunofluorescence microscopy suggested a predominantly mitochondrial localization for the Lon protease in cultured human ceils. A truncated LON gene, in which translation was initiated at Metll8ofthe coding sequence, was expressed in Escherichia coli and produced a protease that degraded a-casein in vitro in an ATP-dependent manner and had other properties similar to E. col Lon protease.

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Section: Resultsmentioning

confidence: 99%

A human mitochondrial ATP-dependent protease that is highly homologous to bacterial Lon protease.

Wang

Gottesman²,

Willingham³

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

178

124

View full text Add to dashboard Cite

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“…For construction of pBADlonS679A, carrying a mutation at the serine active site (Fig. 1), the SalI-SphI fragment at the 3Ј end of the lon gene was amplified from pBR327-S679A (26), by using primers Oli1 (5Ј-GCTGACCGTCGACGATAG) and Oli2 (5Ј-ACATGCAT-GCGGTCACTATTTTG). On sequencing, an unexpected SphI site was present 23 bp from the stop codon of lon, leading to a frame-shift mutation at the carboxyl-terminal end of the S679A Lon.…”

Section: Methodsmentioning

confidence: 99%

“…In Lon, a consensus site for ATP binding and hydrolysis has been shown by mutation to be necessary for proteolysis and ATPase activity; mutations in the active site serine ( Fig. 1) block proteolysis but do not fully abolish ATPase activity (14,(26)(27)(28)(29)(30). How and where substrate binding takes place and how substrates are presented to the active site, however, have not been clarified, and very few mutations other than those in the serine or ATPase sites have been described.…”

mentioning

confidence: 99%

Substrate sequestration by a proteolytically inactive Lon mutant

Melderen

Gottesman

1999

Proc. Natl. Acad. Sci. U.S.A.

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Lon protein of Escherichia coli is an ATPdependent 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 ⌬lon mutant. These in vivo results suggest that the pathway of degradation by Lon couples ATPdependent 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. Intracellular protein degradation is responsible for the rapid turnover of specific, unstable regulatory proteins and the clearing of abnormal proteins from the cytoplasm. The high molecular weight, ATP-dependent proteases that are primarily responsible for this degradation must recognize and select their protein substrates from among an enormous pool of other proteins that should not be degraded. Recent studies suggest that binding of substrate is followed by unfolding of the substrate or, at least of a portion of it, sufficient to gain access to and be cleaved by the proteolytic sites (1, 2). Processive degradation of the substrate then proceeds, leading to the release of short peptides of 6-15 aa.

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“…The Lon monomer includes an N terminus of unknown function, a central ATPase containing a typical ATP-binding motif, and a C-terminal proteolytic domain with a catalytically active residue at Ser-679 (5)(6)(7)(8). The N-terminal region also contains a "basic-acid-basic" region (9).…”

mentioning

confidence: 99%

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

Nomura

Kato

Takiguchi

et al. 2004

Journal of Biological Chemistry

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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.

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Site‐directed mutagenesis of La protease

Cited by 91 publications

References 15 publications

A human mitochondrial ATP-dependent protease that is highly homologous to bacterial Lon protease.

A human mitochondrial ATP-dependent protease that is highly homologous to bacterial Lon protease.

Substrate sequestration by a proteolytically inactive Lon mutant

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

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